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The Human Testis
Current Reviews WILLIS
E.
BROWN,
editor-in-charge
The Human Testis The testes are obligated to perform two vital tasks-to produce spermatozoa for perpetuation of the speci3s and to elaborate hormones which are utilized by the germinal and somatic cells. This dichotomy of function resides within the two compartments of the testes, each of which is composed of tissue which is totally dissimilar in structure. The gametogenic and the hormonal portions of the testes, however, have parallel functional interrelationships, for many of the conditions that stimulate or depress spermatogenesis likewise modify hormone production. 1 In other respects, these tissues exhibit certain degrees of autonomy so that it is convenient, at times, to regard the testes as actually being two separate organs confined in one physical unit. 2 The amazing capacity of testicular cells to alter their form and function according to the age of the individual is a salient feature of their phYSiology. Normal function of the adult testis is dependent upon the successful accomplishment of an orderly transformation from the embryonic to the mature organ. The mechanisms involved in the growth and development of the testis thereby become a foundation for the knowledge of its function in the adult individual, especially as it pertains to matters of reproduction. It would then appear profitable to recount some facts and theories of this metamorphosis as they relate to fertility.
THE PRENATAL TESTES The primary gonocyte, or the primordial germ cell, must migrate from an independent position in the embryonic mesenchymal tissue to unite with the cells which are to compose the interstitial portions of the testis. 3 A failure to accomplish this union is thought by some to explain the total lack of germinal epithelium seen in testicular biopsies of some infertile men. During the fifth week of embryonic life the urogenital ridge may be recognized as a protrusion along NOTE. The voluminous mass of rapidly expanding knowledge requires culling from the accumulated data those findings which, to the reviewer, approximate facts and are applicable to the thesis of reproduction. An attempt at inclusiveness within the limitations of this space is impossible and the guilt of omission of many details is unavoidable.
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the dorsal mesentery of the celom. At this point the embryo is a bisexual organism awaiting sex differentiation as is preordained by genetic determinators, carried by the sex chromosomes and autosomes. However, an undetermined number of forces operate to influence complete sexual differentiation. Exogenously administered androgens and estrogens have modified the development of the ductal systems but never have reversed the sex. 4 There is, however, a constant tendency of the embryo to feminize which infers that the embryonic testes secrete a hormone which has a local action on the ductal development. Jost removed the embryonic testes in rabbits and returned the embryos to the uterus for development. 5 • 6 He demonstrated that, if embryonic castration is done at an early, specific time the embryo will develop female ducts and external genitalia. The removal of the embryonic ovary did not prevent normal differentiation of the female genital system. The primary sex cords, which are the anlage of the seminiferous tubules, appear at the seventh week, followed one week later by the Leydig cells. At this time the wolffian duct structures are seen which later become the epididymides, ductus deferens, and seminal vesicles. Failure of fusion of the epididymides to the testes or incomplete development of the epididymides and/or vasa is encountered with sufficient frequency to require routine consideration in every case of azoospermia. 7• 8 At the end of the third month the external genitalia are sufficiently developed to allow recognition as a male or as a female. The testicle is suspended on the peritoneal fold opposite the first and second lumbar vertebrae. Its descent into the scrotum at birth is influenced by the hormones of the pituitary or the androgens (or both), and by the gub~rnaculum attachments coexistent to straightening of the body of the fetus. 9 • 1o Failure to achieve complete scrotal descent by the age of puberty results in partial or complete atrophy of the germinal epithelium. l l The maternal hormones exert a profound effect upon the fetal testis. Prior to birth the seminiferous tubules are small solid cords of cells packed with primary germ cells. Surrounding these are an abundance of polyhedral epithelioid Leydig cells which dominate the microscopic appearance of the fetal testis. 12 It is probable that the luteinizing hormone (LH) factor of the maternal gonadotropins has stimulated the interstitial cells illustrating their susceptibility to this hormone. 13 COMMENT. Complete or partial absence of the epididymides or vasa offers a therapeutic challenge, for spermatogenesis is usually quite normal as determined by testis biopsy. If the entire epididymis is not palpated on each side and azoospermia is present, a biopsy is indicated. It is wise to inspect the adnexa by enlarging the incision when the biopsy is obtained, thereby confirming or discarding the suspicion of incomplete development. A proximal segment of the epididymis is usually present, situated as a nubbin at the superior pole of the testis. A subsequent exploration may be done once the biopsy confirms that spermatogenesis is normal, in the hope that the vas may be found and anasto-
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mosed to the remnant of the epididymis. My experience has been discouraging, for the vas is usually either absent or, if found, has too small a lumen to permit anastomosis. On a number of occasions I have scheduled the second operation at the time of ovulation with the intent of securing spermatozoa from the stump of the epididymis for artificial insemination if a corrective operation was impossible. Although motile spermatozoa have thus been obtained, and mixed with the patient's ejaculate collected just prior to the operation, a conception has not followed.
THE PREPUBERAL TESTES At birth the maternal hormones are abruptly withdrawn and involution of the Leydig cells consequently takes place. They are not again seen in the interstitial areas for the next 16 or more years. In their place the interstitial areas are sparsely occupied by fibroblast-like cells. Some traces of lipids have been found in these cells but their androgenic activity is exceedingly low. For instance, the young adult will average 3.7 colorimetrically measured international units per milligram of androgenic steroids; the child excretes only 0.214 The tubules slowly enlarge and contain two layers of germinal epithelium, having slight mitotic activity. Save for a gradual elongation and some increase in the tortuosity of the seminal tubules, together with the development of partial lumina, the testicle appears to be dormant for about 10 years. 15 It enlarges in size proportionate to the general body growth but is relatively unimportant to the immediate welfare or development of the young individual. This preadolescent phase of development, however, is not without significance to the future destiny and function of the testis. There is considerable evidence to support the opinion that the preadolescent germinal epithelium is even more susceptible than the adult to some harmful agents such as: roentgen rays, toxins, and vitamin deficiencies. 16 • 17 Contrariwise, the child's testis is immune to the destructive effects of the virus of mumps. lB. 19 A number of observers believe that the cryptorchid testis does not escape some injury if allowed to reside in an ectopic location.2o • 21 Others believe that such is not true and argue that undescended testes of the adult can better withstand body heat, so that orchiopexy can be safely postponed until just before puberty. This is substantiated by an 80 per cent salvage rate as proved by sperm counts or paternity of individuals who had bilateral orchiopexy performed at about the tenth year. 22 • 23. 24 It becomes apparent that few precise facts are known about the importance of illnesses that occur in preadolescence and their capacity to affect future fertility. There is sufficient justification, however, to guard the reproductive organs of the young boy against unnecessary exposure to x-rays, insist on adequate and well-balanced diet, avoid unnecessary surgical trauma, and hope that the child will acquire mumps before the age of testicular susceptibility. COMMENT. Numerous articles have appeared within recent years questioning
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the usefulness or desirability of orchiopexy. Three accomplishments may follow the operation if properly performed. The inevitable aspermatogenesis of the adult ectopic testis may be avoided. The immense psychologic value of a scrotum containing both testes should not be disregarded. Finally, in the event a testis tumor develops, the organ is in an accessible position for prompt examination and early treatment. The operative risks are minimal and there appears little to justify an attitude of resignation.
ADOLESCENCE Maturity does not begin with any given age but rather may occur at any time from 9 to 19 years. 25 There is no way to predict the time of onset of this "period of ripening" of the primary and secondary sex organs but once the process is started an average of 3 years is required for its completion. These mechanisms are highly complex, involving genetic influences, endocrine function, capacity of somatic and germinal cells to respond to growth stimuli, and matters of cell differentiation and activation. The anterior pituitary is involved in all ramifications of endocrine function and is at least one of the prime activators of a series of coordinated and reciprocal events characterizing puberty. The gonadotropic hormones appear in the urine at about the age of 12 and thereafter in increasing amounts. 25 Folliclestimulating hormone (FSH) may be the initial secretion, for tubular development precedes interstitial activity, thus indicating a lag in the production of LH. The diameter of the tubules enlarges and the testicular weight increases disproportionate to body growth. It is not generally recognized that 18 per cent of boys do not begin pubescence until the age of 14 or later, and the erroneous diagnosis of the Froehlich's syndrome is commonly made. 26 The spermatogonia increase in number, followed by the sequential production of the various stages of spermatogenesis, finally culminating months later in the production of mature spermatozoa. POSSibly the adrenocortical hormones contribute to the ripening of the Sertoli cells. 2 At about 16 years of age recognizable Leydig cells are found and hormonal activity is suggested by an abundance of lipid in the interstitial cells. Within a year the Leydig cells gain adult structure and androgens are produced in appreciable amounts, as determined by extracts of urinary ketosteroids. Coincident to the response of the Leydig cells and androgenic production, the other familiar signs of the adult male habitus appear; penile enlargement, pubic and axillary hair, enlargement of the prostate and seminal vesicles, lowering of the voice, scrotal rugae, and some acne. Likewise the output of the pituitary gonadotropins is probably checked by testicular secretions.27 This critical period of excitation involves hazards which may have grave consequences to fertility. Factors of nutrition, trauma, liver damage, infection, and irradiation have been speculated upon as adverse influences at a time of special sensitivity. Better documented are the results of failure of (1) pituitary
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gland, (2) seminiferous tubules, and (3) Leydig cells, separately or conjointly.25 If the pituitary does not release gonadotropins, the tubules and Leydig cells will fail to develop and sterility and eunuchoidism will follow. If FSH fails the tubules are immature, but the LH will afford normal male habitus by stimulation of Leydig cells and production of testicular androgens. Failure of Leydig-cell stimulation results in eunuchoidism and yet may not necessarily result in tubular failure, thereby accounting for the "fertile eunuchs."28 Evidence of dependency of spermatogenesis on Leydig cell function is, however, suggested by the development of spermatogenesis in hypogonadism treated with androgens over a period of years. 29 Clinical entities are now established by evaluation of testicular biopsies, hormonal assays, combined with the facts from the history and the physical examination. COMMENT. An exasperating realization exists that new technics are almost at hand which will more accurately chart physiologic events characterizing adolescence. It is possible that minor deviations from the normal pattern could then be detected which, if corrected, might spare the individual from germinal-cell deficiencies in later life. Until such refinements are devised the clinician must labor under the uncertainty that some types of hormonal treatments may possibly do more harm than good.
