Historical Aspects of Testicular Function: Virility, Androgen Production, and Spermatogenesis

Abstract

From antiquity, man has been fascinated by at least 2 processes of testicular function: virility and reproduction; their biological basis was uncovered beginning in the mid-19th century.

We have divided the search into 3 epochs: the speculative and observational, the experimental, and the biochemical/physiological. The first begins with Susruta, approximately 3000 years ago, and winds its way through the Greek, Roman, the Christian Bible, Arabic, Chinese, and Indian pathways before coalescing in Europe at the dawn of the Renaissance. The second began with. Thomas Willis, who postulated a virilizing factor from the testis. A century later de Bordeu hypothesized a neurosecretory function for the hypothalamus/pituitary. After John Hunter began to study testis implantation, it was Berthold who showed a secretory function of the testis following implantation. Charles-Éduard Brown-Séquard focused the medical and lay communities on testis secretion with self-experimentation with animal testis extracts leading to more than 4 decades of uncertainty in the newly launched science of endocrinology. Multiple series of testicular implants and vas deferens ligations for the purposes of rejuvenation of older men followed.

The medical experimentation continued in the biochemical/physiological epoch where androgenic steroids were isolated, purified, identified, synthesized, and used in clinical trials. The effects of castration, some known from antiquity, were placed on a modern scientific basis with studies of the Skoptzy, a self-castrating sect from Russia and the castrati opera singers. Details of hypothalamic-pituitary-gonadal axis function as well as the embryology of male sexual differentiation and spermatogenesis were defined during this epoch.

Graphical Abstract

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There are at least 2 fundamental biological processes of testicular function: the first is the maturation of the germ cells, and the second is the secretion of 1 or more substances known as testis hormone(s) (1). The historian VC Medvei described the testis as “the oldest key to the endocrine treasure trove” (2). According to the age of the loss of function of the testis, one notes lack of development of the secondary sexual characteristics, changes in body composition, or the loss of sexual capacity, desire, and fertility (3). In this historical review we focus on the observations and then the experimental evidence that permitted the extraction, purification, identification, and chemical synthesis of the primary androgenic hormone testosterone as well as the physiology of the hypothalamic-pituitary-testicular axis and spermatogenesis. The word testosterone is derived from the stems of testicle, sterol, with the suffix from ketone, once the structure of the molecule was determined (4). Along the way, other androgens and a few testicular peptide hormones are mentioned, especially as they affect the physiology (and pathology) of the hypothalamic-pituitary-gonadal axis focusing on males: children, adolescents, and adults.

Over time several words have been used to indicate what we now would call internal secretions or hormones, at least since 1855, when in a lecture at the Collège de France Claude Bernard described the phenomenon: the liver has an external secretion of bile and an internal secretion of sugar (5). More recently paracrine, autocrine, and intracrine secretions have been described. A paracrine mechanism denotes cells that release a chemical messenger to affect nearby cells; the autocrine mechanism denotes cells that secrete the messenger to affect the same cell through external receptors and intracrine where the secretion and action are within the same cell, never reaching the intercellular space. In 1913 Biedl proclaimed that “the honour of having first proved the existence of an internal secretion and of having realized its significance belongs undoubtedly to Bernard” (6).

The word endocrinology is derived from the Greek words endon (inside or within) and krino (to separate or sift). In 1905 Starling in his Croonian Lecture put forth the word hormone, derived from the Greek hormao, which means to put into quick motion or to excite or arouse (7):

These chemical messengers …, or “hormones” as we may call them, have to be carried from the organ where they are produced to the organ which they affect, by means of the blood stream, and the continually recurring physiological needs of the organism must determine their production and circulation through the body. [(7), p. 6]

Eugène Gley proposed at an international congress of medicine in 1913 a similar word, harmozone, also from the Greek I regulate, for the secretions of the interstitial cells of the testis, the corpus luteum, the thyroid, and the pituitary—those factors that are morphogenic and slowly influence growth and nutrition (8-10). The word hermetick (with the property of stimulating) of the same derivation had been used in 1666 by John Smith to describe the power and contraction of muscle (11).

Speculative and Observational Epoch

Early efforts to understand either hypogonadism or loss of virility with aging or, for that matter, any disease, centered around curses of the gods or witches (Malleus Maleficarum, Hammer of the Witches) and Divine punishment, including those due to deviant practices; the lack of a substance that existed in plants or animal organs; or, perhaps, the aging process itself (12-14):

morbus … ad iram deorum imortalium relatos esse (that diseases were attributed to the wrath of the immortal gods). [(12), p. 4]

The extant knowledge about a putative male factor was gained from observations of animals and men who had been castrated or were born hypogonadal, as indicated in the literature as eunuchs, men who had been castrated or born without testicular function, and their habitus as eunuchoid, for which the arm span is at least 5 cm greater than the height. The present understanding is that estrogens, whether derived from testosterone or secreted, are responsible for epiphyseal closure. Modern endocrinology has described this state (disproportion of the skeleton with elongation of the legs and arms due to lack of estrogen to close the long bone epiphyses) as exemplified by 2 genetic conditions: aromatase deficiency and lack of estradiol β receptor function (15, 16).

Early information about therapy for this condition came from Sushruta (also Susruta) beginning in approximately 600 Bce, who recommended the ingestion of animal testicular tissue for the amelioration of impotence (Fig. 1) (17).

Hippocrates (460-370 Bce) was an Asclepiad best known for creating medicine as an art, independent of either philosophy or religion, and emphasized clinical reasoning based on direct observation and disruption of the normal. Among his Aphorisms are that

“… eunuchs are not subject to gout nor do they become bald” [(18) Aphorisms VI, 28] and “… a youth does not get gout before sexual intercourse.” [(18) Aphorisms VI, 30]

The latter meant in all likelihood before full sexual maturation. In a book that was continuously in print until the 19th century, Pedacius Dioscorides of Anazarbus (∼60 Ad), a Greek who prepared and studied the effectiveness of natural drugs, noted testis tissue as a therapeutic agent (aphrodisiac) for male hypogonadism, although the vast majority of his treatise, De Materia Medica, was dedicated to plant-based remedies (19).

Aristotle (384-322 Bce), also an Asclepiad, noted the effects of castration in male birds, animals, and man. In the Generation of Animals [(20), Book 9, vol. 4], he observed the effects of castration of roosters caused the secondary sex characteristics (comb and wattles) to regress. Song birds lost their song upon castration. Pre- and peripubertal males did not develop secondary sex characteristics, and adult castrates were unable to reproduce but also did not become bald with advancing age (21) He also described an added characteristic of castration of the young animal:

all animals, if operated on when young, become bigger than their unmutilated fellows [(22), pp. 981-982]

The Old Testament (King James version) states in Deuteronomy 23:1 that

He that is wounded in the stones, or hath his privy member cut off, shall not enter into the congregation of the LORD.

However, in the New Testament, Matthew states (with reference to Christ describing the 3 kinds of people for whom the injunction to marry does not apply):

But He said to them “Not all men can accept this statement, but only those to whom it has been given.” For there are some eunuchs, which were so born from their mother's womb: and there are some eunuchs, which were made eunuchs of men: and there be eunuchs, which have made themselves eunuchs for the kingdom of heaven's sake. He that is able to receive it, let him receive it. [Matthew 19:11-12]

This led to the cult of the Mother goddess Cybele and many centuries later to the Skoptzy sect in Russia whose members practiced self-castration in the 19th century as a mechanism to enter the Kingdom of God (see later discussion) (23).

Pliny, the Elder (23-79 Ce) was a nonmedical, renaissance man whose major work was writing what was then the largest book ever, Naturalis Historia (Natural History) for which Volume II, Book 7 is of most import to endocrinology; for he suggested eating animal organs as general remedies, but he advised men with impotence or the need for the improvement of sexual function to ingest the testicles (aphrodisiac) (24).

Aretaeus of Cappadocia (first and second centuries, Ce) made some observations that may have anticipated the dual function of the testis. Around 150 Ce, when writing a chapter on gonorrhea, he stated

For it is the semen, when possessed of vitality, which makes us to be men, hot, well-braced in limbs, well-voiced, spirited, strong to think and act. For when the semen is not possessed of its vitality, persons become shriveled, have a sharp tone of voice, lose their hair and their beard, and become effeminate, as the characteristics of eunuchs prove. [(25), pp. 346-347]

Next came the rise of Arabic medicine after the fall of Rome in 410 Ce. The Greek, Roman, and Egyptian texts were preserved through translation into Arabic, only to be retranslated to Latin and other languages as the Medieval times turned to the Enlightenment. Between the 9th and 16th centuries, medicine and pharmacy were carried on and advanced mainly by members of the Muslim Empire. Baghdad became the capital of the Eastern Caliphate and Cordoba of the Western Caliphate. Although a Christian, Johannes Mesuë (∼777-857), also known as Yuhanna ibn Musawayh, with some monks who copied the manuscripts, not only translated many of the Greek and Latin texts to Arabic but likely saved these classics from extinction. Mesuë suggested decoction (the liquor resulting from concentrating the essence of a substance by heating or boiling, especially for a medicinal preparation) of animal testes for the same purpose as Pliny, the Elder (26). Modern steroid chemistry would point to the futility of these products for their stated purpose, but we await Charles-Édouard Brown-Séquard in the 19th century to accelerate the trajectory of organotherapy, or the administration of tissue derived from the body for medical purposes, here specifically that related to the testis and rejuvenation, whether by testis extract or implantation/transplantation or ligation of the spermatic duct (27, 28).

Albertus Magnus (?1193 to November 15, 1280) was a German Dominican Friar, philosopher, scientist, and later bishop of the Catholic Church. He suggested ingestion of powdered hog testis in wine for men with poor sexual power (29). Others have used the term opotherapy [opo(s), juice + therapy], when the substance administered is in the form of an extract to replace absent or insufficient hormone production with organic substances that serve the same purpose, but often without knowledge of the molecular basis (30). Treating an organ with itself, whether as an implant, transplant, or extract, is also known as similia similibus (“like cures like”), although that has more recently been co-opted by the practice of homeopathy.

The Pȇn Ts’ao (herbal of the Chinese Pharmacopoeia, 1596), likely based on Emperor Shȇn Nung, 27 centuries Bce, described the ingestion of semen from young men for the treatment of sexual weakness (31). From the 11th century, but entirely independently, Chinese alchemists, physicians, and iatro-chemists sought the healing power of extracts of urine because of its connection to the blood. Classical Chinese medicine envisaged a constant interaction among all of the organs of the body, mediated through the blood. Since urine was considered an extension of the blood, then what might be found in the blood would also be in the urine. Urine was far more available, without high concentrations of protein and thus a more desirable starting point of a pharmacological preparation. We focus on androgenic activity as presaging the later theories of de Bordeu, which reminded one of the Greek theory (Hippocrates) of pangenesis, a developmental theory of heredity, suggesting that all cells in an organism are capable of shedding minute particles, called gemmules, which are able to circulate throughout the body and finally congregate in the gonads of both males and females (expressed by Darwin as a proposed mechanism to explain heredity) (32). The first experimental indication of that was by Legallois (see later discussion), who observed the constancy of the arterial blood but differences between venous blood among the organs and arterial blood.

Multiple methods were tried only partially successfully to find extracts with androgenic activity in relatively purified form, described as white or crystalline, and to use them for the treatment of male hypogonadism. Most started with hundreds of liters of male urine, which was evaporated; heated again, often with charcoal; ground; and sublimated. The retained residue was mixed with dates to make pills that could be taken orally with soup or warm wine (33, 34). Many centuries later it was considered likely that these sublimates contained steroid glucuronides and sulphates, as these would be intact at the usual temperatures of sublimation 140 to 280 °C (33, 34). Other Chinese physicians in the 12th century Ce suggested desiccated or raw animal testicular tissue for a host of symptoms compatible with male hypogonadism.

