Gary R. Marshall

Experimental induction of puberty in the infantile female rhesus monkey

Normal ovulatory menstrual cycles were initiated in prepubertal female rhesus monkeys by the infusion of gonadotropin-releasing hormone for 6 minutes once every hou;. When this regimen was discontinued, the animals promptly reverted to an immature state. These findings permit the conclusion that neither adenohypophysial nor ovarian competence is limiting in the initiation of puberty and suggest that this process depends on the maturation of the neuroendocrine control system that directs the pulsatile secretion of gonadotropin-releasing hormone from the hypothalamus.

Control of the rhesus monkey menstrual cycle: permissive role of hypothalamic gonadotropin-releasing hormone

In rhesus monkeys with hypothalamic lesions (which appear to abolish the endogenous production of gonadotropin-releasing hormone), normal ovulatory mestrual cycles were reestablished by an unvarying, long-term replacement regimen consisting of one intravenous pulse of synthetic gonadotropic-releasing hormone per hour. This finding is in accord with the hypothesis that the pattern of pituitary gonadotropin secretion throughout the menstrual cycle (basal secretion interrupted, once every 28 days on the average, by a preovulatory surge) is not directed by alterations in hypothalamic gonadotropin-releasing hormone secretion but by the ebb and flow of ovarian estrogens acting directly on the pituitary gland.

Dynamics of the Follicle-Stimulating Hormone (FSH)-Inhibin B Feedback Loop and Its Role in Regulating Spermatogenesis in the Adult Male Rhesus Monkey (Macaca mulatta) as Revealed by Unilateral Orchidectomy1

The purpose of this study was to document the morphological changes in the seminiferous epithelium that underlie the compensatory testicular hypertrophy observed in response to unilateral orchidectomy (UO) in the adult rhesus monkey and to describe the concomitant response in the endocrine feedback loops controlling testicular function in this species. Adult male monkeys were implanted with indwelling venous catheters; seven animals were then subjected to UO (data are presented from six) and three to sham UO. Profiles of circulating concentrations of FSH, LH, testosterone (T), inhibin B, and pro-alpha-C were monitored in 12-h series of sequential blood samples collected before, on the day of UO (day 0), and on days 1, 2, 4, 8, 16, 32, and 42 or 43 after UO. In the UO monkeys, the remaining testis was taken on day 44. Sertoli and germ cells in the removed and remaining testes were counted and expressed either as number per testis or, in the case of the differentiated spermatogonia (B1, B2, B3, and B4), as number per cross-section of the seminiferous tubule. UO was associated with a marked increase in the number of all germ cells more mature than undifferentiated spermatogonia (Ap) in the remaining testis. Sertoli cell number, on the other hand, did not change, and it is therefore reasonable to propose that the primary locus of the spermatogenic compensation was the differentiated spermatogonia. The additional finding that the relationship between the number of Sertoli cells and total germ cells in the remaining testis became robust (r = 0.92; P 0.05 for the removed testis) indicated that in the monkey, spermatogenesis does not normally operate at its ceiling. The increased drive to the seminiferous tubule of the remaining testis is hypothesized to be mediated by the sustained increase in FSH secretion that was observed after UO, although a role for increased testicular T production cannot be excluded. The stimulus for increased FSH secretion was presumably provided by the abrupt, 50% decline in circulating inhibin B levels. Interestingly, inhibin B secretion by the remaining testis was not dramatically affected by UO, and therefore, the deficit in circulating levels of this hormone and thus the error signal to FSH secretion were maintained for the duration of the experiment. In contrast, the changes in circulating LH and T concentrations were only transient, and within 48 h of UO, these hormonal parameters had returned to control values. The mechanisms by which the remaining testis rapidly acquires the capacity to double T production in the face of an unchanging LH drive remains to be determined. The foregoing body of evidence suggests that sperm output by the monkey testis is regulated by the circulating concentration of FSH and that in physiological situations, FSH secretion is insufficient to stimulate spermatogenesis to its ceiling. The results also indicate that FSH secretion is controlled by a feedback system in which the feedforward arm (FSH-inhibin B) is less robust than the feedback loop (inhibin B-FSH). Thus, a decrease in the inhibin B feedback signal results in a sustained increase in FSH secretion that drives the testes toward their spermatogenic ceiling, which is presumably set by Sertoli cell number.