THE ADULT TESTIS By the age of 22 all normal males have completed the maturing processes of the body and any retardation evident in the germinal or somatic tissues must be regarded as abnormal and not due to a delay of adolescence. Normalcy implies full and complete function of the testis and accessory sexual glands. Therefore the production of the full complement of androgens, spermatozoa, and normal amount of seminal vesicular and prostatic secretions, in turn, becomes a fairly reliable index of adequacy of the pituitary-gonadal axis.
THE GERMINAL EPITHELIUM The kinetics of spermatogenesis in rodents has been quantitatively studied, but the human testis does not completely match the regularity of periodic cell division as observed in the lower animal. Consequently, there are always areas in the normal human testes where sperm formation is in apparent abeyance and search and selection is required to demonstrate a "typical tubule" which shows all the stages of active spermatogenesis. Leblond and Clermont have divided stages of spermatogenesis in the rat into 4 phases with subdivisions giving 19 specific synchronized steps in cell mitosis. 3 0,31 Spermatocytes appear in the epithelium of a rat following 5 successive stages of spermatogonial division. After a second division 1 spermatogonium out of 4 stops dividing, while the others go through 3 stages of division, leading to spermatocyte formation. The nondividing spermatogonium resumes mitosis in the next cycle when it acts as a stem cell for the successive divisions. Roosen-Runge demonstrated 7 divisions in the human
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evolution of the spermatogonia before differentiating into spermatocytes, but observed "a puzzling lack of uniformity" in the regular periodic divisions of spermatogenesis in the human as compared to the rat.82 • 88 Nature's lavishness prompted one individual to estimate the total number of spermatozoa produced by a typical male between the ages of 25 and 55 and arrived at the astronomical figure of 339,385,500,000. 34 Two thirds of the seminal tubule is occupied by germinal epithelium, thereby leaving a clear central lumen usually devoid of debris and containing few, if any, free, spermatozoa. Radially disposed in the germinal epithelium are the supportive Sertoli cells to which a majority of mature sperm are attached. The phenomenon of this affinity has earned the designation of "trophic cells of Sertoli," for it would appear that all the spermatozoa seek some elements from the Sertoli cells before they leave the testis on their journey through the ductal system. Cytochemical stains have demonstrated lipids, glycogens, and alkaline phosphatase within the Sertoli cells. 35 • 86 The spermatids contain no glycogen, but after detachment as spermatozoa from the Sertoli cell they are rich in this substance, and it would appear that a perfusion of glycogen had occurred.37 It also has been noted that the amount of lipids increases in defective spermatogenesis and with age. In this respect some young infertile men resemble senile men and the theory of nonutilization by the developing spermatozoa lends support that the Sertoli cell is, in fact, trophic to the germinal epithelium.3s • 39. 40
PERITUBULAR MEMBRANE The peritubular or basal membrane surrounds each tubule. This collagenous tunica contains glycoproteins and alkaline phosphatase in the basal membranes. 37 Likewise fibroblasts are identified having a histochemical nature somewhat similar to that of fibroblasts found in granulation tissue of wounds. All the blood vessels in the testis are located within the interstitial area, and therefore the peritubular membrane must be permeable to nutritive elements for spermatogenesis, and likewise must permit an exodus of the waste products of cell metabolism. Peritubular fibrosis is a common finding in the testes of men with reduced spermatozoa and is seen more commonly in old age. The degree of atrophy of the germinal epithelium is directly proportionate to the thickness of the fibrosis, so it is conjectured that a thick tunica acts as a barrier to the interchange of nutrient elements, thereby blockading spermatogenesis.
THE INTERSTITIAL AREA The intertubular connective tissue is composed of Leydig cells, fibroblasts, and blood vessels. Two types of Leydig cells have been identified; one is immature and without steroids, while the other, mature type contains lipids, steroids, and ascorbic acid, but no alkaline phosphatase.37 The lipids do not appear in material amounts until puberty and are first found in the Leydig cells and subsequently within the Sertoli cells. With advancing age intensification of lipid drop-
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lets is seen in the Sertoli cells at the expense of Leydig cells. There is uniformity of opinion that the lipids and cholesterol are the precursors of the testicular steroids, but positive identification of the relative source of testicular estrogens and androgens has not been positively established. The Leydig cell is strongly implicated as a source of androgen and the Sertoli cell has been suspected as the elaborator of the estrogens. New evidence suggests that the Leydig cell may be the sole source of both steroids.41 COMMENT. Histochemical studies upon the testis of animals and man have been employed for over 40 years. This tool has been useful in identifying precise responses in the testis to extra gonadal hormones and has localized the source of testicular steroids. Animals which have seasonal estrus are particularly valuable for demonstrating the cytologic response to hormonal stimuli as contrasted in the active and passive periods. In recent years the staining technics and interpretations have been perfected and extended and histochemical methods promise much for the future.
VASCULAR SUPPLY Interference with the blood supply to the testes for as short a time as 2 to 3 hours may result in irreversible damage. 42 The clinical inference is obvious, for the cord is exposed to trauma in such common operations as hernia repair. 43 The testicular artery runs an extensive course over the surface of the testis before sending penetrating branches into the parenchyma. Sutures placed in the tunica albuginea of the rat has caused a decrease in the size of the testicle to one third normal size. 44 Suturing of the testis to the fascia lata is done in the performance of the Keetley-Torek operation for cryptorchidism. It is possible that this may account for lack of satisfactory reports regarding ultimate fertility in the Torek operations as compared to those methods of orchiopexy not involving sutures in the lateral walls of the testes. 45 INNERVATION Severe degeneration of the seminiferous tubules results from denervation of the lumbar spinal nerves which probably pass through the sympathetic trunk, which include the third, fourth, fifth, and sixth lumbar ganglia. The pain and thermoregulatory centers may reside at about the level of D-lO to D_12.46,47 The atrophy of the germinal epithelium may be due to paralysis of the blood vessels, thereby causing alterations of testicular temperature incompatible with spermatogenesis. Hypertrophy of the Leydig cells has been noted as a parallel development to spermatic failure and increased amounts of androgens and estrogens appear in the urine of paraplegic men. 48 The inference has been raised that one consequence of spermatic failure is the lack of a check on the pituitary gland, resulting in increased elaboration of LH, thereby stimulating the steroid-producing cells.