It was not until the late 16th century that Thomas Vicary (1490?-1561) stated a rationale for organotherapy:

in what part of the body the faculty which you would strengthen lies, take the same part of the body of another creature in whom the faculty is strong, as a medicine. (35)

Théophile de Bordeu (February 22, 1722-November 24, 1776) was a French physician who espoused a medical philosophy, vitalism (he founded the Vitalistic School at Montpellier), which stated that life arises from a special kind of force; that is, living organisms are fundamentally different from the nonliving because they contain some nonphysical element or are governed by different principles than are inanimate objects (36). De Bordeu described a double innervation (trophic and functional) of glands and organs with many areas of the brain as the initiators to stimulate individual organs and thus regulate them. This implied the existence of secretory nerves based on a local increase in circulation when a specific gland is active. The activated gland secretes a new product that reaches the general circulation from the veins draining the specific gland to affect the rest of the organism. De Bordeu developed the principles of constitutional medicine, as originally stated by Hippocrates. These are as follows:

• Principle of Integration in Health and Disease: the organism is a whole and acts as a whole; disease is a total, psychosomatic reaction.

• Principle of Ætiological Constellation: disease represents the reaction of our constitution to adverse environmental factors; it is determined by a combination of various environmental factors and individual constitutional features.

• Principle of Natural Healing Powers: cure is based on the natural, self-regulating power of the organism to restore its balance disturbed by the adverse environmental factors.

He did not do experiments but reasoned in the manner of Hippocrates for the forces of integration of the entire organism. To explain how the circulating humors (Hippocrates) integrate an organism to a unified whole, De Bordeu wrote (37) [in translation based on M. Neuburger (38)]

Each of the organs serves as a factory and laboratory of a specific humor which it returns to the blood after having prepared it within itself and imparted to it its own intrinsic character… The blood has peculiar qualities which it has acquired in the tissues from where it comes. Every organ sends to it continuous emanations and thus the blood carries in its torrent extracts of all organs, which are indispensable for the life of the whole. [(39) Œvres complète, II p. 942) as cited by Cawadias, 1949]

Specifically, for the testes [in translation based on M. Neuburger (38)]:

The testes give a male tonality to the organism … set a seal upon the animalism of the individual … not only each gland, but each organ of the body, is the workshop of a specific substance or secretion, which passes into the blood [our emphasis], and upon whose secretion the physiological integration of the body as a whole depends [(39) Œ, II p. 942), as cited by Cawadias, 1949]

The statement shows quite remarkable insight into modern-day endocrinology and perhaps even neuroendocrinology. Neuburger considered him the forerunner of the theory of internal secretions, and today he seems the closest of the 18th-century scientists to the modern theory of it (38).

Thus, he hypothesized that internal secretion was a general mechanism to integrate the various functions of the organism and is in keeping with the concept of Thomas Willis (40). More than a century after de Bordeu, the internal secretion concept was specifically stated by Brown-Séquard and d’Arsonval in 1891 (41), becoming a basic pillar of the newly distinguished discipline of endocrinology.

The transition to the experimental epoch did not come quickly or easily to medicine, for there were those who adhered to the ancient art of medicine where the observational aspects were key (Hippocrates) and the practitioners offered a prognosis and comforted their patients. Many of the diseases were self-limited. The more scientific aspects began slowly, well before the middle of the 19th century, but it was at that time that there was a real tension between art and the new scientific discoveries in the practice of medicine. This concept was put into sharp focus by Oliver Wendell Holmes, Jr., the American Jurist, in 1860 in an address to the Massachusetts Medical Society titled Currents and Countercurrents in Medical Science. Holmes was a strict believer in medicine as an art and stated

I firmly believe that if the whole materia medica, as used now, could be sunk to the bottom of the sea, it would be all the better for mankind—and all the worse for the fishes. (42)

The burgeoning science, which would become designated endocrinology in the early 20th century, was a prime example of this schism. Sir Edward Sharpey-Shäfer saw this not only as a problem for endocrinology but for physiology in general. He was among the first to define criteria upon which this new field ought to be established to sort out conflicting observations and interpretations using information from both the clinic and the laboratory. He firmly fell on the side of rational scientific medicine (43). His contemporary, Ernest Starling, stated this principle succinctly:

Only … by way of experiment, can we hope to attain to a comprehension of the wisdom of the body … and thereby to the mastery of disease and pain, which will enable us to relieve the burden of mankind. [(44), p. 865]

Experimental Epoch

Although the experimental epoch for what is now known as endocrinology has often been considered to begin with Berthold, there are a number of physiologists whose work, relevant to more modern endocrinology, began much earlier; we start with Thomas Willis.

Thomas Willis (January 27, 1621-November 11, 1675) was a pioneer investigator of the anatomy of the brain. In fact, it was his pupil Richard Lower, a brilliant anatomic dissector, who honored his teacher by naming the circle of Willis and defining blood flow within it. In 1664, Willis postulated that something from the testis flowed into the blood to permit vitality in man, marking an inauspicious beginning to modern testis endocrine physiology. This was in addition to the production of semen. His words in translation from Latin by Samuel Pordage are

“The blood pours out something-through the spermatic arteries to the genitals, so also it receives a certain ferment from these parts-to wit certain particles imbued with a seminal tincture” which “carried back into the bloody mass make it vigorous and inspire to it a new and lively virtue.” [(40) De Cerebri Anatomne, 1664]

Perhaps this concept is the germination of de Bordeu's a century later, and it is compatible with modern neurophysiology—neurosecretory material formed in hypothalamic nuclei passes down nerve fibers through the infundibulum to the posterior pituitary, eg, oxytocin and vasopressin. Hypothalamic nerve terminals liberate releasing hormones into the portal vessels in the infundibulum and are carried down the pituitary stalk to the anterior pituitary with the anterior pituitary hormones released into the general circulation.

In the 18th century, functional correlations among parts of the body were considered consensus or sympathies, distant action within the body, whether through the nervous system or blood borne. There were universal sympathies, in which the whole constitution sympathizes with some sensation or action of a part, and partial sympathies, where 1 or more parts sympathize with some local sensation or action, including remote actions. There appears to be no physical connection between the parts, save for the vital principle. John Hunter (February 13, 1728-October 16, 1793) sought to study the sympathies of the testes and was likely the first person to attempt testis transplantation/implantation in 1762; however, there are virtually no detailed records of his experiments. Apparently, Hunter was more concerned with his interest in the vital force (also noted as living principle or spirits), which permitted the implants to secrete, rather than testicular function itself (45). Thus, a disease was caused by an alteration in a vital property or principle of the body. He did not interpret his experiments with a humoral (or nervous) etiology in mind. Although it was common knowledge that Hunter kept meticulous records, what may have been written has been destroyed. Much of the available information is gleaned from notes taken by others during some lectures delivered by John Hunter (46). Directly quoting 1 of William Hunter's lectures (John's brother and colleague in research), William Mercer wrote

I have also frequently taken out the Testis of a cock and replaced it in his belly, where it has adhered, and has been nourished; nay, I have put the Testis of a cock into the belly of a hen with the same effect. (47)

Further information from a student's notes indicated that using an injection technique, Hunter described a direct connection between blood vessels of the hen's liver and the implanted testis (48). Quoting an additional source, Hunter described the consequences of an incomplete castration: noting hypertrophy of the residual fragment over a long period (8 months), presaging the biological activity of pituitary gonadotropes (49). Hunter did not postulate that the male secondary characteristics were maintained by a blood-borne substance from the testis. That was the revelation of Arnold Berthold. Hunter made meticulous observations of testicular size (function) in sparrows whose testicular size changes as the breeding season approaches:

If a cock sparrow is killed in the winter, before the days have begun to lengthen, the testicle will be found very small; but if that organ is examined at different times in other sparrows, as the warmth of the weather increases and if the examination is continued to the breeding season, the difference in size of the testicle will be very striking. [(50), p. 66]

The sparrows that he studied remain on display at the Museum of the Royal College of Surgeons in London.

Hunter's interpretation of the sum of his experiments was

These appearances shew that the living principle exists in several parts of the body, independent of the influence of the brain, or circulation [emphasis added], and that it subsists by these or is indebted to them for its continuance; and in proportion as animals have less of a brain and circulation, the living power has less dependence on them and becomes a more active principle, in itself. [(51) vol. 1, p. 128]

John Hunter left an indelible mark on investigation, however, by the Hunterian triad (aka, tripod): clinical observation, use of comparative physiology, and finally, experimentation. Thus, John Hunter showed the first experimental proof of the concept of internal secretion of the testis, to be solidified by the work of Berthold (1849) and finally definitely shown by Pézard (52). This property of the testis first required the general concept of internal secretion by Bernard, Addison, and Brown-Séquard in the early 1850s.

In France, Antoine-Laurent Lavoisier and Pierre Simon LaPlace powerfully showed that the methods of experimental science were far better to solve chemical and biological problems than those previously applied. They were the founders of experimental biology. César Julien Jean Legallois (February 11, 1770-February 10, 1814), an experimental physiologist and physician, anticipated the concept of internal secretion, originally stated as de Bordeu's speculative in his doctoral thesis: Le sang est-il identique dans tous les vaisseaux qu’il parcourt? [Is the blood the same in all vessels it moves through?]. Legallois described that arterial blood was the same for all of the organs, but its composition changed in its course through that organ, and thus each organ has its influence upon all of the others through the blood (53). In fact, he devised an equation to define the product of the gland:

[(54), p. 209]

However, at this time there was an alternative hypothesis. Georg Prochaska (April 10, 1749-July 17, 1820), a Viennese physiologist, proposed that a sympathetic mechanism was responsible both for the maturation of the secondary sex characteristics and strength and courage (55). The testes were responsible for the harmonious interactions among the various parts of the body, here due to nervous system reflexes rather than a humoral mechanism.

Friedrich Wöhler (July 31, 1800-September 23, 1882) was a German chemist, the first to synthesize an organic compound, urea, from inorganic building blocks carbon, hydrogen, oxygen, and nitrogen. That convincingly destroyed the concept of vitalism and noted that there really were no differences between the structural chemistry of life and that of the inanimate nature (56). Wöhler's teacher and mentor was Jöns Jakob Berzelius (August 20, 1779-August 7, 1848), the well-respected Swedish chemist, who had formulated the difference between inorganic and organic compounds and had stated that organic compounds must have their origins in living organisms. He argued for the dualistic nature of organic compounds; that is, organic molecules could be interpreted by the principles of inorganic chemistry but also had vital origins. Berzelius never accepted the dismissal of the concept of vitalism, even after Wöhler. Berzelius also defined the concept of isomerism, for which more than 1 compound has the same chemical formula but different chemical structures. It is ironic that that is what Wöhler showed when he used the salt ammonium cyanate to transform into urea through a reaction sequence that went ammonium cyanate to ammonia and cyanic acid, which rearranged to urea:

Arnold Adolf Berthold (February 26, 1803-January 3, 1861) castrated roosters, turning them into capons. Upon castration, Berthold observed very quickly decreased aggressiveness and far less fighting among them. Shortly after transplantation of testis into the abdominal cavity, the aggressive nature of the former rooster returned, along with crowing and chasing hens. His conclusion from these observations was quite simple—that the transplanted testis with new vascular connections produced a substance into the blood that acted throughout the body (57). He also specifically noted that the nerve supply of the grafted and revascularized testis had not been reestablished. In his own words [in translation from German to English by D. P. Quiring (58)] the biological effects

must be caused by the productive relationship of the testes, that is to say, through their action on the blood on the organism as a whole. [(58), p. 401]

From these experiments one could postulate a humoral connection from the testis with effects on distant organs, or in fact, the rest of the body, although Berthold himself did not. In any case, this work may be considered the experimental birth of the discipline of endocrinology and the experimental proof of de Bordeu's hypothesis. This small set of experiments is the first clear-cut evidence for internal secretion by a transplantation (implantation) of an endocrine organ. Although this was truly groundbreaking, Berthold's colleague at the University of Göttingen, a very well-respected physiologist, Rudolf Wagner, tried to repeat these experiments, but he could not get the same result and declared them invalid (59). Due to Wagner's reputation, his opinion was accepted, leaving Berthold's critical experiments fallow for more than 50 years until others were able to repeat them in frogs and rats (60, 61). Later scientists, eg, Arthur Foges, considered Berthold's experiments revolutionary (62).

