FSH is a key regulator of testis function, required for the establishment of full complements of Sertoli and germ cells during postnatal testis development and for the maintenance of spermatogenesis in the adult. FSH plays an important role in germ cell survival rather than proliferation, in the window between 14 and 18 days of testicular development, which coincides with the cessation of Sertoli cell proliferation and the onset of germ cell meiosis during the first wave of spermatogenesis. This study aimed to identify the pathway(s) of apoptosis regulated by changes in FSH levels in 14 - to 18-day-old rats, using a model of in vivo FSH suppression by passive immunoneutralization with a rat anti-FSH antibody. Apoptotic pathways were identified by immunohistochemistry using pathway-specific proteins as markers of the intrinsic (activated caspase 9) and extrinsic (activated caspase 8) pathways, followed by quantification of cell numbers using stereological techniques. In addition, RT-PCR was used to assess the expression of pathway-specific genes. We previously reported a 2.5-fold increase in spermatogonial apoptosis in these samples after 4 days of FSH suppression, and now show that this increase correlates with a 9.8-fold (P<0.001) increase in the frequency of caspase 9-positive spermatogonia in the absence of caspase 8 immunoreactivity. By contrast, spermatocytes exhibited both increased caspase 9 (7.5-fold; P<0.001) and caspase 8 (5.7 fold; P<0.001) immunoreactivities after 4 days of FSH suppression. No significant change in the transcription levels of candidate genes required for either pathway was detected. This study demonstrates that, in the seminiferous tubules, FSH suppression induces spermatogonial apoptosis predominantly via the intrinsic pathway, while spermatocyte apoptosis occurs via both the intrinsic and extrinsic pathways.

The roles of testosterone (T) and its metabolites on hamster spermatogenesis are poorly defined. This study assessed the effects of T, dihydrotestosterone (DHT) and oestradiol (E) on the re-initiation of spermatogenesis in the adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16 h light:8 h darkness) were exposed to short photoperiods (SD, 8 h light:16 h darkness) for 11 weeks to suppress gonadotrophins. Groups of eight animals then received T, DHT and E for 5 weeks. Cell numbers were determined using the optical disector (sic). The number of Sertoli cells was suppressed in SD controls to 48% (P < 0.001) of LD control and restored either fully or partially by exogenous DHTand E (2.6- and 1.8-fold above SD levels) respectively, corresponding with a twofold elevation of serum FSH. The number of germ cells in SD animals was reduced (all P < 0.001) to levels reported. The number of type A spermatogonia increased in line with the rise in Sertoli cell number, by 2.6-fold (P < 0.01) and 1.8-fold (NS) above SD controls after DHT and E treatments respectively. DHT increased the number of type B spermatogonia/preleptotene spermatocytes, leptotene/zygotene and pachytene spermatocytes by 3.5-, 5.7- and 21-fold above SD (all P < 0.01) respectively, compared with a 2.2-fold (P < 0.01), 2.4-fold (not significant, NS) and 6-fold (NS) in E-treated animals respectively. Exogenous T had little effect on cell numbers or serum FSH compared with SD controls. Spermatids were rarely observed after steroid treatment. We believe this study suggests that steroids can regulate the re-initiation of early spermatogenic cells via a mechanism which includes FSH.

Growth differentiation factor 9 (GDF9) produced within the ovary plays an essential role during follicle maturation through actions on granulosa cells, but extra-ovarian expression, signalling and actions of GDF9 are less well characterised. The present studies confirm GDF9 expression in the mouse testis, pituitary gland and adrenocortical cancer (AC) cells, and establish its expression in LβT2 gonadotrophs, and in mouse adrenal glands, particularly foetal and neonatal cortical cells. AC, LβT2, TM3 Leydig and TM4 Sertoli cells express the requisite GDF9 binding signalling components, particularly activin receptor-like kinase (ALK) 5 and the bone morphogenetic protein (BMP)/GDF type II receptor, BMPRII (BMPR2). We therefore compared GDF9 activation of these potential extra-ovarian target cell types with its activation of granulosa cells. Recombinant mouse GDF9 stimulated expression of activin/transforming growth factor-β-responsive reporters, pGRAS-luc or pAR3-lux, in TM4 and AC cells (IC50=145 ng/ml in the latter case), and two granulosa cell lines, KGN and COV434. The ALK4/5/7 inhibitor, SB431542, blocked GDF9 activity in each case. By contrast, GDF9 lacked specific effects on TM3 cells and rat primary pituitary and mouse LβT2 gonadotrophs. Our findings show that GDF9 regulates the expression of R-SMAD2/3-responsive reporter genes through ALK4, 5 or 7 in extra-ovarian (adrenocortical and Sertoli) cells with similar potency and signalling pathway to its actions on granulosa cells, but suggest that expression of BMPRII, ALK5 (TGFBR1) and R-SMADs 2 and 3 may not be sufficient for a cell to respond to GDF9.

Oestrogen is a metabolite of testosterone, but its role in spermatogenesis is ill-defined. Oestrogen may exert its effects on spermatogenesis, as oestrogen receptor (ER)-β has been localised to both germ and somatic cells. This study sought to establish whether the restoration of early germ cell numbers in spermatogenesis by high-dose exogenous testosterone was influenced by its metabolite, oestrogen. The ER antagonist (ICI 182780) was administered, at a dose known to impair oestrogen action in the male reproductive tract, during testosterone treatment of gonadotrophin-releasing hormone (GnRH)-immunised rats, and germ cell numbers were determined. GnRH-immunised adult Sprague–Dawley rats (n=7–8 per group) received two doses of testosterone, either as a Silastic implant (24 cm (T24 cm)) or an injectable ester for 10 days alone or in combination with ICI 182780 (2 mg/kg, s.c. injection daily). Control rats received vehicle alone. Testes were perfusion-fixed and germ cells were quantified by the optical disector technique.

GnRH-immunisation reduced (P<0.001) both type A/ intermediate spermatogonial and type B spermatogonial/ preleptotene spermatocyte number (56% of control) and leptotene/zygotene spermatocyte number (63% of control). Pachytene spermatocyte and round spermatids were reduced to 12% and l% (P<0.01) of control respectively. Testosterone treatment did not increase type A/intermediate spermatogonial number compared with GnRH-immunised controls over the 10-day study period. Treatment with testosterone-esters increased type B spermatogonial/preleptotene spermatocytes and leptotene/zygotene spermatocyte numbers (both being ~83% of control, P<0.05), while T24 cm treatment did not significantly increase their numbers (~73% of control) compared with GnRH-immunised controls. Both treatments increased pachytene spermatocyte and round spermatid numbers to 55% and 8% of control respectively. Co-administration of ICI 182780 had no effect on any of these germ cell numbers. We conclude that oestrogen action plays no role in the short-term restoration of spermatogenesis by testosterone in the GnRH-immunised rat.