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ADDITIONAL FACTORS INFLUENCING TESTIS FUNCTION AS RElATED TO FERTILITY Heredity
Observations upon mice, flies, and certain animals indicate that one cause of infertility in the male may be genetic in origin. 49 Likewise, there are records of human families in which the male offspring were uniformly sterile. 50 It is possible that certain disturbances of mitoses seen in biopsies of some subfertile men are inherited chromosomal aberrancies.'51 The Endocrines
Pituitary Gland. A normal pituitary is a necessary requisite to complete testicular function. All 6 or more hormones of the pituitary may be involved directly or indirectly with growth, development, and maintenance of testicular function in adult life. Three of these are so intimately associated with the testis that they are collectively designated as the gonadotropic hormones. Their complete identity, individual action, and composition are still debatable but follicle-stimulating hormone (FSH) is considered by many to stimulate the germinal and the Sertoli cells. The leuteinizing hormone LH influences the Leydig cells and may act in conjunction with FSH on the tubular epithelium. 25 The role of the lactogenic gonadotropic hormone (LTH) has not been clearly defined but it may act in conjunction with LH. The other pituitary hormones-the thyrotropin (TTH), the adrenotropin (ACTH) and the growth hormone-may have an indirect action through stimulation of the respective target organs. In the absence of the pituitary hormones, testicular function regresses but pituitary transplants, extracts, or substitutions will sustain spermatogenesis and steroid production. Chorionic gonadotropin (FSH) has been utilized extensively in man, and three effects are generally recognized and agreed upon1: (1) It is capable of stimulating growth and endocrine function in the immature human testes. (2) It may produce descent of the cryptorchid testis. (3) It may produce growth of the Leydig cells and stimulate androgen secretion in some hypogonadal states. Extracts of animal pituitary glands induce antihormone response in other species, thereby limiting the full utilization of potential activity. The gonadotropins now available for clinical administration to humans have been disappointing in the treatment of most infertile men. A few proved cases of pituitary failure with prepuberal types of testicular dysfunction have responded to treatment. 52 Some hitherto-unexplained responses following the administration of gonadotropins might be due to alterations in the estrogen-androgen levels as explained further on. At this time progress awaits the development of more specific and highly sensitive methods of quantitative tests for determining titers of circulating FSH and LH and the availability of purer products for administration. SterOid-Producing Glands of Internal Secretion. The androgenic steroids are
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derived from the adrenals and the testes. Approximately thirty steroids have been isolated from the adrenals, of which 3 have androgenic properties. The adrenals contribute more than one half the total steroids, but doubts are raised as to whether their action is similar to the testicular androgens. The latter are probably produced only by the Leydig cells. Estrogens likewise may have origin in the adrenal tissue, but Maddock and Nelson offer evidence that only the Leydig cell is concerned with estrogen production. 41 Administration of FSH resulted in an increase of 16 times the estrogen production, with concomitant slight elevation of androgens. There was no estrogen response in castrates, which indicated to these investigators that the testis is the sole source of estrogens. Furthermore, cytochemical stains indicated that the Leydig cell and not the Sertoli cell was the site of this steroid. Spermatogenesis was temporarily decreased following FSH administration, presumably by inhibition of the pituitary by the estrogens and androgens. The reciprocal activity of the pituitary and the steroids has recently gained much attention. The circulating steroids regulate the production of gonadotropins, for castration results in an outpouring of the pituitary hormones. 53 In tum, estrogens and androgens are capable of checking their production. Hypophysectomized rats are protected against the loss of spermatogenesis by androgen administration, so that a dual action of inhibition and stimulation is involved. 54 The degree of the effect is somewhat dependent on dosage. In sufficient dosage both androgens and estrogens inhibit spermatogenesis. Estrogens never stimulate maturation of the germinal epithelium, and in balanced amounts androgens can counteract the harmful effects of estrogens. The liver destroys excessive amounts of circulating estrogens except when this organ is incapacitated by disease. 55 Accordingly adequate liver function appears important to the health of the testis. The implications of these discussions are pertinent to the clinical utilization of testosterone for stimulation of spermatogenesis. There are those who believe that once the depression has been produced recovery exceeds the rate of impaired spermatogenesis existing prior to treatment. The usefulness in oligospermia of the so-called rebound principle of clinical therapy has both its advocates and its skeptics.56 • 57. 58 Testosterone therapy has with very rare exceptions failed to convert azoospermic men to individuals capable of having spermatozoa in any appreciable numbers in their semen. 59 In rare cases of eunuchoidism, testosterone not only developed the secondary sexual characteristics and libido but actually resulted in viable spermatozoa with subsequent pregnancy in one patient's wife following years of androgenic therapy.29 It has been stated that approximately 1 of 5 oligospermic men, selected on the basis of favorable testicular biopsies, will show some improvement in the semen following large intramuscular dosage of testosterone (3000 mg.) .59 Failure of sperm to reappear in the semen after testosterone therapy has been reported. 60 The role of adrenal androgens in testicular physiology remains obscure. Precocious spermatogenesis and virilism in boys have been observed in adrenal
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hyperplasia. Reports of both increases and reductions of spermatogenesis have been made following the use of cortisone for the treatment of oligospermia in men. 60 Increased production of gonadotropins following administration of cortisone is known, and some presume that cortisone disturbs the antigonadotropin formation. 6l Testicular biopsies on men before and after total adrenalectomy for prostatic cancer did not reveal significant changes in the character of spermatogenesis. 62 Unpublished data on total adrenalectomy in dogs suggest that the testes are not dependent upon the adrenals for continued spermatogenesis.63 Thyroid Gland. Thyroid medication has probably been employed more frequently than any other drug for the treatment of male infertility. The evidence of a truly specific relationship between the thyroid and the gonads is, however, very conflicting. Some experienced clinicians are so convinced of its efficacy that the statement has been made, "Successes following thyroid treatment are now numerous enough to remove all doubt about cause and effect relationship of that trouble to infertility."64 Others have shown that poor semen quality is not a characteristic of low metabolic states. 65 Thyroid administration has restored the ovarian cycle in myxedematous women, and the 17-ketosteroid output is said to be increased in the treatment of men with hypothyroidism. 66 Animal experimentation, likewise, is inconclusive and not in agreement. Removal of gonads in dogs, rabbits, and rats causes slow involution of thyroid. 67 Thyroidectomy at birth prevents testis development, but shortly after birth thyroidectomy retards but does not prevent spermatogenesis. 6s Thyroxine may cause atrophy of the seminal vesicles and probably suppresses gonadotropin production. Some authors believe that thyroid causes testicular hypertrophy, and others report atrophy. Numerous theories have been advanced to explain the relationship between the thyroid and the gonad, among which are that "the thyroid may sensitize peripheral tissues," "the gonadal hormones have a direct effect on gonadotropin production," "[the thyroid has] indirect effect by way of adrenal alterations," and "[the thyroid regulates] oxygen metabolism of the cell." The net result of a perusal of the literature is that despite therapeutic popularity much remains to be known about thyroid-gonadal interrelationships, and the clinical use of thyroid substances must be regarded as largely empirical at this time.
Nutrition Inadequate intake, assimilation, and utilization of food may affect the testes of the immature male more severely than comparable deficiencies in the adult. 69 Rats maintained on an inanition diet from age 3 to 4 weeks may not produce cells above spermatocyte stage during the entire first year of life.70 Other deprivation experiments in the growing animal likewise support increased concern about the remote effects of improper nutrition in childhood.71 Recent surveys demonstrate that dietary deficiencies are common regardless of social and financial status. Impaired spermatogenesis may result not only from avitaminosis but also from deficiency in caloric intake, lack of such minerals as magnesium, zinc, sodium,
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potassium, and phosphorus, and from insufficient proteins. 72 • 73 Impaired sexual performance has been reported after fat-free diet. Tubular atrophy, sloughing, sperm agglutination, and even complete loss of germinal epithelium, except for the Sertoli cells, is known to follow exclusion of vitamins A and/or E. The effects of the latter may be irreversible. The testis of the mature individual is fairly resistant to deficiencies. Weight losses of considerable proportions can take place in the experimental animal before loss of spermatogenesis is noted. The severe nutritional deficiencies endured by prisoners of war and men confined to concentration camps often caused complete loss of libido at the time of starvation. It is likely that spermatogenesis was also greatly reduced, yet postwar examinations at the time of weight recovery have repeatedly demonstrated that many of these men had normal seminal specimens. Vitamin therapy for oligospermia and poor motility seems justified when indicated by poor dietary habits and when there is evidence that liver function is impaired. Specific therapeutic effects are difficult to identify for reasons similar to those discussed under thyroid therapy. Temperature
An extensive literature exists upon the adverse effects of heat upon the adult testis. Local or systemic elevations in temperature may cause disappearance of germinal cells in the reverse order of spermatogenesis, indicating that the primary cells are most resistant to the effects of elevation of temperature. A 10minute exposure of the scrotum of a guinea pig to water warmed (6° C.) above body temperature results in great damage to the tubules. 74 Recovery requires some weeks but repeated treatment results in permanent sterility. The thermoregulatory function of the testes is well known and recently interest has again developed in varicoceles as they might interfere with heat loss. Improvement in semen has been reported following varicocelectomy, but additional observations are required before conclusions are to be reached. 75 Radiation
The biologic action of ionizing radiation has been the subject of many studies directly upon sperm, after localized irradiation of the testes, and more recently on whole body exposure. 76 , 77 The results have been analyzed in terms of cellular destruction, inability of sperm to fertilize the ova, the effects on fetal mortality, and the emergence of mutants in the progeny.7S-S1 The production of detrimental mutants is predicted in 3 of every 5 children of men who had received a total of 225 r to their gonads in prepuberal years. Serious consideration should be given to the needs for diagnostic x-ray and, especially, for therapeutic radiation, in boys.82 Irradiation of the adult testis may require years for reconstruction of germinal epithelium. 77 The damage is apparently proportionate to the dosage received,
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and this probably causes azoospermia in men with poor spermatogenesis much more quickly than when spermatogenesis is normal. COMMENT. Any consideration of factors which influence testis function have therapeutic connotations. Such implications are worthy and justified, for perhaps the principal objective of such thinking is to provide effective treatment for infertility in man and animals. This discipline is still very young and has had serious attention by only a very few until 30 or 40 years ago. The treatment methods leave much to be desired and, by force of circumstance, are sometimes based upon empiric formulas and may, at times, be unscientific and of little value. Clarification of the basic principles of the physiology of the male reproductive system affords a foundation for sound treatment. Encouragement and support for fundamental investigation appears to be the logical approach for reasonably efficient forms of treatment.
