David R. Simorangkir

Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques

BACKGROUND The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined. METHODS To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. Adark and Apale spermatogonia). RESULTS FACS analysis identified a THY-1+ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFRα1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFRα1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1+, PLZF+, GFRα1+, NGN3+/−, cKIT−) comprises most Adark and Apale and is considerably larger in primates than in rodents. It is noteworthy that the majority of Adark and Apale share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from Adark, but is expressed by some Apale and may mark the transition from undifferentiated (cKIT−) to differentiating (cKIT+) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF+, GFRα1+, NGN3+, cKIT+/−) is smaller in primates than in rodents. CONCLUSIONS These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.

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.

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.

Sertoli cell differentiation in rhesus monkey (Macaca mulatta) is an early event in puberty and precedes attainment of the adult complement of undifferentiated spermatogonia.

In primates, the time course of Sertoli cell proliferation and differentiation during puberty and its relationship with the expansion of undifferentiated type A spermatogonia that occurs at this critical stage of development are poorly defined. Mid and late juvenile and early and late pubertal male rhesus monkeys were studied. Testes were immersion fixed, embedded in paraffin, and sectioned at 5 μm. Sertoli cell number per testis, S-phase labeling (BrdU), and growth fraction (Ki67 labeling) were determined and correlated with corresponding parameters for undifferentiated type A spermatogonia (A dark and A pale). Dual fluorescence labeling was used in addition to histochemistry to monitor spermatogonial differentiation during the peripubertal period using GFRα-1 and cKIT as markers. While the adult complement of Sertoli cells/testis was attained in early pubertal monkeys after only a few weeks of exposure to the elevated gonadotropin secretion characteristic of this developmental stage, the number of undifferentiated type A spermatogonia several months later in mid pubertal monkeys was only 50% of that in adult testes. Both A dark and A pale spermatogonia exhibited high S-phase BrdU labeling at all stages of juvenile and pubertal development. Spermatogonial differentiation, as reflected histochemically and by relative changes in GFRα-1 and cKIT expression, was not observed until after the initiation of puberty. In the rhesus monkey and maybe in other higher primates including human, the pubertal proliferation of undifferentiated spermatogonia is insidious and proceeds in the wake of a surge in Sertoli cell proliferation following termination of the juvenile stage of development.