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

Abstract

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 mm. 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 GFRa-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 GFRa-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.

Time course and role of luteinizing hormone and follicle‐stimulating hormone in the expansion of the Leydig cell population at the time of puberty in the rhesus monkey (Macaca mulatta)

“…As reported previously (Simorangkir et al, 2012), late-juveniles had a combined testis volume of <5 ml with no evidence of increased nighttime circulating testosterone levels. Early-pubertal animals had a combined testis volume of 5 ml and nighttime testosterone concentration in excess of 1 ng/ml for several weeks.…”

“…Five μm sections (mounted on Superfrost Plus glass slides, Fisher Scientific, Pittsburgh, PA, USA) of Bouins fixed, paraffin embedded testes that had been generated in three previously published studies (Ramaswamy et al 2000; Simorangkir et al 2003 and 2012) were used.…”

“…Five μm sections of Bouins fixed paraffin embedded testes from adults (N=3; 8.1±1.95 years of age; 10.9±2.22 kg) that had been previously deposited in our tissue bank were also used. All animals in this experiment had been injected with an iv bolus of BrdU (33 mg/kg BW) 2 to 3 h before castration (Simorangkir et al 2003 and 2012). …”

“…For both experiments, the animals were maintained under a controlled photoperiod (lights on, 07.00–19.00 hours), and the experimental protocols were conducted in accordance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals and with approval from the Institutional Animal Care and Use Committee (Ramaswamy, 2000; Simorangkir et al 2003 and 2012). …”

“…For the analysis of S phase (BrdU) labeling of interstitial cells, sections from the spontaneous puberty study (Simorangkir et al, 2012) that were stained for BrdU were examined. Briefly, the primary antibody (mouse monoclonal anti-BrdU antibody, diluted 1:33; Roche Diagnostic Corp, Indianapolis, IN, USA) and the secondary antibody (biotinylated horse anti-mouse antibody, diluted 1:200; Vector Laboratories Inc, Burlingame, CA, USA) were used followed by treatment with Vector ABC kit and DAB kit to detect BrdU labeled cells in testis sections.…”