SUMMARY The dual function of the mature testis is dependent upon an extraordinary number of diversified influences which govern its growth and development from fetal to postadolescent life. Spermatogenesis is far more delicate and labile than hormone production, for both the Sertoli and the Leydig cells can withstand many adverse provocations that prove to be detrimental to the germinal epithelium. The primary spermatogenic cells are more robust than the final products of cell division, for injury is almost always first recorded in the late stages of spermatogenesis. Recent advances in knowledge have clarified many mechanisms related to testicular physiology, yet many of the therapeutic manipulations based on these inferences have, so far, failed to stimulate faulty spermatogenesis. ROBERT S. HOTCHKISS, M.D. New York City
REFERENCES 1. ALBERT, A., UNDERDAHL, L. 0., GREENE, L. F., and LORENZ, N. Male hypogonadism: VI. The testis in gonadotropic failure in adults. Proc. Staff Meet. Mayo Glin. 29:368, 1954. 2. LUDWIG, D. J. The effect of androgens on spermatogenesis. Endocrinology 46:453, 1950. 3. TRABUCCO, A. Congenital male sterility. J. Urol. 80: 156, 1948. 4. MOORE, C. R. The influence of hormones on the development of the reproductive system. ]. Urol. 45:369, 1941. 5. JOST, A. Recherches sur la differen-
ciation sexuelle de l'embryon de lapin. Arch. anat. micro et morphol. expo 36: 271, 1947. 6. GRUMBACH, M. M., VAN WYK, J. J., and WILKINS, L. Chromosomal sex in gonadal dysgenesis: Relationship to male pseudohermaphrodism and theories of sex differentiation. J. Glin. Endocrinol. & Metab. 15:1161, 1955. 7. DEAN, A. L., MAJOR, J. W., and OTTENHEIMER, E. J. Failure of fusion of the testis and epididymis. ]. Urol. 68:754, 1952. 8. MICHELSON, L. Congenital anomalies of ductus deferens and epididymis. J. Urol. 61 :384, 1949.
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9. BEACH, K W. Undescended testes: Cause and treatment. J. Urol. 60: 623,1948. 10. LEWIS, L. G. Cryptorchidism. N. Y. State]. Med. 54:3078, 1954. 11. PACE, J. M., and CABOT, H. Histologic study of 24 cases of retained testes in the adult. Surg. Gynec. & Obst. 63:16, 1936. 12. ENGLE, E. T. The life history of the human testis. J. Urol. 74:379, 1955. 13. SNIFFEN, R. C. Histology of the normal and abnonnal testis at puberty. Ann. N. Y. Acad. Sc. 55:609, 1952. 14. HAMILTON, J. B. Androgenic activity per milligram of colorimetrically measured ketosteroids in urine: An index of the respective contributions from testicular and extra-testicular sources. J. GUn. Endocrinol. & Metab. 14:452, 1954. 15. CHARNY, C. W., CONSTON, A. S., and MERANZE, D. R. Testicular developmental histology. Ann. N. Y. Acad. Sc. 55:597, 1952. 16. STURKIE, P. D., PINO, J. A., WEATHERWAX, J. L., DONNELLY, A. J., and DORRANCE, G. M. Effect of x-rays on fertility of male chickens treated before puberty. Radiology 52:112, 1949. 17. ESSENBERG, J. M., and ZIKMUND, A. An experimental study on the effects of roentgen rays on the gonads of developing chicks. Radiology 31 :94, 1938. 18. WERNER, C. A. Mumps orchitis and testicular atrophy. Ann. Int. Med. 32: 1066, 1950. 19. CHARNY, C. W., and MERANZE, D. R. Pathology of mumps orchitis. Proc. Gonf. Ster. & Inf. III:167, 1947. 20. SMITH, D. R. The treatment of cryptorchidism. Galifornia Med. 81: 379, 1954. 21. ROBINSON, J. N., and ENGLE, E. T. Some observations on the cryptorchid testis. J. Urol. 71 :726, 1954. 22. PRENTISS, R. J., MULLENIX, R. B., WmSENAND, J. M., and FEENEY, M. J. Medical and surgical treatment of
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cryptorchidism. A. M. A. Arch. Surg. 70:283, 1955. MACCOLLUM, D. W. Clinical study of spermatogenesis in undescended testis. Arch. Surg. 31 :290, 1935. GROSS, R. K, and JEWETT, T. C. Surgery for undescended testis; Experiences from 1222 operations. Read at the Annual Meeting of the A. M. A. June 8,1955. J.A.M.A.160:634; 1956. ALBERT, A., UNDERDAHL, L. 0., GREENE, L. F., and LORENZ, N. Male hypogonadism: VII. The testis in partial gonadotropic failure during puberty (lack of luteinizing hormone only). Proc. Staff Meet. Mayo GUn. 30:31, 1955. HURXTHAL, L. M. Hypogenitalism during the usual time of puberty. J.A.M.A. 136:12, 1948. NELSON, W.O., and HELLER, C. G. Hyalinization of seminiferous tubules associated with nonnal or failing Leydig cell functions. J. Glin. Endocrinol. 5:13, 1945. MCCULLAGH, E. P., BECK, J., and SCHAFFENBURG, C. A. A syndrome of eunuchoidism with spermatogenesis. J. GUn. Endocrinol. & Metab. 13:489, 1953. WERNER, S. C. Spennatogenesis and apparent fertility in a eunuchoid male in the eleventh year of androgen therapy. J. GUn. Endocrinol. 6:612, 1951. LEBLOND, C. P., and CLERMONT, Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N. Y. Acad. Sc. 55:548, 1952. CLERMONT, Y., and LEBLOND, C. P. Renewal of spennatogonia of the rat. Am. J. Anat. 93:475, 1953. ROOSEN-RuNGE, E. C., and BARLOW, F. D. Quantitative studies on human spennatogenesis. Am. J. Anat. 93: 143, 1953. ROOSEN-RuNGE, E. C. Kinetics of spennatogenesis in mammals. Ann. N. Y. Acad. Sc. 55:574, 1952. MOTT, F. M. Normal and morbid
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conditions of testes from birth to old male paraplegics. Am. ]. Glin. Pathol. 20:24, 1950. age in 100 asylum and hospital cases. 48. BORS, E., ENGLE, E. T., ROSENQUIST, Brit. M. J. 2:655, 1919. R. C., and HOLLIGER, M. D. FertilLONG, M. E., and ENGLE, E. T. Cyity in paraplegic male. J. Glin. Entochemistry of the human testis. docrinol. 10:381, 1950. Ann. N. Y. Acad. Sc. 55:619,1952. ARZAC, J. P. Glycogen in human 49. SMELSER, K. Spontaneous sterility in male rat. Anat. Rec. 64: Supp. 1, testicular biopsy material. J. Glin. 1935. Endocrinol. 10:1465, 1950. MANCINI, R. E., NOLAZCO, J., and DE 50. BIBEN, R. L., and GoRDON, G. S. Familial hypogonadotropic eunuchLA BALZE, F. A. Histochemical oidism. Glin. Endocrinol. & Metab. study of the normal adult testis. 15:931, 1955. Anat. Rec. 114:127, 1952. PERLMAN, P. L. The functional sig- 51. ENGLE, E. T. The cytological problem in spermatogenic arrest. Ann. nificance of testis cholesterol in the N. Y. Acad. Sc. 55:703, 1952. rat: Effects of hypophysectomy and 52. HOWARD, R. P., SNIFFEN, R. C., SIMcryptorchidism. Endocrinology 46: MONS, F. A., and ALBRIGHT, F. Tes341, 1950. ticular deficiency: A clinical and MONTAGNA, W. Some cytochemical pathologic study. ]. Glin. Endocriobservations on human testis and epinolo 10:121, 1950. didymis. Ann. Acad. Sc. 55:629, 53. HAMILTON, J. B., HAMILTON, H. B., 1952. and MESTLER, G. E. Aging in apLYNCH, K. M., and SCOTT, W. W. parently normal men: II. Androgenic The Sertoli cells as related to age of activity of urinary ketosteroids and man and experimental alterations of other alpha and beta fractions; the the pituitary-gonadal axis in the anirelationship of androgenic titres to mal. Fertil. & Steril. 3:35, 1952. values obtained by colorimetric assay. MADDOCK, W.O., and NELSON, W. O. J. Glin. Endocrinol. & Metab. 14: 139, The effects of chorionic gonadotropin 1954. in adult men. J. Glin. Endocrinol. & 54. PULLEN, R. L., WILSON, J. A., Metab. 12:985, 1952. HAMBLEN, E. c., and CUYLER, W. K. HINMAN, F., and SMITH, G. I. SperClinical reviews in andrologic endomatogenesis following experimental Crinology: I. PhYSiology, functional testicular ischemia. Fertil. & Steril. pathology and diagnosis. ]. Glin. 6:443, 1955. Endocrinol. 2:577, 1942. BAKER, J. W. Insult to the testicle 55. GLASS, S. J. The importance of the in herniorrhaphy. Surg. Gynec. & liver in reproductive physiology. Tr. Obst. 75:285, 1942. Am. Soc. Study Steril. Western HARRISON, R. G., and BARCLAY, A. E. Journal of Surgery Publishing Co., Distribution of the testicular artery to 1946, p. 169. the human testis. Brit. J. Urol. 20: 56. HECKEL, N. J., Rosso, W. A., and 57, 1948. KESTEL, L. Spermatogenic rebound phenomena after testosterone therapy. REA, C. E. Fertility in cryptorchids. Minnesota Med. 34:216, 1951. J. Glin. Endocrinol. 11 :235, 1951. TALBOT, H. S. A report on sexual 57. SWYER, G. I. M. Effects of testosterone implants in men with defective function in paraplegics. J. Urol. 61: spermatogenesis. Brit. M. J. 2:1080, 265,1949. 1953. STEMMERMANN, G. N., WEISS, L., AUERBACH, 0., and FRIEDMAN, M. 58. GETZOFF, P. L. Clinical evaluation A study of the germinal epithelium in of testicular biopsy and the rebound