A set of experiments done by Strasburg physiologist Friedrich Goltz (1834-1902) was also ignored. Here Goltz mated a spinal cord-transected female dog with a normal male. A pregnancy ensued with the delivery of 3 pups. This clearly showed that sexual function, at least in the female, although controlled by the gonads, was not through the mechanism of sensory nerves ascending to the brain as had been previously postulated. Thus, the sex glands, here the ovaries, exerted their effect on the brain by a humoral mechanism through the blood (63). Eighty years after the publication of the 1849 report, C.R. Moore noted that Berthold established the modern concept that, wherever located, the testis produces a substance that is distributed throughout the body through the blood circulation (64). One should consider Berthold's contribution as a major precursor to the discipline of endocrinology (65).

Claude Bernard (July 12, 1813-February 10, 1878) was a preeminent French physiologist considered 1 of the founding scientists of experimental biology. He began in Paris as a laboratory assistant to Professor Franҫois Magendie and then succeeded him in 1855 as a full professor at the Collège de France. Although perhaps better known for his work showing the stability of the internal environment (milieu intérieur), he was also 1 of the first scientists to note the glycogenic function of the liver as an endocrine process. The liver, through an internal secretion, prepares sugar at the expense of the elements of the blood passing through it (glucose and glycogen). A key experiment was feeding a dog a noncarbohydrate meal and then analyzing the sugar content of both the portal and hepatic veins. After this meal, there was very little glucose in the portal vein (directly from the intestine) but glucose in the hepatic vein (exiting the liver). His simple conclusion was that the liver secreted (produced) the glucose. In 1856 he discovered the origin of the glucose, glycogen, and considered it a storage reserve. It could be synthesized and degraded to glucose to maintain the sugar content of the blood. When glucose is in excess in the blood, the liver stores it as glycogen. If the level of glucose in the blood is too low, then glycogen is broken down within the liver, and its level in effluent blood increases. His monumental book Introduction à l’étude de la Medicine (An Introduction to the Study of Medicine) put experimental science on the map for medicine (65). He described many issues concerning good scientific theories and good scientists. One of these, Authority vs Observation, noted [in translation, by Henry Copley Greene, 1927]

When we meet a fact which contradicts a prevailing theory, we must accept the fact and abandon the theory, even when the theory is supported by great names and generally accepted. (66)

He also described good science, especially relevant to endocrinology: observations triumphed over theory, irrespective of how authoritative the theorist. Observation poses a problem (question) from which a hypothesis is derived. Experiments are performed to confirm or refute it; the results are interpreted to form conclusions:

A skilled hand, without the head to direct it, is a blind instrument; the head without the realizing hand remains powerless. [noted in his book Introduction à L’étude de la Médecine Expérimentale (66)]

The original experiments of Berthold in roosters/capons were finally repeated successfully by Albert Pézard (April 1, 1875-November 21, 1927) who took a lipoid extract from approximately 1/10 of a cryptorchidic hog testis that had only Leydig cells, for the Sertoli cells and the seminiferous tubules had degenerated. He injected the extract into capons twice a week for 5 months and noted growth of the comb and wattles, crowing, and the sexual instincts of a rooster (52, 67, 68). These all regressed with cessation of injections of the extract. Although technically a bioassay for this testicular substance, the protocol was hardly appropriate for a practical and useful bioassay. Pézard also determined that the continuous presence of the substance from the testis was necessary for masculinizing activity, since that diminished and then disappeared soon after castration and required repeated administration to keep the animal masculinized. He noted

The continuity of endocrine function of the testis is an indispensable condition for the maintenance of the conditional characters. The morphogenic action of the testicle presents in addition a remarkable constancy which is dissembled by the arrest of growth in the adult.” [(69), p. 531]

Pézard remarked on 2 other laws, the first of minimum efficacy, which was noted by partial castration or castration adding back minute fragments of testicles scattered within the peritoneum. He showed an approximately 30-fold difference between the minimal dose required and the physiological one from the intact testis. The second he called the all or none law (Fig. 2):

As soon as the functional threshold value is passed, whatever may be the mass of the active gland, a cock takes on as a whole its secondary sexual characteristics. [(69), p. 535]

Bouin and Ancel (70) followed Franz Leydig's (1821-1908) description of the interstitial cells of the testes in 1850 by characterizing a relative or absolute increase in these cells compared to the seminiferous tubules and Sertoli cells after ligating and resecting the ductus deferens in adult rabbits (71, 72). They called the group of cells the interstitial gland (la glande interstitielle) or endocrine part. Degeneration and atrophy of the seminiferous epithelium and tubules or generative part followed ligation, although the Leydig cells remained. The secondary sexual characteristics remained until sacrifice, indicating that the masculinizing substance from the Leydig cells was present, despite the diminished testicular volume. These investigators added a second experiment to prove this point; after 3 to 4 months, the atrophied testis was removed and the secondary sexual characteristics declined. The investigators ascribed that to the loss of the remaining Leydig cells from the now-removed testis (72). They summarized their work as follow:

In numerous previous studies we have assembled a group of morphological, physiological and chemical facts that, taken together, allow us to formulate the following hypothesis: that the general action of the testes on the organism, ascribed in the past to the testes as a whole, is actually due to the interstitial gland. [70, translated by Christensen (73)]

In 1908 Tandler and Grosz irradiated the testes of roebucks who showed none of the typical changes of castration, such as loss of antlers; however, microscopic examination of the irradiated testes showed near total destruction of the tubules with preservation of the interstitial cell mass (74).

Massaglia (75) repeated the experiments of Ancel and Bouin but in the rooster rather than the rabbit. He chose the rooster because it was much easier to determine changes in the secondary sexual characteristics in that animal than in the rabbit. Massaglia described marked degeneration and atrophy of the seminiferous tubules following ligation of the ductus deferens, but the secondary sexual characteristics did not diminish until the atrophic testis was removed. In addition, there was now hypertrophy of the anterior pituitary to the extent of a capon, when it was only slightly increased in size following the ligation of the ductus deferens with intact Leydig cells. A second set of experiments included the castration of the roosters with seeding of the peritoneum with bits of 1 of the testes. This set of experiments gave variable results based on the remaining amount of viable testicular tissue (Leydig epithelium). From both sets of experiments, Massaglia concluded that the testis produces an internal secretion that masculinizes the animal; the postcastration changes in both the secondary sexual characteristics and the anterior pituitary did not change with enough viable testicular tissue in situ or in the peritoneal cavity (75). Further, he considered that the Leydig cells produced the masculinizing substance because they had the characteristics of secretory cells. Animals remained normal from the point of view of the secondary sexual characteristics even with atrophy of the seminiferous epithelium; removal of the atrophic testis, which still contains Leydig cells, led to the loss of these characteristics, and then the animal had the same changes in the pituitary that follow castration (75).

P.E. Smith demonstrated that hypophysectomized, immature mice or rats failed to mature and had atrophy of the sex organs. Adult animals showed rapid regression of sexual characteristics and loss of reproductive function as demonstrated by collapse of spermatogenesis, decreased diameter of the seminiferous tubules, and decreased testicular weight (76, 77). The testes are not self-regulating organs. Smith and Engle clearly showed pituitary control of gonadal function by daily administration of anterior pituitary extracts from adult castrate animals into immature mice and rats. The immature animals showed precocious maturation of their intact gonads with swift increases of 5- to 10-fold those of the control animals (78, 79). Functional gonadal activity could be restored in the hypophysectomized animals with grafted, fresh, living anterior pituitary lobes. Smith (76) also defined a tuberal syndrome where injury to the tuber cinereum rather than the anterior pituitary was characterized by severe adiposity and moderate genital atrophy, as had previously been described in the human by Fröhlich (adipose-genital syndrome) (76, 80).

More than a decade later, the hormonal theory was revived by Lillie and Tandler and Keller who studied the freemartin, a masculinized bovine female fetus with stunted ovaries, female ducts inhibited, and partial development of the Wolffian ducts (81, 82). In fact, it was John Hunter who presaged the “patho-physiology” of the freemartin, writing in 1786:

… when a cow brings forth two calves, and one of them is a bull-calf, and the other to appearance a cow, that the cow-calf is unfit for propagation; but the bull-calf becomes a very proper bull. [(50) J. Hunter 1786]

With an exchange of blood between the male and female fetus, the Müllerian duct derivatives are absent or vestigial with some Wolffian structures but with minimal masculinization of the external genitalia. The mechanism was considered to be that hormones secreted by the male fetus reach the female fetus through the multiple vascular anastomoses of the bovine placenta and masculinize the female fetus (82). In addition, there was no hormone or combination of hormones that could reverse the basic phenotype of the freemartin or in the pouch marsupial, in which primary genes may be important (83, 84). With these data, Professor Jost foresaw the existence of a second fetal testicular hormone, generically denoted as Müllerian inhibiting substance and now defined as anti-Müllerian hormone (85).

Alfred Jost established that testicular function in the fetus is critical for sexual development along the male line. His meticulous, microsurgical experiments removing the gonads before the onset of sexual differentiation inevitably led to a female reproductive tract irrespective of the genetic sex of the embryo (86, 87). The phenotype was regressed Wolffian structures with the maintained Müllerian ducts. If a fetal testis was grafted adjacent to an ovary of an XX embryo, the female pattern of internal ducts reversed to that of a male; the Müllerian duct regresses but only on the ipsilateral side. However, if a pellet of crystalline testosterone, rather than testicular tissue, was implanted, the Wolffian ducts were stimulated, but the Müllerian structures did not regress (86, 87). The conclusion from these dissection experiments was inescapable: masculinization of the reproductive tract was imposed by fetal testicular hormone(s), a hypothesis first noted by Bouin and Ancel in 1903, who identified the testicular interstitial cells as the reservoir of the driving force for male sexual differentiation (88). This occurred several years before the word hormone was defined.

Rejuvenation (Revitalization)

Rejuvenation or revitalization is the action or process of giving new energy or vigor (physical strength and good health) to someone. An elixir vitae (elixir of life), a hypothetical substance believed to maintain life indefinitely, had been a goal of some men from the very earliest of times. Organotherapy and prolonged survival were the product of superstition and magic, not based on objective data. However, even in 1936, much of the common use of endocrine preparations, stimulated by the advertisements of pharmaceutical/supplement companies, was very little advanced from the magic and other medical remedies of primitive peoples (89). One of the first recordings of that with reference to the gonadal axis came nearly 3 millennia ago when Shusruta recommended the ingestion of animal testicular tissue for the amelioration of impotence (17).

This concept became more prominent with the self-experiment of Charles-Édouard Brown-Séquard, formerly a preeminent neurology professor at the Collège de France. In 1856 he pushed forward the concept of internal secretion by noting that the adrenal gland produces substances that affect the circulation of blood. These glands are essential to life itself, as his adrenalectomy experiments in several species led to the death of the animals (90). He then noted the similarities between the adrenalectomized animals and the newly described Addison's disease, progressive adrenal failure with deficiency of adrenal cortical hormones (91, 92). It was not until 1869 that Brown-Séquard suggested that the glands (adrenals) “have internal secretions and furnish to the blood useful, if not essential principles” that act at distant sites from their production.