Orchiopexy: An Experimental Study of the Effect of Surgical Technique on Testicular Histology

Orchiopexy was performed in 35 mature Sprague-Dawley rats using absorbable suture, nonabsorbable suture or a classical dartos pouch technique without suture fixation. Examination of the chromic-fixed testes revealed severe acute inflammation of the suture site and most of the parenchyma in 76 per cent, with abscess formation in 65 per cent, complete absence of spermatogenesis in 82 per cent and evidence of tubular necrosis in 88 per cent. Nylon-fixed testes were adherent only at the sutures, with absent spermatogenesis in 29 per cent, moderate to severe tubular necrosis in 29 per cent and tubular atrophy in 58 per cent. Dartos-fixed testes demonstrated complete circumferential adherence, normal spermatogenesis in 94 per cent and minimal focal tubular atrophy in 23 per cent. Contralateral testes appeared normal in all animals.

Pulsatile Stimulation with Recombinant Single Chain Human Luteinizing Hormone Elicits Precocious Sertoli Cell Proliferation in the Juvenile Male Rhesus Monkey (Macaca mulatta)

Abstract In this study, we determined the relative role of LH and FSH in initiating the pubertal proliferation of Sertoli cells in primates. Sixteen juvenile male rhesus monkeys (Macaca mulatta) bearing venous catheters received intermittent intravenous infusions of single chain human LH (schLH) or recombinant human FSH (rhFSH) or a combination of both for 11 days. The schLH infusion elicited a physiological testosterone response. On Day 11, monkeys were castrated, and one-half of a testis was fixed in Bouins fluid. Infusion of the gonadotropins, either alone or in combination, effected a significant increase in testicular weight, seminiferous cord diameter, and the number of Sertoli cells per testis (schLH, 295 ± 46 × 106; rhFSH, 342 ± 64 × 106; LH+FSH, 298 ± 26 × 106 versus vehicle, 204 ± 26 × 106). The latter finding indicated that LH, in addition to FSH, plays a critical role in the initiation of the pubertal proliferation of Sertoli cells in primates. Moreover, combined gonadotropin treatment led to the appearance of germ cells as mature as early primary spermatocytes, indicating that initiation of spermatogenesis had been set in motion. Because the duration of hormone stimulation was only 11 days, the latter result suggests that Leydig and Sertoli cells of the juvenile monkey testis can immediately transduce a gonadotropin signal to the germ cell.

Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells

OBJECTIVE To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs). DESIGN Laboratory study using human tissues. SETTING Research institute. PATIENT(S) Healthy adult human testicular tissue. INTERVENTION(S) Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). MAIN OUTCOME MEASURE(S) Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay. RESULT(S) Immunohistochemistry costaining revealed the relative expression patterns of SALL4, UTF1, ZBTB16, UCHL1, and ENO2 in human undifferentiated spermatogonia as well as the extent of overlap with the differentiation marker KIT. Whole mount analyses revealed that human undifferentiated spermatogonia (UCHL1+) were typically arranged in clones of one to four cells whereas differentiated spermatogonia (KIT+) were typically arranged in clones of eight or more cells. The ratio of undifferentiated-to-differentiated spermatogonia is greater in humans than in rodents. The SSC colonizing activity was enriched in the THY1dim and ITGA6+ fractions of human testes sorted by FACS. ITGA6 was effective for sorting human SSCs by MACS; THY1 and EPCAM were not. CONCLUSION(S) Human spermatogonial differentiation correlates with increased clone size and onset of KIT expression, similar to rodents. The undifferentiated-to-differentiated developmental dynamics in human spermatogonia is different than rodents. THY1, ITGA6, and EPCAM can be used to enrich human SSC colonizing activity by FACS, but only ITGA6 is amenable to high throughput sorting by MACS.

Postnatal and Pubertal Development of the Rhesus Monkey (Macaca mulatta) Testis

Abstract: This review examines the neurobiology, endocrinology, and cell biology underlying the development of the testis from birth until puberty in the rhesus monkey, a representative higher primate.