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phenomena. Fertil. & Steril. 6:465, 1955. CHARNY, C. W. Treabnent of male infertility with large doses of testosterone. I.AM.A 160:98, 1956. MICHELSON, L., ROLAND, S., and KOETS, P. The effects of cortisone on the infertile male. Fertil. & Stem. 6:493, 1955. WILKINS, L., and CARA, J. Further studies on the treatment of congenital adrenal hyperplasia with cortisone: V. Effects of cortisone therapy on testicular development. I. Clin. Endocrinol. & Metab. 14:287, 1954. HOTCHKISS, R. S. Observations upon the testes following total adrenalectomy in man. Ann. ¢stet. e ginec. Third Special Number, No.6, June, 1953. HOTCHKISS, R. S., and MORALES, P. Unpublished data. MEAKER, S. R., and VOSE, S. N. Thyroid and spermatogenesis. I.AMA. 115:1426, 1940. HAMBLEN, E. C., PULLEN, C. A., and CUYLER, W. K. Clinical studies of thyrogonadal correlates: II. Basal metabolic rates and seminal findings. I. Clin. Endocrinol. 1 :528, 1941. LERMAN, J. Thyroid physiology: Gland, physiology and therapy. AM.A Council on Pharmacy and Chemistry, Symposium (ed. 2). Chicago A.M.A., 1942. SALMON, T. N. Effect of pituitary growth substance on the development of rats thyroidectomized at birth. Endocrinol. 29:291, 1941. MARINE, D. Physiology and principal interrelations of thyroid. I.A.M.A 104:2250, 1935. GOETTSCH, M. The relationship between vitamin C and some phases of reproduction in the guinea pig. Am. I. Physiol. 95 :64, 1930. MASON, K. E. Histologic study of sterility in rats due to dietary defi-
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ciency. Proc. Nat. Acad. Sc. 11 :377, 1925. BISKIND, M. S., and WILLIAMS, R. R. Nutritional survey at a children's institution: Incidence of avitaminotic lesions and effects of therapy. Am. I. Digest. Dis. 14:121, 1947. SHETTLES, L. B. The relation of nutrition to spermatogenesis. Proc. Third Ann. Conf. on Biol. Spermatozoa, Nat. Comm. Matern. Health. 1942. FOLLIS, R. H., DAY, H. G., and McCOLLUM, E. U. Histologic studies of tissues of rats fed a diet extremely low in phosphorus. ]. Nutrition 20: 181, 1940. MOORE, C. R. Temperature and scrotal function, in CAMPBELL, M. Urology. Philadelphia, Saunders, 1954. Vol. I, Sect. I, Chap. 5, p. 132. DAVIDSON, H. A. Treabnent of male subfertility. Practitioner 173:703, 1954. MURPHREE, R. L., WmTAKER, W. M., WILDING, J. L., and RUST, J. H. Effect of whole body exposure to irradiationsupon subsequent fertility of male rabbits. Science 115 :709, 1952. ROBINSON, J., and ENGLE, E. T. Effect of neutron radiation on human testis. ]. Urol. 61 :781, 1949. CHARNY, C. W. Low dosage irradiation in male infertility. Fertil. & Steril. 1: 150, 1950. EVANS, T. C. Effect of small daily doses of fast neutrons on mice. Radiology 50:811, 1948. MULLER, H. J. The manner of dependence of permissible dose of radiation on the amount of genetic damage. Acta radial. 41 :5, 1954. MULLER, H. J. Damage to posterity caused by irradiation of the gonads. Am. I. Obst. & Gynec. 67:467, 1954. KIRSH, 1. E. Radiation dangers in diagnostic radiology. I.AM.A 158: 1420, 1955.
E.
BROWN,
editor-in-charge
The Human Testis The testes are obligated to perform two vital tasks-to produce spermatozoa for perpetuation of the speci3s and to elaborate hormones which are utilized by the germinal and somatic cells. This dichotomy of function resides within the two compartments of the testes, each of which is composed of tissue which is totally dissimilar in structure. The gametogenic and the hormonal portions of the testes, however, have parallel functional interrelationships, for many of the conditions that stimulate or depress spermatogenesis likewise modify hormone production. 1 In other respects, these tissues exhibit certain degrees of autonomy so that it is convenient, at times, to regard the testes as actually being two separate organs confined in one physical unit. 2 The amazing capacity of testicular cells to alter their form and function according to the age of the individual is a salient feature of their phYSiology. Normal function of the adult testis is dependent upon the successful accomplishment of an orderly transformation from the embryonic to the mature organ. The mechanisms involved in the growth and development of the testis thereby become a foundation for the knowledge of its function in the adult individual, especially as it pertains to matters of reproduction. It would then appear profitable to recount some facts and theories of this metamorphosis as they relate to fertility.
THE PRENATAL TESTES The primary gonocyte, or the primordial germ cell, must migrate from an independent position in the embryonic mesenchymal tissue to unite with the cells which are to compose the interstitial portions of the testis. 3 A failure to accomplish this union is thought by some to explain the total lack of germinal epithelium seen in testicular biopsies of some infertile men. During the fifth week of embryonic life the urogenital ridge may be recognized as a protrusion along NOTE. The voluminous mass of rapidly expanding knowledge requires culling from the accumulated data those findings which, to the reviewer, approximate facts and are applicable to the thesis of reproduction. An attempt at inclusiveness within the limitations of this space is impossible and the guilt of omission of many details is unavoidable.
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the dorsal mesentery of the celom. At this point the embryo is a bisexual organism awaiting sex differentiation as is preordained by genetic determinators, carried by the sex chromosomes and autosomes. However, an undetermined number of forces operate to influence complete sexual differentiation. Exogenously administered androgens and estrogens have modified the development of the ductal systems but never have reversed the sex. 4 There is, however, a constant tendency of the embryo to feminize which infers that the embryonic testes secrete a hormone which has a local action on the ductal development. Jost removed the embryonic testes in rabbits and returned the embryos to the uterus for development. 5 • 6 He demonstrated that, if embryonic castration is done at an early, specific time the embryo will develop female ducts and external genitalia. The removal of the embryonic ovary did not prevent normal differentiation of the female genital system. The primary sex cords, which are the anlage of the seminiferous tubules, appear at the seventh week, followed one week later by the Leydig cells. At this time the wolffian duct structures are seen which later become the epididymides, ductus deferens, and seminal vesicles. Failure of fusion of the epididymides to the testes or incomplete development of the epididymides and/or vasa is encountered with sufficient frequency to require routine consideration in every case of azoospermia. 7• 8 At the end of the third month the external genitalia are sufficiently developed to allow recognition as a male or as a female. The testicle is suspended on the peritoneal fold opposite the first and second lumbar vertebrae. Its descent into the scrotum at birth is influenced by the hormones of the pituitary or the androgens (or both), and by the gub~rnaculum attachments coexistent to straightening of the body of the fetus. 9 • 1o Failure to achieve complete scrotal descent by the age of puberty results in partial or complete atrophy of the germinal epithelium. l l The maternal hormones exert a profound effect upon the fetal testis. Prior to birth the seminiferous tubules are small solid cords of cells packed with primary germ cells. Surrounding these are an abundance of polyhedral epithelioid Leydig cells which dominate the microscopic appearance of the fetal testis. 12 It is probable that the luteinizing hormone (LH) factor of the maternal gonadotropins has stimulated the interstitial cells illustrating their susceptibility to this hormone. 13 COMMENT. Complete or partial absence of the epididymides or vasa offers a therapeutic challenge, for spermatogenesis is usually quite normal as determined by testis biopsy. If the entire epididymis is not palpated on each side and azoospermia is present, a biopsy is indicated. It is wise to inspect the adnexa by enlarging the incision when the biopsy is obtained, thereby confirming or discarding the suspicion of incomplete development. A proximal segment of the epididymis is usually present, situated as a nubbin at the superior pole of the testis. A subsequent exploration may be done once the biopsy confirms that spermatogenesis is normal, in the hope that the vas may be found and anasto-
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mosed to the remnant of the epididymis. My experience has been discouraging, for the vas is usually either absent or, if found, has too small a lumen to permit anastomosis. On a number of occasions I have scheduled the second operation at the time of ovulation with the intent of securing spermatozoa from the stump of the epididymis for artificial insemination if a corrective operation was impossible. Although motile spermatozoa have thus been obtained, and mixed with the patient's ejaculate collected just prior to the operation, a conception has not followed.