He considered the following, before undertaking self-experimentation:

the testicles, apart from their role in reproduction, have a dynamic function of the greatest importance in man. Weakness in old men is dependent on two causes: organic changes and the lack of stimulation exercised on the nervous centres by active testicles [(93), p. 431]

As a neurologist, he considered the nervous system of great importance in the androgenic aspects of testicular function. In an uncontrolled series of experiments Brown-Séquard, at age 72 years, injected himself daily for 20 days with a mixture of aqueous extracts (liquid testicularie) of 1 part testicular vein blood, 1 part semen, and 1 part juice extracted from dog or guinea-pig testes (94). He described

A radical change took place in me … I had regained at least all the strength I possessed a good many years ago. I was able to make experiments for several hours. After dinner I was able to write a paper on a difficult subject. My limbs, tested with a dynamometer, gained 6 to 7 kg in strength. The jet of urine and the power of defecation became stronger. [(94), p. 106]

However, the improved state of health returned to the preinjection state within 4 weeks of discontinuing the orchitic liquor. In that same paper Brown-Séquard reported similar salutary results in 3 additional men. Not all physicians and scientists accepted these anecdotal data. In fact, in an editorial titled “The Pentacle of Rejuvenescence” in the British Medical Journal its editors noted

The statements he [Brown-Séquard] made which have unfortunately attracted a good deal of attention in the public press—recall the wild imaginings of medieval philosophers in search of an elixir vitae [(95), p. 1416]

It was not at random that the word pentacle—a 5-pointed star, the symbol in occult terms, of the perfect man—was chosen as part of the title.

Irrespective of these being placebo effects, similar extracts became popular for aging men and were sold all over the world for more than 40 years. Brown-Séquard's were widely advertised and sold under the names of Sequarine or Spermine in the United States and Sequardine Serum in the United Kingdom, although the commercial enterprise was unrelated to Brown-Séquard himself (Fig. 3) (95, 96). So began the rise in organotherapy, the hypothesis that if a given condition (disease) could be successfully treated with an extract of an organ or gland, then that condition was likely caused by an insufficiency of an internal secretion of that gland. Organotherapy had its proper medical introduction with injectable and oral administration of extracts from thyroid tissue for the significant amelioration of myxedema or the physical signs of cretinism (97-99). Not all physicians embraced the Brown-Séquard form of elixir of life; there were editorial reports of the veracity of the original Lancet article (100-103) and physiological objections that

• there is no proof that the testis produces the vital fluid or that its loss is the cause of enfeeblement.

• all artificial excitation of the testis is debilitating to the aged.

• the exhaustion that follows sexual activity is not due to the loss of a secretion but rather to excitation of the nervous system (94, 104).

Before his death, Brown-Séquard, admitted that perhaps he had fooled himself and that the self-described findings may have been due to suggestion (placebo). Perhaps the lesson here is that without a control group as modern clinical trials require, observed results may be due to a strong placebo effect. What is required is a careful definition of the problem, random allocation of subjects, relevant control group(s), careful measurement techniques, and reserve in drawing conclusions from the data.

Within a few years, especially after the reports of Murray (97, 98) the tide began to turn toward the salutary effects of some of the organ extracts, even to the point that the editors of the journals who had severely criticized Brown-Séquard began to note their benefits: that work on gland and organ extracts has led to a new category of biological substances now considered chemical messengers (105, 106):

It is now some years since Brown-Séquard announced the wonderful effects which followed the subcutaneous injections of testicular extracts as exemplified in his own person, and though many jeered at him as the discoverer of the secret of perpetual youth, the notion has steadily gained ground that there is, after all, something in it. Since also, the success that has followed the injection of thyroid extract in myxoedema, we can hardly wonder that this belief has increased. Physiologists have recently been making a number of observations, which show that many organs do more than what was formerly regarded as their functions. [(107), p. 1279]

Jacques-Arsène d’Arsonval (June 8, 1851-December 31, 1940), a French scientist, was a key assistant to Claude Bernard before moving to assist Brown-Séquard, especially for the preparation of the extracts from testis used to treat aging men. He was well known for his laboratory abilities, especially for the biological and technical applications of electricity. He built an apparatus to sterilize the testicular extracts using high pressure CO₂ (108).

Several years later, Brown-Séquard presented details of his method of extraction—not only of the testis principle but also organic extracts of multiple other organs (109, 110). Specifically for the testis extract, he noted multiple additional successes of rejuvenation of men even in their 10th decade! By this time, he claimed that more than 1600 men were treated with the orchitic liquid, not only with 90% efficacy for the ravages of aging but also with great success for those with locomotor ataxia and cancer (but not for those with epilepsy). He also made an oblique comment that ovaric liquid was not as potent as the orchitic liquid. He then summarized his experience with organotherapy:

… that to give to the organism out of order from the lack of certain principles, and believing that the morbid phenomena of old age are due to the deficiency of a certain internal secretion, I resolved to try to give the missing elements of that secretion by means of injections of a liquid extracted from a healthy gland of the kind of the one which age had rendered faulty. [(110), p. 1212]

The sum of the experiments with extracts of testis and many other organs led to the generalized concept of internal secretions (endocrinology) to be a property of all tissues (108). From the scientific aspect, the experiments of Brown-Séquard—at least the production of the extracts—was meticulously repeated a bit more than a century later by Cussons and colleagues (111). These investigators prepared dog testicular extracts precisely as noted from the original work of Brown-Séquard (93) from several different breeds of dogs to determine whether they contained biologically relevant amounts of testosterone. The mean testosterone concentration in the testicular extracts was 390 nmol/L, equivalent to 112 μg/L. Using the administration protocol of Brown- Sèquard (that is, approximately 20 days of 1 mL extracts of dog or guinea-pig testis), he would have injected the equivalent of 186 ng/day. This should be contrasted to the physiological secretion of 6 to 8 mg/day and modern-day treatment regimens of 5 to 10 mg/day (111). The authors concluded that their results were consistent with testicular physiology, for testosterone is largely secreted as it is synthesized, without a significant storage pool. Steroid biochemistry notes that they are not soluble in water.

The study of enhancement of physical performance with testicular extracts began in 1896, when Zoth and Pregl in independent trials investigated whether such extracts could increase muscle strength and, perhaps, alter physical performance. They administered bull testis extract to themselves and measured the strength of their middle fingers using a fatigue curve derived from Mosso's ergograph (112, 113). They noted that strength was increased but without any control experiment. Their data showed that subcutaneous injection of orchidic extracts was unable to increase strength, but when combined with muscular exercise, the ergographic data showed an increase by as much as 50% (112, 113). Zoth concluded “The training of athletes offers an opportunity for further research in this area and for a practical assessment of our experimental results” (112). As early as 1889, the same year as the first Brown-Séquard report, an American professional baseball player, James “Pud” Galvin, the first pitcher to win more than 300 games in his career, administered a similar extract to enhance his athletic performance. The Washington Post contemporaneously noted

If there still be doubting Thomases who concede no virtue of the elixir, they are respectfully referred to Galvin's record in yesterday's Boston-Pittsburgh game. It is the best proof yet furnished of the value of the discovery. (114)

However, the history of doping with “supplements” reverts back to at least ∼700 Bce when Olympic athletes ingested supplements from animal hearts or testicles or even figs in search (and perhaps wishful thinking) of a performance advantage (115). Given the limited knowledge and therapeutic temptations of this era, these treatments are better described as contemporary medicine than as fraud (116).

As stated by Aretaeus:

But if any man be continent in the emission of semen, he is bold, daring, and strong as wild beasts, as is proved from such of the athletæ as are continent. … but an animal becomes strong from nothing else than from semen. Vital semen, then, contributes much to health, strength, courage, and generation. [(25), p. 346]

Testicular Transplants (Implants) and Ductus Deferens Ligation in Man as Part of the Rejuvenation Movement

At the turn of the 20th century, there was heightened interest in the United States considering the topic of androgen “therapy” involving testicular transplantation and rejuvenation and later with the newly discovered androgens. The underlying hypothesis for the success of an endocrine organ transplant concerned the prevention of signs and symptoms of the nonfunctional or removed organ by the one added to a distal site. This provided strong evidence of internal secretion by the transplanted organ. Lewellys Barker stated the principle of substitution in endocrine deficiency as

1. An organ derived from another person or from an animal that will readily yield that product to the body of the patient (organ transplantation)

2. Fresh or dried glandular substance derived from animals

3. The active principle of the gland concerned, extracted from the glands of animals or prepared synthetically [Read at the Sixth Annual Scientific Session of the Association for the Study of Internal Secretions, St. Louis, MO, May 22, 1922 (117)]

In fact, the Journal of the American Medical Society convened a group to determine what was known about the subject, especially the work of Eugen Steinach. The following was stated in an editorial in JAMA concerning the development of male secondary sexual characteristics:

Great possibilities in the application of these findings loom up; but it is too early for anything more than interesting speculations. [(118), p. 484]

Before considering testicular transplantation in man, the Viennese physiologist Eugen Steinach (January 28, 1861-May 14, 1944) performed such experiments in animals as part of his work studying both sexual differentiation and the endocrine function of the testis. The early experiments with ductus deferens ligation permitted him to hypothesize that an increase in hormonal production follows the cessation of gonadal secretion (semen) after surgical ligation of the ductus deferens (119). He showed that extracts of testes from frogs that were able reproduce differed from those that could not. The former were able to elicit male sexual behavior in castrated frogs, but the latter were not (61). This was strong evidence for an internal secretion of the testis. Later he moved on to the issue of which cell was responsible for the biological effect. He considered the Leydig cell because its histology was secretory; however, not all agreed, and an alternate hypothesis designated a connective tissue function for the Leydig cells, stating that it was the sperm that was the source of the testis hormone. Steinach followed the convincing work of Ancel and Bouin, as detailed earlier, and considered the Leydig cell mass as the interstitial gland. His most consistent work showed that the male behavioral changes were accompanied by the proliferation of the interstitial cells in the transplanted tissue, upon careful histological analyses of his experiments. However, these experiments were flawed by the lack of appropriate controls because the apparent increase in interstitial cells was likely due to the shrinkage of the much greater mass of tubular cells and thus the appearance of a greater mass of Leydig cells. He also stated the anatomic, physiologic, and behavioral changes that the animals underwent mimicked some of those of senility in man. These could be reversed by reintroducing testicular tissue. Even though the reproductive part quickly degenerated, the puberty gland [Pubertätsdrüse (119)] part did not, and either pubertal maturation was started in those castrated before puberty or the secondary sexual characteristics reappeared in those castrated as adults. Old rats implanted with testicular tissue not only showed general improvement but also an increase in sexual activity. That was his academic work, however flawed. He was far more recognized for its purported clinical application. He directly extrapolated these results to man, leading to more than a decade of Steinach operations that permitted the mobilization of the body's own resources.

Steinach had a theory of senility that he considered a state of functional castration, since organs and tissues that had involuted with aging could be rejuvenated; that is, the signs and symptoms of aging could be ameliorated by stimulating the pubertal gland of the aging individual. To attain that, he considered combatting aging (alter-sbekämpfung) with endocrine rejuvenation (Verjüngung). Perhaps both could be “cured” with male hormone. He was a strong advocate for an operation (autoplastic implantation, or strengthening or attenuating particular body parts, here, unilateral vas deferens ligation) that he had popularized along with the urologic surgeon Karl Lichtenstern to increase male sex hormone secretion and thereby rejuvenate elderly men. The key element was that vasectomy increased the blood supply to the interstitial cells (interstitial gland). The first patient was operated on in 1918, and a large number followed. The results were described in a book by Steinach, not primarily in the medical/scientific literature (120). Operations were predominantly 1-sided, but occasionally both vasa were ligated. In the vernacular, it was “being Steinached.” In essence, one doubly-ligated the ductus deferens and severed the duct between the 2 ligatures. The seminiferous tubules degenerated, and the testis became smaller with an abundance of Leydig cells, although one was not sure if there was an overall increase or a relative one because of the smaller volume of the testis.

Thus, destruction of the sperm-producing cells, whether in a cryptorchid testis, unilateral vas deferens ligation, or even external radiation, did not impair masculinization (121). The conclusion from these disparate research trials was that the generative tissue assumed no part in the masculinization paradigm via internal secretion.

