Gonadotrophin-releasing hormone antagonist arrests premeiotic germ cell proliferation but does not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry

Abstract

Meiosis constitutes a crucial phase of spermatogenesis since the recombination of genetic information and production of haploid round spermatids need to be achieved. Although it is well established that gonadotrophic hormones are required for completion of the spermatogenic process, little is known about the dynamic and kinetic aspects of development of spermatocytes into spermatids and its endocrine control in the primate. In this study, S-phase germ cells were labelled using 5-bromodeoxyuridine (BrdU) incorporation and were then followed throughout meiosis under normal conditions and following GnRH antagonist (ANT)-induced gonadotrophin withdrawal in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis). Adult animals received either vehicle (VEH, n=4) or the ANT cetrorelix (n=5) throughout 25 days. On day 7 all animals received a bolus injection of BrdU. A biopsy was performed after 3 h, one testis was removed 9 days later (day 16 of treatment) and the other testis after 18 days (day 25 of treatment). Serum testosterone and inhibin levels, and testis weight were reduced (P<0·05) by ANT treatment. BrdU localized to pachytene spermatocytes 9 days after BrdU and to round spermatids 18 days after BrdU in both groups, demonstrating that BrdU-labelled pachytene spermatocytes had undergone meiosis. Flow cytometric analysis revealed that the relative number and number per testis of BrdU-tagged 2C and 4C cells were reduced significantly (P<0·05) within 16 days of ANT treatment. Numbers of 1C cells were lowered by day 25. The cell ratio for 1C:4C was similar with VEH and ANT (P>0·05). These findings indicate that ANT reduced the number of cells available for meiosis but did not alter the rate of transition into round spermatids. Unexpectedly, however, the stagedependent progression of BrdU-tagged round spermatids was significantly (P<0·05) retarded under ANT as seen from the frequency of tubules containing BrdU-labelled round spermatids. The average duration of spermatogenic cycle was slightly prolonged (9·8 days in the VEH group and 10·8 days in the ANT group (P=0·09)). Since no atypical germ cell associations could be found, it remains unclear whether this slight prolongation is entirely due to altered spermatid progression or whether earlier phases are affected. We conclude for the nonhuman primate that (1) BrdU-labelling of premeiotic germ cells is suitable for tracing their meiotic transition into postmeiotic cells, (2) unlike in the rat, gonadotrophin suppression initially affects premeiotic cell proliferation and thus the number of cells available for meiosis, (3) the meiotic process continues quantitatively despite gonadotrophin deficiency and (4) prolonged gonadotrophin deficiency might alter the timing of germ cell development.

Norethisterone enanthate has neither a direct effect on the testis nor on the epididymis: a study in adult male cynomolgus monkeys (Macaca fascicularis)

“…A similar approach to the study of feedback mechanisms has been reported recently using a 'testicular clamp' in which endogenous gonadotropin secretion was abolished with a GnRH antagonist, and the gonadotropin was replaced by infusion of recombinant human FSH and LH (21). The efficacy of Cetrorelix in suppressing endogenous gonadotropin secretion in cynomolgus monkeys has been established previously (16). The effect of administration of FSH on spermatogenesis in the GnRH antagonist-suppressed nonhuman primate model (22) and the combined treatment using FSH and hCG in the primate have also been reported previously (23).…”

“…The investigators were blinded to the experimental status of the animals. In addition, at study day 0 and on the last study day (day 38) testicular biopsies were performed as previously described (16).…”

“…Testis volume was determined as previously described by measuring the length and width of the testis using Vernier callipers (16). The formula for an ellipsoid was used to estimate testicular volume.…”

“…Tissue was fixed in Bouin's solution, embedded in paraffin and cut to 3 mm sections for light microscopical analysis as described previously (16). Sections were stained with periodic acid Schiff 's base and hematoxylin and the most advanced germ cell types were evaluated in all tubules.…”

Experimental endocrine manipulation by contraceptive regimen in the male marmoset (Callithrix jacchus)

“…Testicular volume was calculated applying a formula for an ellipsoid from length and width measures of the testis using Vernier callipers as described previously (Weinbauer et al 1998, Junaidi et al 2005.…”