Gonadotropin-Independent Proliferation of the Pale Type A Spermatogonia in the Adult Rhesus Monkey (Macaca mulatta)

Abstract The goal of the present study was to examine the relative roles of testosterone (T) and FSH in the proliferation and differentiation of pale type A (Ap) spermatogonia in the rhesus monkey (Macaca mulatta). Twenty adult male monkeys were treated with daily injections of a GnRH-receptor antagonist, acyline, to suppress endogenous gonadotropin secretion during an experiment comprising three phases. Phase 1 established a chronic hypogonadotropic state marked by a profound decrease in testicular size. During phase 2, half the monkeys were implanted with T-filled capsules, and the other half received control implants. Treatment with T produced circulating T levels of approximately 15 ng/ml and normal testicular T content. At the end of phase 2, monkeys were fitted with indwelling i.v. catheters and housed in remote sampling cages for the final phase. During phase 3, five monkeys from the T- and non-T-treated groups were stimulated with recombinant human FSH. The remaining five monkeys from each group received an infusion of vehicle. On the last day of FSH or vehicle infusion, monkeys were bilaterally castrated after receiving an i.v. bolus of bromodeoxyuridine (BrdU). The BrdU labeling of Ap spermatogonia was robust in the hypogonadotropic group and was uninfluenced by treatment with T and FSH, either alone or in combination. In contrast, both T and FSH stimulated spermatogonial differentiation, and this effect was amplified by combined treatment. We conclude that marked Ap spermatogonial proliferation occurs constitutively and in a gonadotropin-independent manner and that differentiation of Ap into B spermatogonia is absolutely gonadotropin dependent and may be driven by either T or FSH.

A selective monotropic elevation of FSH, but not that of LH, amplifies the proliferation and differentiation of spermatogonia in the adult rhesus monkey (Macaca mulatta)

BACKGROUND Unilateral orchidectomy in monkeys increases spermatogenesis in the remaining testis in association with elevated follicle-stimulating hormone (FSH) secretion and testicular testosterone. The present study examined the relative importance of FSH and testosterone in driving the primate testis toward its spermatogenic ceiling. METHODS Adult male rhesus monkeys were treated with a gonadotropin-releasing hormone receptor antagonist to inhibit endogenous FSH and luteinizing hormone (LH) secretion. The gonadotrophin drive to the testis was replaced with a pulsatile recombinant human FSH and LH infusion to maintain testicular volume and circulating testosterone and inhibin B at physiological levels. A selective monotropic elevation of FSH or LH that doubled the concentrations of inhibin B or testosterone, respectively, was then imposed for 4 weeks, each in a group of four monkeys. In a third group (n = 4), the gonadotrophin drive remained clamped at physiological levels. Bromo-deoxyuridine was administered 3 h prior to castration, and the effects of the monotropic hormone increments on germ cell number, S-phase labeling and degeneration were determined. RESULTS Increased FSH, but not LH, produced increases in testicular volume (P < 0.05), the proportion of A pale spermatogonia entering the cell cycle and the numbers of differentiated spermatogonia and more advanced germ cells (P < 0.05). Indexes for spermatogonia labeling and germ cell degeneration were not affected. CONCLUSIONS Under physiological conditions, circulating concentrations of FSH directly dictate sperm output of the primate testis by regulating the proportion of Ap spermatogonia in the growth fraction. An effect of FSH on survival of the first generation of differentiated B spermatogonia is not excluded.

Estrogen Promotes Germ Cell and Seminiferous Tubule Development in the Baboon Fetal Testis

Abstract The foundation for development of the male reproduction system occurs in utero, but relatively little is known about the regulation of primate fetal testis maturation. Our laboratories have shown that estrogen regulates key aspects of the physiology of pregnancy and fetal development. Therefore, in the present study, we characterized and quantified germ cells and Sertoli cells in the fetal baboon testis in late normal gestation (i.e., Day 165; term is 184 days) and in baboons administered the aromatase inhibitor letrozole throughout the second half of gestation to assess the impact of endogenous estrogen on fetal testis development. In untreated baboons, the seminiferous cords were comprised of undifferentiated (i.e., type A) spermatogonia classified by their morphology as dark (Ad) or pale (Ap), gonocytes (precursors of type A spermatogonia), unidentified cells (UI), and Sertoli cells. In letrozole-treated baboons, serum estradiol levels were decreased by 95%. The number per milligram of fetal testis (×104) of Ad spermatogonia (0.42 ± 0.11) was 45% lower (P = 0.03), and that of gonocytes (0.58 ± 0.06) and UI (0.45 ± 0.12) was twofold greater (P < 0.01 and P = 0.06, respectively), than in untreated baboons. Moreover, in the seminiferous cords of estrogen-deprived baboons, the basement membrane appeared fragmented, the germ cells and Sertoli cells appeared disorganized, and vacuoles were present. We conclude that endogenous estrogen promotes fetal testis development and that the changes in the germ cell population in the estrogen-deprived baboon fetus may impair spermatogenesis and fertility in adulthood.