A number of prominent men underwent this operation including Sigmund Freud and William Butler Yeats. Stephen Lock proposes that it was the Steinach operation that led to the reawakening of Yeats’s poetic genius when Yeats wrote (122)

Steinach had a passionate follower in the United States, Dr. Harry Benjamin, a German-American endocrinologist and sexologist who made far-reaching claims for the operation:

… with the Steinach operation the patient is given a more or less massive and continuous dose of his own gonadal hormone…. All symptoms due to senility, including sexual impotence, as a rule improve after the operation…. A few other possible indications would be: beginning arteriosclerosis, hypertrophy of the prostate, eunuchoidism, mental depression, and cases of dementia praecox, where we suspect gonadal deficiency as a cause. [(123), p. 688]

These words were written without a shred of data. Although there were few scientific evaluations of the procedure, in 1936 Steinach himself summarized his work over several decades (124).

The earliest testis grafts in the United States were performed by Victor D. Lespinasse (December 2, 1878-December 14, 1946) and G. Frank Lydston (March 3, 1858-March 14, 1923). The former implanted slices of a donor testis among the fibers of the rectus abdominis muscle in a patient who had lost both testes. Within 4 days, the patient had strong erections and marked sexual desire. Libido and erections were reported even 2 years after the transfer of testicular tissue (125). Lespinasse is purported to have said that “a man is as old as his glands.” This finding was soon followed by Lydston, who reported his first clinical case in 1914 (126, 127), although he had done considerable animal work previously and published longer term results in the ensuing years (128). As Lespinasse, Lydston did not perform an orthotopic transplant with blood vessel anastomosis but did implant testicular tissue onto the abdominal wall. Lydston stated that the implantation could retard senility and prolong life because of the hormones secreted by the implant (129).

Serge Abrahamovich Voronoff (July 10, 1866-September 3, 1951), a French surgeon born in Russia, initiated a different track. He studied medicine in Paris and learned the transplantation technique from Alexis Carrel, a French surgeon and Nobel Laureate. Early in his career, he studied the effects of castration on men in Egypt and noted them to be similar to some manifestations of the aging process. It was from these observations that he set his eventual course to that of testicular transplantation. Upon his return to France, Voronoff began to transplant testes from young horses to older ones to improve these beasts of burden. Given that the supply of human testes was vanishingly small, he chose a close phylogenetic relative, the chimpanzee and baboon, to begin his studies in man. The first operation from a chimpanzee to a frail patient occurred in 1920 when he sewed strips of the simian testis into the capsule of the testis of the recipient. Voronoff was an excellent self-promoter, who gained fame by the xenotransplantation of monkey testes into man for rejuvenation and the curing of the aging process in men (130, 131). He cut the donor testis into longitudinal wedges and placed 1 or more under the connective tissue capsule of the recipient's testis, after abrading the host site to stimulate angiogenesis.

His work, although wildly popular, was not without controversy because of the use of simians and because the results were never objectively verified but were widely cited in the secular press as the “famous doctor who inserted monkey glands into millionaires.” Several thousand operations were performed worldwide by the middle 1930s, when the discovery and then synthesis of testosterone was reported:

In the manifestation of his physical and intellectual qualities, varying according to the individual, man himself is worth whatever his sex glands are worth. [(130), p. 58]

He had set up a clinic in Algiers that was quite successful in terms of patients attending for the implants. The Royal Society of Medicine in London was concerned enough to send a medical committee in 1927 to investigate Voronoff and his clinic. Their unequivocal findings were that the claims of Voronoff concerning the rejuvenation of his patients were unsubstantiated (132).

Leo Leonides Stanley (1889-1976) was an American surgeon who served as the chief surgeon at San Quentin State Prison in California from 1913 to 1951. He was hired in 1913 without surgical experience but with the opportunity to study his vision that disease, particularly endocrine disease, was a cause of crime. His practice combined eugenic sterilization with research in endocrinology with a goal of protecting society and rejuvenating an aging and threatened manhood (133).

A prevailing hypothesis at the time was the 19th-century model of Césare Lombroso, who thought that one was racially and biologically predisposed to crime. One's physiognomy, here quantitated by measurements upon the skull (phrenology), was the outer sign of criminal characteristics, gross anatomical, atavistic, and racial typologies (134, 135); that is, the delinquent is constitutionally different from the “norm.” However, this Lombrosian doctrine became the most influential “scientific” theory of his time, despite an inadequate sample size, no control group, comparison of various ethnic and racial groups, and no statistical analysis. Others postulated that criminality could be explained by disruption in 1 or another gland of the endocrine system (136), but there were many unknowns along with poor study design and analysis (137). Stanley also considered that there was no specific criminal type but that it was something internal in the criminal's body, the blood and internal organs. He focused on the endocrine system and considered endocrinology a “science endowed with the power of social reform” [(138), p. 314].

He focused his work on the testicle. He started with transplantation (implantation) as that was considered a mechanism for the rejuvenation and a cure for male aging. In 1918, he began by transplanting strips of testes from executed prisoners to other inmates using Lydston's technique (128):

wishing to prove or disprove some of the assertions of the pioneer physician, Dr. Lydstrom (sic) of Chicago regarding testicular implantation.… [(138), p. 109]

Given the paucity of human testes, he soon branched out to animal testes, particularly those from goats and rams. At first, the animal tissue was not grafted to the recipient but laid on the pampiniform plexus within the scrotum of the recipient. A majority were rejected, which led to a change in procedure in which the implant of the ram's testis was made into the fascia overlying the rectus abdominis muscle. Further alteration included injecting the macerated material subcutaneously above the abdomen, without a surgical procedure. In his own words, “animal testicular substance injected into the human body does exert decided effects,” and he finished with “These observations were undertaken with an unbiased mind and with the endeavor to pursue the truth wherever it may lead” [(139), p. 714].

This last quote refers back to one of Thomas Jefferson and the University of Virginia, the academic home of one of the authors (A.D.R.) of this report:

… this institution (University of Virginia) will be based on the illimitable freedom of the human mind for here we are not afraid to follow truth wherever it may lead, nor to tolerate any error so long as reason is left free to combat it. (140)

Please note that these words were written more than 30 years before the guidance by Claude Bernard in the context of experimental medicine. By 1940, Stanley had performed more than 4000 implants and a large number of equally unethical experiments on inmates of San Quentin (133). A snapshot of a thousand men that he “treated” is shown in Table 1 (141). The terms “benefitted” and “not benefitted” remained largely undefined. Like Voronoff, most of the reporting of outcomes of the procedures of both men occurred in the popular press, not as actual clinical findings in the scientific press.

Following Voronoff came John R. Brinkley (July 8, 1885-May 26, 1942), who showed an entrepreneurial interest in impotence and antiaging therapies immediately after hearing a Serge Voronoff lecture (142). He began a several decades’ long program of goat testicular implantations in Milford, Kansas. He had previously, but fraudulently, claimed to have a medical degree but got that from a diploma mill in Kansas City, Kansas: Kansas City Eclectic Medical University. Brinkley, along with the other rejuvenists noted earlier, appealed to 2 desires of elderly men: youthful vitality and a longer life. This played into Brinkley's entrepreneurial genius; he was a blatant, egocentric super showman (143). His operation consisted of twin incisions in the recipient's scrotum, insertion of donor goat gonads alongside the recipient's testes, and anastomosis of a subsidiary artery and nerve alongside the vas deferens into the epididymis. However, usually all that Brinkley did was deposit the goat testes into a pouch in the recipient's scrotum, and that became the basis of later lawsuits. He used the media, especially his own radio station, to advertise his operation and by 1926 had done more than 4000. His reporting of those was not in the scientific literature but in a self-authored book, The Brinkley Operation (144). He was eventually forced out of practice following multiple articles in the Kansas City Star and loss of his medical license from the Kansas State Medical Board after a committee of doctors from the board watched his surgery. That loss of license was upheld by the Kansas Supreme Court with a blistering decision, in part stating

Being an empiric without moral sense, and having acted according to ethical standards of an imposter, the licensee has perfected an organized charlatanism until it is capable of preying on human weakness, ignorance, and credulity to an extent quite beyond the invention of the humble mountebank who has heretofore practiced his pretensions under the guise of practicing medicine and surgery. [(145) Brinkley v. Hassig, 83 F.2d 351, 356 (10th Cir. 1936)]

Brinkley was also pursued by Morris Fishbein, the long-time editor-in-chief of JAMA, who in 1938 wrote a two-part article in the American Medical Association's Hygeia titled “Modern Medical Charlatans” (146). Brinkley sued Fishbein for libel but lost (147). The Appeals Court ruling indicated

We think above stated facts are sufficient to support a reasonable and honest opinion that plaintiff should be considered a charlatan and quack in the ordinary, well-understood meaning of those words. (148)

Perhaps a fitting summary for the entire era of rejuvenation comes from Morris Fishbein himself:

Unfortunately those wishing for renewed youth suffered inordinately with the will to believe., [(149) The New York Times, May 15, 1944, p. 19]

A more recent attempt at rejuvenation has been the replacement of organ transplants with the injection of viable embryonic cells. Paul Niehans (November 21, 1882-September 1, 1971) was a Swiss surgeon who developed cell therapy for many conditions, including the rejuvenation of older people (mostly men). He determined the levels of protein degradation (“Abderhalden reaction”) within particular organs and tissues. After diagnosis, the patients were injected with cell suspensions from corresponding organs or with embryonic cells from sheep. Although the treatment was considered for many reasons, old age and failing masculinity was a prominent one (150). Its major purpose was the “‘mitigation of the deterioration of aging” (151).

Niehans postulated that “premature senescence is a pathological problem, which can be in a large degree solved through the revitalization of damaged organs, particularly the sex glands.” In Niehans's view, “youthful cells serve the tired, the ill, the old, and the weak. Here science bestows on us a new capital of life. Many valuable people thus avoid a disabled existence and enjoy life afresh, overcoming their depressions” (151). He continued:

The new healing method, that today may still seem revolutionary, employs specific embryonic or youthful fresh cells, cell cultures, conserved cells or cell-rich liquids, to treat the sufferings against which contemporary medical art had been powerless. [(150), p. 1]

Later he perfected a freezing and vacuum drying technique rather than relying on fresh cells. The hypothesis was similar to those noted earlier for testis transplant or vasoligation—that the failing cells would be replaced by viable ones from the embryonic animal. He had a large clinical practice in Clarens, Switzerland, but this method was considered unproven (152).

Dénouement (Testis Transplants, Implants, and Vas Deferens Ligation)

General interest in any of the transplant operations declined in the 1930s, especially in the United States because of the discovery and then commercial synthesis of testosterone and its esters and the Great Depression, which markedly diminished discretionary spending. In addition, in vivo bioassays became practical and sensitive enough to detect the small amounts of testis hormone that might be liberated by the implanted tissue [reviewed in (1, 153)]. In 1930, using multiple in vivo bioassays, Moore unequivocally showed that castration effects could be detected within 5 days and that they could be delayed indefinitely with the administration of lipoid testis extract or could restore the male accessory structures if they had been permitted to atrophy after castration. These were the data that indicated that the body does not store testis hormone for days, that what hormone is made is found in the urine of the animal, and that continuous hormone production was required to maintain the secondary sexual characteristics. None of these conditions held for the human-to-human or the animal-to-human testicular implants. In fact, the nonviable implants quickly undergo autolysis. Moore concluded from the presented experiments that autotransplantation of 2 testes in rats or guinea pigs did not alter the findings of castration itself. Thus, no hormone is liberated from the transplant. In man, there was no objective evidence of testis hormone production (benefit) either from the implanted testis material or from the man's own testes. Moore concludes

… it appears that we shall have to invoke psychic states as material even for discussion, and such provinces are beyond the one considered here. [(153), p. 1915]

Organized Medicine (Endocrinology)

In the second decade of the 20th century, a time when rejuvenation therapy was gaining momentum, an entrepreneurial organotherapist, Dr. Henry Harrower, began to establish a national organization of endocrinologists in the United States (154). In 1916 at the Annual Meeting of the American Medical Society, he organized a meeting of a neophyte group and called it the Association for the Study of Internal Secretions, which incorporated in 1917 and in 1952 was renamed the Endocrine Society. Dr. Harrower edited the first 2 issues of Endocrinology, the official journal of the society. Due to his business interests, which were considered incompatible with the purposes of the society, others, mostly academic physicians, assumed leadership roles in the new organization. What was set up, however, was a marked tension between the organotherapists and the more biochemically oriented academicians. Although widely published in the academic literature, he set up the Harrower Laboratory of Organotherapy, which sold extracts that were not only endocrine in nature but also of multiple other organs. Harrower followed 2 principles:

1. Principle of homostimulation, which held, for example, that thyroid extract could restore thyroid hormone deficiency to normal (155).