THE PREPUBERAL TESTES At birth the maternal hormones are abruptly withdrawn and involution of the Leydig cells consequently takes place. They are not again seen in the interstitial areas for the next 16 or more years. In their place the interstitial areas are sparsely occupied by fibroblast-like cells. Some traces of lipids have been found in these cells but their androgenic activity is exceedingly low. For instance, the young adult will average 3.7 colorimetrically measured international units per milligram of androgenic steroids; the child excretes only 0.214 The tubules slowly enlarge and contain two layers of germinal epithelium, having slight mitotic activity. Save for a gradual elongation and some increase in the tortuosity of the seminal tubules, together with the development of partial lumina, the testicle appears to be dormant for about 10 years. 15 It enlarges in size proportionate to the general body growth but is relatively unimportant to the immediate welfare or development of the young individual. This preadolescent phase of development, however, is not without significance to the future destiny and function of the testis. There is considerable evidence to support the opinion that the preadolescent germinal epithelium is even more susceptible than the adult to some harmful agents such as: roentgen rays, toxins, and vitamin deficiencies. 16 • 17 Contrariwise, the child's testis is immune to the destructive effects of the virus of mumps. lB. 19 A number of observers believe that the cryptorchid testis does not escape some injury if allowed to reside in an ectopic location.2o • 21 Others believe that such is not true and argue that undescended testes of the adult can better withstand body heat, so that orchiopexy can be safely postponed until just before puberty. This is substantiated by an 80 per cent salvage rate as proved by sperm counts or paternity of individuals who had bilateral orchiopexy performed at about the tenth year. 22 • 23. 24 It becomes apparent that few precise facts are known about the importance of illnesses that occur in preadolescence and their capacity to affect future fertility. There is sufficient justification, however, to guard the reproductive organs of the young boy against unnecessary exposure to x-rays, insist on adequate and well-balanced diet, avoid unnecessary surgical trauma, and hope that the child will acquire mumps before the age of testicular susceptibility. COMMENT. Numerous articles have appeared within recent years questioning
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the usefulness or desirability of orchiopexy. Three accomplishments may follow the operation if properly performed. The inevitable aspermatogenesis of the adult ectopic testis may be avoided. The immense psychologic value of a scrotum containing both testes should not be disregarded. Finally, in the event a testis tumor develops, the organ is in an accessible position for prompt examination and early treatment. The operative risks are minimal and there appears little to justify an attitude of resignation.
ADOLESCENCE Maturity does not begin with any given age but rather may occur at any time from 9 to 19 years. 25 There is no way to predict the time of onset of this "period of ripening" of the primary and secondary sex organs but once the process is started an average of 3 years is required for its completion. These mechanisms are highly complex, involving genetic influences, endocrine function, capacity of somatic and germinal cells to respond to growth stimuli, and matters of cell differentiation and activation. The anterior pituitary is involved in all ramifications of endocrine function and is at least one of the prime activators of a series of coordinated and reciprocal events characterizing puberty. The gonadotropic hormones appear in the urine at about the age of 12 and thereafter in increasing amounts. 25 Folliclestimulating hormone (FSH) may be the initial secretion, for tubular development precedes interstitial activity, thus indicating a lag in the production of LH. The diameter of the tubules enlarges and the testicular weight increases disproportionate to body growth. It is not generally recognized that 18 per cent of boys do not begin pubescence until the age of 14 or later, and the erroneous diagnosis of the Froehlich's syndrome is commonly made. 26 The spermatogonia increase in number, followed by the sequential production of the various stages of spermatogenesis, finally culminating months later in the production of mature spermatozoa. POSSibly the adrenocortical hormones contribute to the ripening of the Sertoli cells. 2 At about 16 years of age recognizable Leydig cells are found and hormonal activity is suggested by an abundance of lipid in the interstitial cells. Within a year the Leydig cells gain adult structure and androgens are produced in appreciable amounts, as determined by extracts of urinary ketosteroids. Coincident to the response of the Leydig cells and androgenic production, the other familiar signs of the adult male habitus appear; penile enlargement, pubic and axillary hair, enlargement of the prostate and seminal vesicles, lowering of the voice, scrotal rugae, and some acne. Likewise the output of the pituitary gonadotropins is probably checked by testicular secretions.27 This critical period of excitation involves hazards which may have grave consequences to fertility. Factors of nutrition, trauma, liver damage, infection, and irradiation have been speculated upon as adverse influences at a time of special sensitivity. Better documented are the results of failure of (1) pituitary
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gland, (2) seminiferous tubules, and (3) Leydig cells, separately or conjointly.25 If the pituitary does not release gonadotropins, the tubules and Leydig cells will fail to develop and sterility and eunuchoidism will follow. If FSH fails the tubules are immature, but the LH will afford normal male habitus by stimulation of Leydig cells and production of testicular androgens. Failure of Leydig-cell stimulation results in eunuchoidism and yet may not necessarily result in tubular failure, thereby accounting for the "fertile eunuchs."28 Evidence of dependency of spermatogenesis on Leydig cell function is, however, suggested by the development of spermatogenesis in hypogonadism treated with androgens over a period of years. 29 Clinical entities are now established by evaluation of testicular biopsies, hormonal assays, combined with the facts from the history and the physical examination. COMMENT. An exasperating realization exists that new technics are almost at hand which will more accurately chart physiologic events characterizing adolescence. It is possible that minor deviations from the normal pattern could then be detected which, if corrected, might spare the individual from germinal-cell deficiencies in later life. Until such refinements are devised the clinician must labor under the uncertainty that some types of hormonal treatments may possibly do more harm than good.
THE ADULT TESTIS By the age of 22 all normal males have completed the maturing processes of the body and any retardation evident in the germinal or somatic tissues must be regarded as abnormal and not due to a delay of adolescence. Normalcy implies full and complete function of the testis and accessory sexual glands. Therefore the production of the full complement of androgens, spermatozoa, and normal amount of seminal vesicular and prostatic secretions, in turn, becomes a fairly reliable index of adequacy of the pituitary-gonadal axis.