2. Harrower's hypothesis of hormone hunger, which stated that the body would select what is required to restore health from a multiglandular mixture. Any excess would be harmlessly excreted (156).

Thus, Harrower had theories that specifically led to sales of his mono- and pluriglandular extracts, although the evidence base for salutary effects was meager (Table 2).

The academic and scientific members within the Endocrine Society vigorously put forward that the advances in medical science required laboratory support and assistance. In fact, Lewellys Barker, the second president of the fledgling society, pointed out in his presidential address, titled “Remarks on the Function of the Suprarenal Glands Revealed by Clinicopathologic Studies of Human Beings and Experimental Animals,” that laboratory excellence was a required component of good endocrinologic practice (158). He had previously considered medical laboratories to be critical to both medical practice and medical discovery (159).

The next president of the society was Harvey Cushing, who scathingly called out the organotherapists, regarding them as charlatans or pseudo- or quack endocrinologists practicing “endocriminology” (160). He denigrated the hypothesis of hormone hunger by stating

Surely nothing will discredit the subject in which we have a common interest so effectively as pseudoscientific reports which find their way from the medical press into advertising leaflets, where cleverly intermixed with abstracts from researchers of actual value the administration of pluriglandular compounds is promiscuously advocated for a multitude of symptoms, real and fictitious. [(161), p. 1726]

The first 2 presidential addresses were published in Endocrinology, but Cushing's was published in the higher impact journal, JAMA (161).

Once more the society spoke against organotherapy, as Leonard G. Rowntree noted, not only individual practitioners of organotherapy but also some of the pharmaceutical companies engaged in a “most fraudulent exploitation of the medical profession and the public” (162). Rowntree specifically stated

The practice of administering such mixtures constitutes a disgrace to 20th century medicine, a menace to our profession, and a betrayal of our patients. [(162), p. 190]

In 1925 the American Medical Association concurred and officially published a pamphlet dismissing unfounded claims of the organotherapists (163).

Another of the presidents of the Endocrine Society, E.H. Rynearson, continued on with the concept of endocriminology in support of Harvey Cushing's 1921 Society of Internal Secretions (Endocrine Society) presidential address. Endocriminology was later defined as

A psychological state of clinicians with strong endocrine prejudices, which results in the incrimination of the endocrine glands without adequate objective diagnostic data, and in the practice of organotherapy without clear-cut indications. [(164), p. 506]

and applied clinical endocrinology as

The critical translation and application by capable and well-informed clinicians (“specialists” and “general practitioners”) of facts of the science of endocrinology to the diagnosis and treatment of patients. [(164), p. 506]

Relevant examples for either organotherapy or even for the newly available commercial testosterone preparations included impotence, regardless of cause; sterility or low sperm counts, which is of course odd, given that testosterone suppresses spermatogenesis; and the male climacteric or psychosomatic stresses of mid-life men. Then, as now, the sole valid indication for testosterone therapy in the adult male is as hormonal substitution (replacement therapy) for hypoandrogenism from disturbance of the hypothalamic-pituitary-gonadal axis (primary or secondary hypogonadism).

Organotherapy did not rapidly decline even as purer hormones became available. It morphed from the single extract of Brown-Séquard to the properly employed extracts of the thyroid (97). As the 20th century unfolded, many endocrinologists urged the prescription of monoglandular extracts or, less commonly, pluriglandular extracts based on Harrower's hypothesis of hormone hunger.

Biochemistry and Physiology of the Testis

The definitive era of the discovery of testicular function and the substances responsible for androgen effects and reproduction began in the second decade of the 20th century, based on the few studies in the 19th century, for example, Berthold (57). Pézard noted that all of the effects of the male substance (hormone) from the testis occurred (or not) simultaneously; the secondary sexual characteristics, especially notable in the common laboratory animal utilized (the capon), and the more psychologic, such as sexual impulses toward females, lusty crowing, and intolerance of other males (Fig. 3). Pézard and others independently attributed these changes to internal secretion because the characteristics were abrogated by castration but reappeared on testicular fragment implantation into the peritoneum of a capon. Similar findings followed multiple injections of testicular extracts (67, 68). As noted, in 1920 he specifically stated 3 principles (laws): those of continuity, minimum efficacy, and the all or none law, derived from his work as well as that of others.

The rooster comb and wattles had been a bioassay for androgenic activity; however, that did not seem practical, nor was it quantitative as the bio-organic experiments to isolate and identify the active principle in the testis, which was clearly in a small amount, began. In the United States, a series of experiments was undertaken to provide an assay(s) for the quantification of the androgenic factor. Gallagher and Koch perfected the capon comb assay, although it was still quite variable (Fig. 4) (165) and defined the bird unit as follows:

A bird unit is defined as the amount of the hormone which, when injected daily for five days, yields an average of 5 mm. increase in length and height of the combs of at least 5 Brown Leghorn capons. [(166), p. 339]

Additional bioassays were developed, many by the same University of Chicago group that included a rat prostate cytology test, a rat seminal vesicle cytology test, which depended on the cytological changes in the secreting cells of these organs (167, 168). An additional test of prolongation of spermatozoön motility was devised. All of these were apparently satisfactory; however, some take a long experimental period and all have imprecise endpoints necessitating the use of many animals per experimental point. What was required was a more rapid assay that used few animals per dose level of androgenic substance. Development of the Hershberger bioassay began in the 1930s, and subsequent iterations take advantage of a “modified” assay that employs castrated, peripubertal rats, which have minimal endogenous androgens, and measures the output in terms of the weights of the ventral prostate, seminal vesicles, levator ani/bulbocavernosus muscles, the Cowper's glands, and the glans penis (169)

Before extraction, concentration, and isolation of specific chemical entities with androgenic activity, all that was available were the physiological experiments, mainly in experimental animals:

• Castrated males noted atrophy of the secondary sexual characteristics but had pituitary hypertrophy.

• Fat-soluble testicular extracts could prevent that atrophy, if administered just after castration. In addition, over time atrophied sexual organs would regenerate with continuous administration.

• Preparations from urine of males could reverse the atrophied sexual organs but could neither prevent nor ameliorate the hypertrophy of the anterior pituitary that followed castration.

• An aqueous extract of the testis that contained a minimal amount of sex gland stimulating activity could not only prevent the castration-induced changes in the anterior pituitary following castration but also inhibit the hyperfunctioning of the anterior pituitary.

• Destruction of the seminiferous tubules produced a hyperfunctioning pituitary but did not lead to atrophy of the secondary sexual characteristics.

In sum, as the late 1920s turned into the 1930s, one had the idea that there were 2 testis hormones with very different origins and activities. With the available bioassays within 5 years, the organic chemists were able to isolate, determine the chemical structure, and synthesize several androgenic compounds with testosterone being the most potent.

With the ability to more carefully quantitate the androgenic hormone potency of testis extracts as well as having less cumbersome assays, Lemuel C. McGee, a medical student working in the laboratory of Frederick C. Koch in the Department of Physiological Chemistry and Pharmacology at the University of Chicago, was able to detect androgenic activity in not only a lipoid extract of bull testis utilizing the capon bioassay but also in the guinea pig (spermatozoön motility test) (170-172). This was a major breakthrough in the study of the androgenic hormone of the testis. Then followed the initial and detailed description of the changes from castration and restoration (recovery) of the histology and anatomy of the secondary sexual organs upon administration of their increasingly pure extracts (173). The investigators used similar extraction techniques for beef thymus and brain, and eventually many more tissues, but found no androgenic activity (173, 174).

Funk and colleagues used the urine from young males as starting material for the preparation of the androgenic hormone. Employing the capon bioassay of McGee and Koch to evaluate the androgenic activity obtained from concentrated or extracted urine, Funk and colleagues found significantly greater activity in the urine than in testis extracts, although the material was not pure (175). Further work noted a similarly active biological material extracted from urine from adult human males (176). Koch and colleagues were careful not to name their active substance, for they had shown that it remained a mixture of materials:

It is our feeling that until more is known of the chemical nature of the hormone no name should be given the extract. As yet any name would be valueless and not at all descriptive. Too often a name gives a false sense of security as regards the purity of the product, a fact we wish to emphasize, for it is our firm opinion that the extract is as yet grossly impure even though the minimal daily bird dose be as low as 0.01 mg. [(177), p. 500]

Koch set strict criteria for naming the testis hormone or, for that matter, any impure chemical mixture (178):

• The exact chemical nature of the product should be known.

• It should be pure.

• Its physiological activity should be accurately assayed on mammals.

Contemporaneously, Doisy, working with extracts of the ovary, put forth similar criteria for any endocrine agent (4 stages) (179):

• Recognition of the gland or organ as one producing internal secretion

• Methods of detecting internal secretion

• Preparation of extracts leading to purification of the hormone

• Isolation of pure hormone and determination of its structure and its synthesis

As summarized by Moore and Price (174), the lipoid extracted material (testis hormone) had an effect on both the testis and the pituitary of intact animals with a far greater effect in the pubertal animal when compared to the adult. It was able to exert a depressing effect on the pituitary while stimulating the accessory sex organs. When injected into a gonadally intact male, there was little damage to the seminiferous tubules but robust stimulation of the reproductive accessory structures. However, young males injected with testis hormone had testicular arrest with degenerative changes in the germinal epithelium and an excessive increase in size of the accessory structures. Hypophysectomy, followed by testis hormone injections, permitted the reproductive accessory structures to remain in a normal state but did not produce a normal testis, with the latter undergoing degeneration, as if no testis hormone had been injected. Moore and Price concluded from their own experiments and those of others that many reproductive system alterations could be interpreted as interrelationships between the gonad (testis hormone) and the pituitary (gonadotropins). The gonad could only function with signals from the pituitary, and the latter was at least partially regulated by a secretion from the testis (63, 173, 180, 181). Thus, the experiments informed about the integration and regulation of the pituitary-gonadal axis (the hypothalamic part came later).

During the time when several groups of scientists were unraveling the chemical and biological basis of the androgenic hormone, others pushed forward on a second aspect of testicular physiology. That aimed to investigate the relation of the testis to the anterior pituitary to resolve how the absence of another testicular substance led to significant hypertrophy of groups of cells that become hyperfunctional. Critical to this work were the meticulous hypophysectomy studies in the frog and rat of P.E. Smith as noted earlier (above) and in the human by Cushing. In 1923 Mottram and Cramer irradiated rat testes and noted that the tubular elements rapidly degenerated but that the interstitial cells remained intact (182). The castration cells of the pituitary hypertrophied, but the secondary sexual characteristics did not atrophy (182). Their conclusion was that there were 2 testicular hormones: 1 that maintained the secondary sexual characteristics and a second that maintained the feedback to the pituitary.

In 1926 Nukariya noted that a saline extract of the testes administered to castrated animals prevented the hypertrophic changes in the anterior pituitary but did not reverse atrophy of the secondary sexual characteristics (183). As noted earlier, it was the lipid soluble material that yielded androgenic activity, but, in addition, a water-soluble fraction of less testis tissue, likely to contain only a minimal amount of the androgenic activity, could prevent the cellular changes from appearing in the anterior pituitary. Thus, the conclusion in the 1920s and early 1930s was that a second testicular hormone existed that was not involved in pubertal maturation or maintenance of the secondary sexual characteristics (184). An early potential name was inhibin (inhibere, to check), which maintained the feedback to the pituitary.

Elegant parabiosis experiments to strengthen the hypothesis of a second testicular hormone were conducted by Martins and Rocha (185). Unexpected in their work was the finding that even the prepubertal testes secreted this prehypophyseal material, well before the production of androgens, leading to pubertal maturation. They also noted that the parabiosis of a castrated immature male rat to an infantile female rat led to precocious sexual maturation in the latter. If the castrated partner is male, implantations of testicular strips or injections of macerated testis could prevent the sexual precocity in the female partner (185). Castration of a male with parabiosis to either a normal male or normal female led to marked stimulation of the gonads and accessory sex organs in both but no such changes in the accessory sex organs of the castrated male. The anterior pituitary from the castrated male is not under the control of the gonad of its normal male partner, but anterior pituitary hormones that stimulate gonads pass from the castrated partner into the normal partner. The investigators could prevent these changes by implanting testis tissue or injecting small amounts of an aqueous extract of testis tissue into the castrated partner. The data from these experiments are compelling for a second testicular hormone, a seminiferous tubular product that did not act chemically like a steroid (185).

The Effects of Castration In the Human Male

Through the ages castration had been performed for a variety of reasons (186):

• To retain supremacy of the male elders

• As revenge against an enemy on the battlefield

• To keep slaves to serve as eunuchs in harems or seraglios

• As as punishment for rape, seduction, and adultery

• As a self-inflicted religious rite (see Matthew 19:12; the Cult of Cybele, the Skoptzy)

• To preserve a boy's treble voice into adulthood

• As a therapeutic measure, more recently for prostate cancer

Men have endured castration for more than 3000 years; the historical evidence started in ancient China and Rome. Castration and orchidopexy are described in biblical and ancient mythological texts. In Roman times, only men with testes could testify in court—the witness would place his right hand on the scrotum and say “I hereby testify” (187).

The term castration was usually considered for removal both of testis and penis (188). The effect of this removal to bring to a condition considered with the term eunuch, derived from 2 Greek words, ℇʊʋƞ ℇƛɷʋ, translated as the “guardian of bedchamber” or “chamberlain.” In the Old Testament, some verses describe newborns with syndromic features (nasal bridge absence, blindness) and crushed testicles; this last anomaly is one for which one could not be admitted to the Kingdom of the Lord (Leviticus 21:17-21). The Hebrew word saris, with the classical meaning as protector of the harem, appears several times in the Old Testament and describes that eunuchs or chamberlains had already existed in Egypt, Ethiopia, and Persia (189).

Some examples of self-castration as an expression of Christian chastity have been described. A Christian philosopher, Origen of Alexandria (c. 185 Ce-c. 254 Ce), allegedly castrated himself for the sake of the Lord (see Matthew 19:12). Later, Christianity rejected this practice (Canon 1 of the Council of Nicea, 325 Ce).

Some mythological Greek tales described Atys, who vowed to remain chaste to the Mother Goddess, Cybele. After breaking the vow, it is purported that Cybele drove him mad, causing him to self-castrate. This act led to the practice that all priests of Cybele's cult should be castrated (190, 191). During the annual, frenzied worship, they flagellated themselves with whips while the initiates would self-castrate and offer their testes to Cybele.

In Ancient Rome the Diritto Romano, the ancient Roman law, indicated different types of eunuchs, for example, the Thlibiae, who had their testicles pressed; the Thladiae, whose testicles were crushed; and the Castrati, who had their testicles (and often their penis as well) completely removed (192).

The poets of the time recorded the presence of eunuchs. Decimus Junius Juvenalis (Juvenal 55-140 Ce) was known for his savage wit and biting descriptions of Roman life:

There are girls who adore unmanly eunuchs—so smooth, so beardless to kiss, and no worry about abortions!

… let the testicles ripen and drip, fill out till they hang like two pound weights; then what the surgeon chops will hurt nobody's trade but the barber's. [(193), Juvenal as translated P. Green, p. 499]

Guy De Chauliac (1298-1368) wrote in his book Chirurgia Magna (194), “A satisfactory herniotomy cannot be performed without castration.” This attitude prevailed throughout Europe for a few hundred years, leading to many unnecessary orchiectomies.

It should be emphasized that prepubertal castrates (whether congenital or acquired) differ remarkably from those who lose testicular function after undergoing male pubertal maturation. For the adult who loses testicular function as an adult, the indices of maturity (characteristic hair growth and distribution, attained growth of the genitalia, deepened voice and adult stature) are not markedly changed (1). However, loss of testicular function occurring prepubertally abrogates maturation of the accessory reproductive organs and spermatogenesis.

The Castrati (Opera Vocalists)

Castrations were performed on 5- to 9-year-old boys to preserve the unbroken male voice through adulthood. The young castrate (musico) then undertook intensive voice training under a music master. After multiple years, the late teenager would have the chest of a man but the vocal cords of a woman. The adolescents were usually tall with disproportionately long legs, a large thorax, and an infantile larynx. Many had gynecomastia. Testosterone had been absent at a vital stage of physical maturation (195, 196). The pitch of the voice was that of a child, although somatic growth continued to permit fully developed resonating chambers of the oral cavity, pharynx, and skull, with adult thoracic power made more effective by the intensive voice training (195). Boys or male falsetto voices sang the high-scale roles (197).

Those who provided the procedure are the so-called “Norcini,” from Preci (Norcia) in Umbria in the Apennines. They were mostly unskilled but specialized in castration because they were butchers accustomed to castrating pigs for food use. However, some were considered so skillful that they were permitted to practice surgery and bone setting (198). Today a sign on a store in Norcia, Antico Norcineria, merely means pork butcher. During that time, castration of the boys was performed without disinfection and under primitive conditions with a special set of instruments appropriately called “castratori.” Many died painfully from wound infection or excess blood loss. Those who survived the procedure were given a stringent musical education that generally lasted approximately 8 years. Their musical debut did not occur until they reached more than 18 years with a well-established singing technique learned during the usual mid-pubertal age. The castrati were considered for female roles in opera, for during that historical period it was not acceptable for women to have the roles of females in operas. The prima donna (the chief female singer) was in reality the primo uomo. Both the primo uomo and the secundo uomo, usually also a castrato, were the primary singers in almost all “opera series” (199).

In that same period in the Church, only men were permitted to sing in the choirs of the Papal Chapel. Paul's letter to the Corinthians was interpreted to convey the meaning that women had no say in Church affairs: “Mulier taceat in ecclesia” [let the women be silent in church 1st Corinthians 14:34]. The ecclesiasts themselves decided that women were to be additionally excluded from any kind of music.

Society in the 18th century considered castrati to be an ambiguous phenomenon. That the castrati could enjoy sexual relationships without the obvious consequences of fertilization was not considered a disqualification from society but a definite triumph. Most castrati never even attempted to contradict the rumors on their legendary virility.

Carlo Broschi (1705-1782), more widely known as Farinelli, was undoubtedly the most famous singer (castrato) of his time with a voice of uncommon power, sweetness, extent, and agility (Fig. 5) (195). His skeletal remains were studied after exhumation in 2006. The consequences of lifelong testosterone deprivation were virtually the same as noted for Gaspare Pacchiereotti (Fabriano, 1740-1821, Padua): tall stature, persistence of less than fully fused epiphyseal lines, and severe osteoporosis with crush fractures of the spine; however, in this instance there was the addition of severe hyperostosis frontalis interna, a specific variety of bone accrual on the inner table of the frontal bone (200, 201). The latter is rare in males compared to mostly postmenopausal females, and it increases with aging and changing sex hormone concentrations. Those males mostly had disturbances of testicular function such as the Klinefelter or Kallmann syndromes, although a new pathophysiological condition, androgen deprivation therapy for prostate cancer, has become more prominent recently (201). All have a diminution of the androgen-to-estrogen balance.

Caffarelli was born Gaetano Majorano in 1710 in Bitonto near Bari. At the age of 9, his father took him to the town of Norcia to be castrated. Although he spent most of his career in Italy, Caffarelli, as many other castrati of the first rank, sang in London's Haymarket Theatre, finally settling near Naples.

Alessandro Moreschi (1858-1922) was 1 of the last castrati and the only 1 from whom recordings of his voice exist today. Those recordings, made by Moreschi from 1902 to 1904, permit us to perceive some of the extraordinary virtuosity and extensive range of the castrati voices during the 18th century.

Some of the physical characteristics of the castrati have been described in autopsy reports (202-204). Zanatta and colleagues described in detail the autopsy of Gaspare Pacchierotti, whose remains were exhumed in 2013 to reconstruct his biological profile. Anthropological measurements, computed tomography of the skeleton, and microtomography on some bones were analyzed. The main findings included (204)

• Teeth and bones consistent with old age. However, there were epiphyseal lines on the iliac crest (usually fuse at ∼23 years)

• Cervical vertebrae strongly eroded with signs of osteophytes and lipping because of osteoporosis and of continuous movement of head and neck

• Insertion of 3 important respiratory muscles—scalenus posterior and the serratus anterior and posterior superior—all of which elevate the ribs and aid in deep respiration.

It is likely that the tall stature, open epiphyseal lines, and decreased cortical density indicate the adverse events of prepubertal castration; however, the findings for the respiratory muscle insertions are likely occupational sequellae.

The Skoptzy

The Skoptzy were a Christian sect that followed its leader, Kondratii Selivanov (1730-1832), who literally took the biblical injunction of sexual chastity to physical castration and declared himself the unique reincarnation of Christ (and of the Tsar) (23). Members of the sect moved away from the Orthodox Church because of their literal belief that entry into the Kingdom of God could only occur if they had 1 of 2 operations: castration (lesser seal) or castration plus removal of the penis (greater seal), following strictly on Matthew 19:11-12 as quoted earlier. At first, castrations were done with a red-hot iron bar (baptism of fire) and later with knives or other sharp objects. Fewer women had surgery, either removal of the nipple or of the entire breast and occasionally mutilation of the labia. The Skoptzy were active in Russia in the late 18th and 19th centuries and had many ceremonies quite apart from the state Church. Many moved on to Romania as the Tsar and then Stalin made life difficult for them. An enduring relic of their long-standing hypogonadism was careful autopsy studies with precise measurements of their bodies (74, 203, 205, 206). When castration occurred prepubertally, the men were tall with disproportionately long limbs and mostly thin at least until middle age. If the castration occurred postpubertally, the men were of average size but very commonly obese. Thus, well before the purification of testosterone, Tandler and Groz had shown that the testes were not only important for reproduction but also affected the secondary sex characteristics, body proportions, osteoporosis, pituitary hyperplasia, gynecomastia, and distribution of body fat (74, 202). Those castrated prepubertally often had open long bone epiphyses into their third decade and beyond.

Chemical Identification of the Androgens and Synthesis

Although a number of testicular extracts had been tried in hypogonadal animals and several in man, it was not until 1927 that potent enough extracts could be reliably prepared. McGee (171) and McGee et al prepared a lipoid extract from bull testes and quantitated it by noting the development of secondary sexual characteristics in a validated assay in capons (172). In 1929 Funk, Harrow, and Lejwa extracted androgenic material from the urine of human males that exhibited activity in several validated bioassays (175). By 1934 Gallagher and Koch purified an extract from bulls’ testes that had approximately 10-fold the biological activity of the urine extract. The activity resided in at least 2 distinct substances based on the alkaline lability of the testis-derived, but not the urine-derived, preparation (176).