THE GERMINAL EPITHELIUM The kinetics of spermatogenesis in rodents has been quantitatively studied, but the human testis does not completely match the regularity of periodic cell division as observed in the lower animal. Consequently, there are always areas in the normal human testes where sperm formation is in apparent abeyance and search and selection is required to demonstrate a "typical tubule" which shows all the stages of active spermatogenesis. Leblond and Clermont have divided stages of spermatogenesis in the rat into 4 phases with subdivisions giving 19 specific synchronized steps in cell mitosis. 3 0,31 Spermatocytes appear in the epithelium of a rat following 5 successive stages of spermatogonial division. After a second division 1 spermatogonium out of 4 stops dividing, while the others go through 3 stages of division, leading to spermatocyte formation. The nondividing spermatogonium resumes mitosis in the next cycle when it acts as a stem cell for the successive divisions. Roosen-Runge demonstrated 7 divisions in the human
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evolution of the spermatogonia before differentiating into spermatocytes, but observed "a puzzling lack of uniformity" in the regular periodic divisions of spermatogenesis in the human as compared to the rat.82 • 88 Nature's lavishness prompted one individual to estimate the total number of spermatozoa produced by a typical male between the ages of 25 and 55 and arrived at the astronomical figure of 339,385,500,000. 34 Two thirds of the seminal tubule is occupied by germinal epithelium, thereby leaving a clear central lumen usually devoid of debris and containing few, if any, free, spermatozoa. Radially disposed in the germinal epithelium are the supportive Sertoli cells to which a majority of mature sperm are attached. The phenomenon of this affinity has earned the designation of "trophic cells of Sertoli," for it would appear that all the spermatozoa seek some elements from the Sertoli cells before they leave the testis on their journey through the ductal system. Cytochemical stains have demonstrated lipids, glycogens, and alkaline phosphatase within the Sertoli cells. 35 • 86 The spermatids contain no glycogen, but after detachment as spermatozoa from the Sertoli cell they are rich in this substance, and it would appear that a perfusion of glycogen had occurred.37 It also has been noted that the amount of lipids increases in defective spermatogenesis and with age. In this respect some young infertile men resemble senile men and the theory of nonutilization by the developing spermatozoa lends support that the Sertoli cell is, in fact, trophic to the germinal epithelium.3s • 39. 40
PERITUBULAR MEMBRANE The peritubular or basal membrane surrounds each tubule. This collagenous tunica contains glycoproteins and alkaline phosphatase in the basal membranes. 37 Likewise fibroblasts are identified having a histochemical nature somewhat similar to that of fibroblasts found in granulation tissue of wounds. All the blood vessels in the testis are located within the interstitial area, and therefore the peritubular membrane must be permeable to nutritive elements for spermatogenesis, and likewise must permit an exodus of the waste products of cell metabolism. Peritubular fibrosis is a common finding in the testes of men with reduced spermatozoa and is seen more commonly in old age. The degree of atrophy of the germinal epithelium is directly proportionate to the thickness of the fibrosis, so it is conjectured that a thick tunica acts as a barrier to the interchange of nutrient elements, thereby blockading spermatogenesis.
THE INTERSTITIAL AREA The intertubular connective tissue is composed of Leydig cells, fibroblasts, and blood vessels. Two types of Leydig cells have been identified; one is immature and without steroids, while the other, mature type contains lipids, steroids, and ascorbic acid, but no alkaline phosphatase.37 The lipids do not appear in material amounts until puberty and are first found in the Leydig cells and subsequently within the Sertoli cells. With advancing age intensification of lipid drop-
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lets is seen in the Sertoli cells at the expense of Leydig cells. There is uniformity of opinion that the lipids and cholesterol are the precursors of the testicular steroids, but positive identification of the relative source of testicular estrogens and androgens has not been positively established. The Leydig cell is strongly implicated as a source of androgen and the Sertoli cell has been suspected as the elaborator of the estrogens. New evidence suggests that the Leydig cell may be the sole source of both steroids.41 COMMENT. Histochemical studies upon the testis of animals and man have been employed for over 40 years. This tool has been useful in identifying precise responses in the testis to extra gonadal hormones and has localized the source of testicular steroids. Animals which have seasonal estrus are particularly valuable for demonstrating the cytologic response to hormonal stimuli as contrasted in the active and passive periods. In recent years the staining technics and interpretations have been perfected and extended and histochemical methods promise much for the future.
VASCULAR SUPPLY Interference with the blood supply to the testes for as short a time as 2 to 3 hours may result in irreversible damage. 42 The clinical inference is obvious, for the cord is exposed to trauma in such common operations as hernia repair. 43 The testicular artery runs an extensive course over the surface of the testis before sending penetrating branches into the parenchyma. Sutures placed in the tunica albuginea of the rat has caused a decrease in the size of the testicle to one third normal size. 44 Suturing of the testis to the fascia lata is done in the performance of the Keetley-Torek operation for cryptorchidism. It is possible that this may account for lack of satisfactory reports regarding ultimate fertility in the Torek operations as compared to those methods of orchiopexy not involving sutures in the lateral walls of the testes. 45 INNERVATION Severe degeneration of the seminiferous tubules results from denervation of the lumbar spinal nerves which probably pass through the sympathetic trunk, which include the third, fourth, fifth, and sixth lumbar ganglia. The pain and thermoregulatory centers may reside at about the level of D-lO to D_12.46,47 The atrophy of the germinal epithelium may be due to paralysis of the blood vessels, thereby causing alterations of testicular temperature incompatible with spermatogenesis. Hypertrophy of the Leydig cells has been noted as a parallel development to spermatic failure and increased amounts of androgens and estrogens appear in the urine of paraplegic men. 48 The inference has been raised that one consequence of spermatic failure is the lack of a check on the pituitary gland, resulting in increased elaboration of LH, thereby stimulating the steroid-producing cells.
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ADDITIONAL FACTORS INFLUENCING TESTIS FUNCTION AS RElATED TO FERTILITY Heredity
Observations upon mice, flies, and certain animals indicate that one cause of infertility in the male may be genetic in origin. 49 Likewise, there are records of human families in which the male offspring were uniformly sterile. 50 It is possible that certain disturbances of mitoses seen in biopsies of some subfertile men are inherited chromosomal aberrancies.'51 The Endocrines
Pituitary Gland. A normal pituitary is a necessary requisite to complete testicular function. All 6 or more hormones of the pituitary may be involved directly or indirectly with growth, development, and maintenance of testicular function in adult life. Three of these are so intimately associated with the testis that they are collectively designated as the gonadotropic hormones. Their complete identity, individual action, and composition are still debatable but follicle-stimulating hormone (FSH) is considered by many to stimulate the germinal and the Sertoli cells. The leuteinizing hormone LH influences the Leydig cells and may act in conjunction with FSH on the tubular epithelium. 25 The role of the lactogenic gonadotropic hormone (LTH) has not been clearly defined but it may act in conjunction with LH. The other pituitary hormones-the thyrotropin (TTH), the adrenotropin (ACTH) and the growth hormone-may have an indirect action through stimulation of the respective target organs. In the absence of the pituitary hormones, testicular function regresses but pituitary transplants, extracts, or substitutions will sustain spermatogenesis and steroid production. Chorionic gonadotropin (FSH) has been utilized extensively in man, and three effects are generally recognized and agreed upon1: (1) It is capable of stimulating growth and endocrine function in the immature human testes. (2) It may produce descent of the cryptorchid testis. (3) It may produce growth of the Leydig cells and stimulate androgen secretion in some hypogonadal states. Extracts of animal pituitary glands induce antihormone response in other species, thereby limiting the full utilization of potential activity. The gonadotropins now available for clinical administration to humans have been disappointing in the treatment of most infertile men. A few proved cases of pituitary failure with prepuberal types of testicular dysfunction have responded to treatment. 52 Some hitherto-unexplained responses following the administration of gonadotropins might be due to alterations in the estrogen-androgen levels as explained further on. At this time progress awaits the development of more specific and highly sensitive methods of quantitative tests for determining titers of circulating FSH and LH and the availability of purer products for administration. SterOid-Producing Glands of Internal Secretion. The androgenic steroids are
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derived from the adrenals and the testes. Approximately thirty steroids have been isolated from the adrenals, of which 3 have androgenic properties. The adrenals contribute more than one half the total steroids, but doubts are raised as to whether their action is similar to the testicular androgens. The latter are probably produced only by the Leydig cells. Estrogens likewise may have origin in the adrenal tissue, but Maddock and Nelson offer evidence that only the Leydig cell is concerned with estrogen production. 41 Administration of FSH resulted in an increase of 16 times the estrogen production, with concomitant slight elevation of androgens. There was no estrogen response in castrates, which indicated to these investigators that the testis is the sole source of estrogens. Furthermore, cytochemical stains indicated that the Leydig cell and not the Sertoli cell was the site of this steroid. Spermatogenesis was temporarily decreased following FSH administration, presumably by inhibition of the pituitary by the estrogens and androgens. The reciprocal activity of the pituitary and the steroids has recently gained much attention. The circulating steroids regulate the production of gonadotropins, for castration results in an outpouring of the pituitary hormones. 53 In tum, estrogens and androgens are capable of checking their production. Hypophysectomized rats are protected against the loss of spermatogenesis by androgen administration, so that a dual action of inhibition and stimulation is involved. 54 The degree of the effect is somewhat dependent on dosage. In sufficient dosage both androgens and estrogens inhibit spermatogenesis. Estrogens never stimulate maturation of the germinal epithelium, and in balanced amounts androgens can counteract the harmful effects of estrogens. The liver destroys excessive amounts of circulating estrogens except when this organ is incapacitated by disease. 55 Accordingly adequate liver function appears important to the health of the testis. The implications of these discussions are pertinent to the clinical utilization of testosterone for stimulation of spermatogenesis. There are those who believe that once the depression has been produced recovery exceeds the rate of impaired spermatogenesis existing prior to treatment. The usefulness in oligospermia of the so-called rebound principle of clinical therapy has both its advocates and its skeptics.56 • 57. 58 Testosterone therapy has with very rare exceptions failed to convert azoospermic men to individuals capable of having spermatozoa in any appreciable numbers in their semen. 59 In rare cases of eunuchoidism, testosterone not only developed the secondary sexual characteristics and libido but actually resulted in viable spermatozoa with subsequent pregnancy in one patient's wife following years of androgenic therapy.29 It has been stated that approximately 1 of 5 oligospermic men, selected on the basis of favorable testicular biopsies, will show some improvement in the semen following large intramuscular dosage of testosterone (3000 mg.) .59 Failure of sperm to reappear in the semen after testosterone therapy has been reported. 60 The role of adrenal androgens in testicular physiology remains obscure. Precocious spermatogenesis and virilism in boys have been observed in adrenal
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hyperplasia. Reports of both increases and reductions of spermatogenesis have been made following the use of cortisone for the treatment of oligospermia in men. 60 Increased production of gonadotropins following administration of cortisone is known, and some presume that cortisone disturbs the antigonadotropin formation. 6l Testicular biopsies on men before and after total adrenalectomy for prostatic cancer did not reveal significant changes in the character of spermatogenesis. 62 Unpublished data on total adrenalectomy in dogs suggest that the testes are not dependent upon the adrenals for continued spermatogenesis.63 Thyroid Gland. Thyroid medication has probably been employed more frequently than any other drug for the treatment of male infertility. The evidence of a truly specific relationship between the thyroid and the gonads is, however, very conflicting. Some experienced clinicians are so convinced of its efficacy that the statement has been made, "Successes following thyroid treatment are now numerous enough to remove all doubt about cause and effect relationship of that trouble to infertility."64 Others have shown that poor semen quality is not a characteristic of low metabolic states. 65 Thyroid administration has restored the ovarian cycle in myxedematous women, and the 17-ketosteroid output is said to be increased in the treatment of men with hypothyroidism. 66 Animal experimentation, likewise, is inconclusive and not in agreement. Removal of gonads in dogs, rabbits, and rats causes slow involution of thyroid. 67 Thyroidectomy at birth prevents testis development, but shortly after birth thyroidectomy retards but does not prevent spermatogenesis. 6s Thyroxine may cause atrophy of the seminal vesicles and probably suppresses gonadotropin production. Some authors believe that thyroid causes testicular hypertrophy, and others report atrophy. Numerous theories have been advanced to explain the relationship between the thyroid and the gonad, among which are that "the thyroid may sensitize peripheral tissues," "the gonadal hormones have a direct effect on gonadotropin production," "[the thyroid has] indirect effect by way of adrenal alterations," and "[the thyroid regulates] oxygen metabolism of the cell." The net result of a perusal of the literature is that despite therapeutic popularity much remains to be known about thyroid-gonadal interrelationships, and the clinical use of thyroid substances must be regarded as largely empirical at this time.