The promising results of the early, preliminary studies led to major efforts to purify and identify “the” testis hormone by 3 large European pharmaceutical companies: Organon in Amsterdam (Ernst Laqueur), Schering in Berlin (Adolf Butenandt), and Ciba in Zurich (Leopold Ruzicka). In 1931 Butenandt and colleagues obtained a biologically active, crystalline substance from thousands of liters of urine from adult males (207). In 1932 he suggested the structure of androsterone (andro, man + ster, sterol + one, ketone) and confirmed it in 1934 (208, 209). Soon after, Ruzicka and Tschopp published a method for its synthesis (210). Several investigators reported that extracts from testicular tissue contained a biologically active androgenic substance different from androsterone and more potent (211, 212). David and colleagues in Laqueuer's laboratory purified and identified the more potent androgen as testosterone in 1935 (4). It was soon synthesized by 2 groups of investigators led by Butenandt and Ruzicka (213, 214), both of whom later were awarded Nobel Prizes for their work. All of these efforts and those that followed were accelerated with the use of the newer and more efficient in vivo bioassays in rats that took advantage of the remarkable increase in the weights of the androgen-dependent primary sexual characteristics as well as some androgen-dependent muscles, such as the levator ani and bulbo-cavernosus (1, 169). Once the hormones testosterone and the less potent androsterone, an oxidized metabolite of testosterone, became available synthetically, it was clear that the more potent testosterone was able to maintain the secretory function and normal growth of the prostate and seminal vesicles and repair the castration damage whether done pre- or postpubertally (215). Androsterone was no longer considered clinically pursued.

The Amsterdam group also noted an acidic, fat-soluble substance, although not androgenic itself, enhanced the androgenicity of several of their compounds. That finding became the impetus to Miescher, Wettstein, and Tschopp to produce a series of fatty acid esters, which quickly led to useful pharmacological agents (particularly the propionate ester) for males with hypogonadism (216, 217). The practice of administering a testosterone ester in oily medium became standard to retard the rate of absorption from the intramuscular depot.

Clinical Experience With Testosterone

The usual signs and symptoms of male hypogonadism had been recognized for centuries: pubertal delay or absence, decreased muscularity, and sparse or absent sexual hair. Hypogonadism was strictly a clinical diagnosis with a few causes being known: atrophic or absent testes, hypothalamic/pituitary disease (mainly tumors), and some with normal testicular tissue (218). Even before the extraction, purification, identification, and synthesis of testosterone, there were reliable hints that a potent material was contained in the testis extracts that could reverse the usual signs and symptoms of hypogonadism in prepubertal adolescents or adult men. Gallagher and Koch presented compelling data to show the biological effects of their potent extracts (177).

A single patient, age 26 years, clearly hypogonadal with eunuchoid proportions and a youthful appearance, 166 cm in height and 50 kg in weight, with small testes and delayed closure of the long bone epiphyses and whose urine did not contain the gonadotropic activity noted in castrates was administered the testis extract for 53 days. Although there was no change in the size of the testes, epididymis, or prostate or increase in sexual hair growth, the frequency of erections greatly increased and were seemingly dose dependent as the dose was lowered and raised during this clinical trial. The patient stopped receiving the testicular extracts as the lab ceased to have the ability to produce such large amounts and the positive effects all declined within 30 days of ceasing administration (219). This n = 1 clinical trial was clearly positive, and further exploration of the effects of the male hormone(s) was merely a few years away with proper clinical trials following the extraction, purification, identification, and synthesis of androsterone and testosterone from male urine and animal testes (166, 219, 220).

Prior to this time, treatment of a few hypogonadal adolescents and young men was attempted with the gonadotropins from pregnancy urine. The results were often marginal, but some patients showed remarkable progress from prepubertal genitalia, even some older than 18 years, with enlargement of the testes and penis and increase in sexual hair (221). However, the responses were unpredictable, and the urinary extract did not show biological activity in those with primary hypogonadism.

In 1940 Aub reviewed the treatment of men with hypogonadism and noted

The best therapy for hypogonadism would be to stimulate the anterior pituitary gland to greater activity and so indirectly stimulate the gonads. [(222), p. 878]

Proper drugs for hypogonadism were not widely available, and Aub went on to describe the specific effects of testosterone therapy, concluding

From this report of the practical [emphasis added] use of testosterone in clinical medicine it is obvious that the drug is a potent one, particularly in hypogonadal males. In this condition its effectiveness as substitution therapy is uniformly accepted. [(222), p. 880]

Rather than launch into a contemporary review of testosterone therapy for hypogonadism and possibly other conditions, we have chosen to selectively review the early clinical experience to show the efficacy of treatment only in hypogonadal males of pubertal age and adults. Many of the studies after the late 1930s involved multiple additional forms of testosterone, longer periods of treatment and follow-up, and treatment related to other medical conditions [eg, Arndt, 1939, (218)] some in women. One of the more controversial conditions was called the male climacteric because of reports that aging men develop symptoms compatible with hypogonadism. In 1944 Heller and Myers considered that this occurred in men whose testis biopsy was compatible with hypogonadism and who excreted excessive (compared to younger men) amounts of gonadotropins. The investigators claimed that the symptoms of hypogonadism as well as the levels of gonadotropins in the urine declined to within the normal range with testosterone propionate therapy (223). This harkens back to the times from antiquity to the rejuvenation era, recapitulating the thinking about revitalization at the end of the 19th and early 20th centuries when the méthode séquardienne was so popular. In addition, it also anticipates the huge increase in testosterone administration after the turn of the 21st century despite no reproducible, objective, evidential basis for classifying male aging as a treatable disease per se (224, 225). It is more likely that adverse endogenous or exogenous factors (e.g., the accumulation of comorbidities) lead to the reproductive system returning (regressing) to its prepubertal state. Many of these are modifiable by diet and exercise, suggesting that preventive measures might maintain higher testosterone levels in (healthy) aging men (226, 227).

One of the first to receive synthetic testosterone propionate was a 27-year-old medical student who was prepubertal with a eunuchoid habitus. He had previously been treated with urinary gonadotropins but without effect on the secondary sexual characteristics. Within 3 days of the first injection of testosterone propionate, he experienced his first erection. Over the next few months, the secondary sexual characteristics including a deepened voice developed, and his body composition changed remarkably. When, unknown to the subject, a placebo was substituted, hot flashes reappeared and erections were less frequent (228). With reintroduction of the active hormone, the salutary effects returned; this is another instance where an n = 1 experiment gave remarkable information.

Vest and Howard treated 8 males with hypogonadism with testosterone propionate (229). Their patient population included 2 boys. The men, who had never gone through pubertal maturation, responded like Hamilton's patient (229). Roughly calculated, a dose of 50 to 75 mg per week seemed appropriate, in the range used presently. As mentioned, Koch treated a single, congenitally hypogonadal male with less pure androgenic material and described similar changes (219). The boys in this clinical trial showed proportionate growth of the external genitalia and laryngeal enlargement (229).

The use of testosterone in boys began slowly for fear of premature epiphyseal closure and perhaps disordered spermatogenesis. Studies in rats had shown both inhibition and stimulation of the testes with testosterone propionate—the former the effect on the pituitary and the latter on the immature germinal epithelium (230). To determine the physiology of androgen secretion in children and adolescents, Nathanson and colleagues measured the daily excretion of androgens in the urine using the colorimetric method of Oesting and Webster (231), which involved several extraction and purification steps but was standardized to the earlier described capon assay (232). Previous investigators had measured single samples of 24-hour urinary androgen excretion with various assays, only some of which were related to a biological assay. Measurement of daily urinary excretion of total androgens was quite variable in the adult; therefore, Nathanson and colleagues measured multiple samples from prepubertal and pubertal boys and girls (232). Although there was considerable intraindividual variability in the daily excretion of the androgens, the investigators concluded that androgen excretion increases with age, with males being slightly higher than females at the younger ages, but that the levels from the males increase greatly with physical maturation. They considered that this assay could yield information about endocrine abnormalities.

Substitution therapy became available with the commercial production of testosterone—at first the propionate ester and then many other forms including intramuscular, subcutaneous, oral, cutaneous, and implantation of crystalline pellets, when pharmaceutical companies began mass production. To avoid having to continuously administer a substance (hormone) that is secreted and used immediately, clinicians attempted to decrease the rate of absorption. For the steroid hormones androsterone and the more potent testosterone, this was accomplished by aliphatic esterification. The aliphatic esters are less soluble in body fluids than the free hormone and are more slowly and often more effectively absorbed (216). Deansely and Parks noted that the esters lose their effectiveness when administered intraperitoneally or intravenously, indicating that their prolongation of biological action occurs because of delayed absorption rather than slow hydrolysis of the compound after absorption (233). Additionally, they discovered an augmented effectiveness of the free hormones when given in dry form by the subcutaneous implantation of tablets or crystals (233). At that time, the propionate appeared to have the optimum combination of the intensity of the biologic effect coupled with duration of action (216). Later it was supplanted by the more useful and longer acting enanthate ester (234). Others delivered testosterone as crystalline methyl testosterone or testosterone propionate from pellets placed subcutaneously (235).

Multiple studies were reviewed by Pullen et al who noted not only genital growth (weeks) but also alterations in blood volume and pigmentation of the skin, appearance of spontaneous penile erections, increased excretion of androgens in the urine, the appearance of sexual hair (weeks), deepening of the voice (months), increased height and weight velocities, and advanced maturation of the epiphyseal growth plates (months) (236). Complications were encountered including peripheral edema (mostly in adults and at higher doses), bilateral gynecomastia, excessive erections, and priapism, which usually could be diminished by reducing the dose of testosterone. Concern was noted for premature epiphyseal closure and disrupted spermatogenesis, but these did not seem important issues in the late 1930s and early 1940s.

In 1942 Pullen et al reviewed the administration of testosterone to adolescent and adult males. These investigators noted the previously well-described signs and symptoms of hypogonadism in the adolescent and categorized them in what would today be called primary, secondary, and functional hypogonadism. The diagnosis of the cause of the delayed maturation in the male adolescents was not as straightforward for some, given the extant state of knowledge and laboratory assays—thus the use of gonadotropin formulations for both diagnosis as well as therapy (237). Multiple small studies spanning the years 1935 to 1940 are reviewed and may be summarized: the various protocols led to growth and maturation of the secondary sexual characteristics, including gynecomastia in some; increased height velocity with maturing of the epiphyseal growth plates; and redistribution of body fat from the feminine to the more masculine habitus (237). Concern was noted for precocious sexual maturation, if the dose was too great with an excessive number of erections. Adult short stature was also a concern but only in those with secondary hypogonadism and GH deficiency.

It should be noted that testosterone is considered substitution (replacement) therapy and was required long term. Eidelsberg and Orenstein investigated some longer term effects including adverse events (238). The authors report the usual responses as described earlier but the necessity to raise or lower the dose based on efficacy or adverse events; the average weekly dose able to maintain the secondary sexual characteristics and well-being was in the range of 50 to 75 mg per week, not unlike that for the more recent (longer acting) testosterone esters. As expected, a few of the adolescents were able to discontinue testosterone therapy, for they likely had severe constitutional delay of growth and puberty (eg, case 3) (238).

In 1942 Jaffe and Brockway used testosterone ointment and oral methyl testosterone to treat 13 males, the younger ones for undescended testicle and the adolescent-age ones for failure to display secondary sexual characteristics, some with eunuchoid proportions (239). The response rate in the former was very low, but the adolescents showed a growth spurt as well as maturation of the secondary sexual characteristics. None had fused epiphyseal growth plates.

Epilogue

It has been a long journey from the ancients observing the effects of castration in domestic animals and man to our present-day knowledge of the diagnosis and treatment of primary, secondary, and functional hypogonadism in adolescent and adult males. Along the way we have learned the physiology and pathophysiology of the integrated hypothalamic-pituitary-gonadal axis as well as a number of methods for the treatment of hypogonadism with testosterone in its many forms, gonadotropins, and hormone antagonists (feedback inhibition), such as clomiphene citrate, to augment gonadotropin release. The specter of male menopause and many forms of “pseudo”-hypogonadism remains as men continue to seek the fountain of youth for its purported dual benefits of greater virility and a longer life span.

Funding

None declared.

Disclosures

None related to the content of this historical review.

References