Nutrition Inadequate intake, assimilation, and utilization of food may affect the testes of the immature male more severely than comparable deficiencies in the adult. 69 Rats maintained on an inanition diet from age 3 to 4 weeks may not produce cells above spermatocyte stage during the entire first year of life.70 Other deprivation experiments in the growing animal likewise support increased concern about the remote effects of improper nutrition in childhood.71 Recent surveys demonstrate that dietary deficiencies are common regardless of social and financial status. Impaired spermatogenesis may result not only from avitaminosis but also from deficiency in caloric intake, lack of such minerals as magnesium, zinc, sodium,
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potassium, and phosphorus, and from insufficient proteins. 72 • 73 Impaired sexual performance has been reported after fat-free diet. Tubular atrophy, sloughing, sperm agglutination, and even complete loss of germinal epithelium, except for the Sertoli cells, is known to follow exclusion of vitamins A and/or E. The effects of the latter may be irreversible. The testis of the mature individual is fairly resistant to deficiencies. Weight losses of considerable proportions can take place in the experimental animal before loss of spermatogenesis is noted. The severe nutritional deficiencies endured by prisoners of war and men confined to concentration camps often caused complete loss of libido at the time of starvation. It is likely that spermatogenesis was also greatly reduced, yet postwar examinations at the time of weight recovery have repeatedly demonstrated that many of these men had normal seminal specimens. Vitamin therapy for oligospermia and poor motility seems justified when indicated by poor dietary habits and when there is evidence that liver function is impaired. Specific therapeutic effects are difficult to identify for reasons similar to those discussed under thyroid therapy. Temperature
An extensive literature exists upon the adverse effects of heat upon the adult testis. Local or systemic elevations in temperature may cause disappearance of germinal cells in the reverse order of spermatogenesis, indicating that the primary cells are most resistant to the effects of elevation of temperature. A 10minute exposure of the scrotum of a guinea pig to water warmed (6° C.) above body temperature results in great damage to the tubules. 74 Recovery requires some weeks but repeated treatment results in permanent sterility. The thermoregulatory function of the testes is well known and recently interest has again developed in varicoceles as they might interfere with heat loss. Improvement in semen has been reported following varicocelectomy, but additional observations are required before conclusions are to be reached. 75 Radiation
The biologic action of ionizing radiation has been the subject of many studies directly upon sperm, after localized irradiation of the testes, and more recently on whole body exposure. 76 , 77 The results have been analyzed in terms of cellular destruction, inability of sperm to fertilize the ova, the effects on fetal mortality, and the emergence of mutants in the progeny.7S-S1 The production of detrimental mutants is predicted in 3 of every 5 children of men who had received a total of 225 r to their gonads in prepuberal years. Serious consideration should be given to the needs for diagnostic x-ray and, especially, for therapeutic radiation, in boys.82 Irradiation of the adult testis may require years for reconstruction of germinal epithelium. 77 The damage is apparently proportionate to the dosage received,
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and this probably causes azoospermia in men with poor spermatogenesis much more quickly than when spermatogenesis is normal. COMMENT. Any consideration of factors which influence testis function have therapeutic connotations. Such implications are worthy and justified, for perhaps the principal objective of such thinking is to provide effective treatment for infertility in man and animals. This discipline is still very young and has had serious attention by only a very few until 30 or 40 years ago. The treatment methods leave much to be desired and, by force of circumstance, are sometimes based upon empiric formulas and may, at times, be unscientific and of little value. Clarification of the basic principles of the physiology of the male reproductive system affords a foundation for sound treatment. Encouragement and support for fundamental investigation appears to be the logical approach for reasonably efficient forms of treatment.
SUMMARY The dual function of the mature testis is dependent upon an extraordinary number of diversified influences which govern its growth and development from fetal to postadolescent life. Spermatogenesis is far more delicate and labile than hormone production, for both the Sertoli and the Leydig cells can withstand many adverse provocations that prove to be detrimental to the germinal epithelium. The primary spermatogenic cells are more robust than the final products of cell division, for injury is almost always first recorded in the late stages of spermatogenesis. Recent advances in knowledge have clarified many mechanisms related to testicular physiology, yet many of the therapeutic manipulations based on these inferences have, so far, failed to stimulate faulty spermatogenesis. ROBERT S. HOTCHKISS, M.D. New York City
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9. BEACH, K W. Undescended testes: Cause and treatment. J. Urol. 60: 623,1948. 10. LEWIS, L. G. Cryptorchidism. N. Y. State]. Med. 54:3078, 1954. 11. PACE, J. M., and CABOT, H. Histologic study of 24 cases of retained testes in the adult. Surg. Gynec. & Obst. 63:16, 1936. 12. ENGLE, E. T. The life history of the human testis. J. Urol. 74:379, 1955. 13. SNIFFEN, R. C. Histology of the normal and abnonnal testis at puberty. Ann. N. Y. Acad. Sc. 55:609, 1952. 14. HAMILTON, J. B. Androgenic activity per milligram of colorimetrically measured ketosteroids in urine: An index of the respective contributions from testicular and extra-testicular sources. J. GUn. Endocrinol. & Metab. 14:452, 1954. 15. CHARNY, C. W., CONSTON, A. S., and MERANZE, D. R. Testicular developmental histology. Ann. N. Y. Acad. Sc. 55:597, 1952. 16. STURKIE, P. D., PINO, J. A., WEATHERWAX, J. L., DONNELLY, A. J., and DORRANCE, G. M. Effect of x-rays on fertility of male chickens treated before puberty. Radiology 52:112, 1949. 17. ESSENBERG, J. M., and ZIKMUND, A. An experimental study on the effects of roentgen rays on the gonads of developing chicks. Radiology 31 :94, 1938. 18. WERNER, C. A. Mumps orchitis and testicular atrophy. Ann. Int. Med. 32: 1066, 1950. 19. CHARNY, C. W., and MERANZE, D. R. Pathology of mumps orchitis. Proc. Gonf. Ster. & Inf. III:167, 1947. 20. SMITH, D. R. The treatment of cryptorchidism. Galifornia Med. 81: 379, 1954. 21. ROBINSON, J. N., and ENGLE, E. T. Some observations on the cryptorchid testis. J. Urol. 71 :726, 1954. 22. PRENTISS, R. J., MULLENIX, R. B., WmSENAND, J. M., and FEENEY, M. J. Medical and surgical treatment of
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