Summary. Unravelled tubules from the monkey caput and cauda epididymidis were perfused through the lumen in vitro during immersion in an organ bath kept at scrotal temperature and containing [3H]carnitine and [14C]inulin. The specific transport of carnitine from the bath to the lumen was constant for 4 h and reached a steady-state value of about 90 pmol/30 min per cm perfused length in the cauda and about 30 pmol/ 30 min/cm in the caput. These regional variations in carnitine transport differ from those found in the rat epididymis but may be relevant to human epididymal physiology.
T. G. Cooper, C. H. Yeung and G. F. Weinbauer
M. H. Brinkworth, G. F. Weinbauer, S. Schlatt and E. Nieschlag
The possible role of apoptosis in spontaneous or induced germ cell death was investigated by treating adult male rats with either a GnRH antagonist (112.5 μg kg−1 day−1 for 14 days) or methoxyacetic acid (650 μg kg−1; single dose) or sham-treated with either of the vehicles (n = 3 per group). The antagonist virtually abolished gonadotrophin secretion, while methoxyacetic acid reduced serum testosterone concentrations and slightly increased those of FSH (neither significantly). Bands of low molecular mass characteristic of apoptotically degraded DNA were detected by electrophoresis in both treatment groups but not in the controls. Sectioned, Carnoy-fixed testes were screened for degenerating cells with periodic acid–Schiff's base and haemalaun or examined for apoptotic cells using a modified in situ end-labelling procedure. Periodic acid–Schiff's-stained dying cells were found in low numbers in control animals with a distribution and frequency that matched that of apoptotic cells. Degenerating germ cells identified by histology were present at certain stages of spermatogenesis after 2 weeks of antagonist treatment. A comparison of their distribution with that of end-labelled cells identified the cell death as apoptotic. Methoxyacetic acid caused a massive depletion of spermatocytes at stages IX-II, which was also found to be apoptotic. It is concluded that spontaneous germ cell death in adult rats is apoptotic and that both gonadotrophin ablation and administration of methoxyacetic acid can cause apoptosis in the germ cells of adult male rats, but via different routes.
Y. Zhengwei, N. G. Wreford, S. Schlatt, G. F. Weinbauer, E. Nieschlag and R. I. McLachlan
This study examined the effect of GnRH-antagonist (GnRH-A)-induced gonadotrophin withdrawal on numbers of germ cells in adult cynomolgus monkeys and aimed to identify the site of the earliest spermatogenic lesion(s) produced. Animals received either GnRH-A (Cetrorelix; 450 μg kg−1 day−1 s.c.; n = 5) or vehicle (control, n = 4) for 25 days. One testis was removed on day 16 and the other testis on day 25. The optical disector stereological method was used to estimate germ and Sertoli cell numbers per testis. After GnRH-A treatment for 16 days, the number of type A spermatogonia was unchanged; however, type B spermatogonia (15% of control), preleptotene + leptotene + zygotene (15% control) and pachytene (55% control) spermatocytes were all reduced (P <0.05). By day 25, these cells were further reduced together with step 1–6 spermatids (38% control; P < 0.05). More mature germ cells were unaffected. The proportion of type A pale spermatogonia at stages VII–XII was reduced (P <0.05) in GnRH-A-treated groups (52% on day 16, 43% on day 25) compared with control (67%). After 25 days of GnRH-A treatment, the number of Sertoli cells was unaltered but nuclear volume was reduced (66% control, P < 0.05). Tubule length was unchanged but volume (50% control), diameter (62% control) and epithelial thickness (59% control) were reduced (P < 0.05). GnRH-A treatment suppressed serum testosterone concentrations into the castrate range, and testicular testosterone concentrations to 21–36% of control values. Serum inhibin (as an index of FSH action) was suppressed in GnRH-A-treated animals by day 16, declining to 38% of control concentrations at day 25. Therefore, the primary lesion produced by GnRH-A induced gonadotrophin withdrawal is the rapid and profound reduction in the number of type B spermatogonia. The time course of germ cell loss suggests the inhibition of type A pale spermatogonial mitosis as the primary mechanism. Later germ cell maturation, specifically meiosis and spermiogenesis, appears to proceed unaffected. The decline in late spermatocytes and spermatids by 25 days of GnRH-A treatment is attributed to a 'depletional wave' from the spermatogonial lesion. The fact that such marked spermatogenic disruption occurs in the face of substantial testicular testosterone concentrations implies a significant role for FSH in spermatogonial development.
G. Rosiepen, G. F. Weinbauer, S. Schlatt, H. M. Behre and E. Nieschlag
The stability of the duration of the cycle of the seminiferous epithelium was determined by investigating incorporation of 5-bromodeoxyuridine into S-phase germ cells of normal and hemicastrated standard laboratory rats (Sprague–Dawley) and feral Brown/Norway rats (Rattus norvegicus). Feral rats were trapped on farms in the surroundings of Münster. The duration of the cycle of the seminiferous epithelium, determined at intervals of 12 days (3 h versus 12 days 3 h after 5-bromodeoxyuridine injection), was remarkably constant and similar in intact laboratory rats (12.49 ± 0.05 days, n = 13, mean ± sem) and feral rats (12.44 ± 0.06 days, n = 8). In hemicastrated laboratory and feral rats the duration of the cycle was similar to that in intact animals, indicating that hemicastration did not influence the kinetics of the seminiferous epithelium cycle. However, the coefficients of variation of the estimated duration of the cycle of the seminiferous epithelium were at least three times lower in hemicastrated rats (one testis from the same animal serving as reference point) compared with that of intact rats (the reference point based on the average staining frequency at 3 h). Overall, no significant differences between laboratory and feral rats could be observed with regard to testis weight and serum concentrations of FSH and testosterone. The number of cells per testis, determined by flow cytometry, was similar in laboratory and feral rats, except for a slight but significant difference in the haploid:tetraploid cell ratio (6.3 ± 0.2 versus 7.5 ± 0.3, P< 0.05). It is concluded that the duration of the cycle of the seminiferous epithelium is identical in feral Brown/Norway rats and their descendent laboratory rat strain, Sprague–Dawley rats. Hemicastration (each animal being its own reference point) profoundly increased the precision of the determination of duration of the cycle of the seminiferous epithelium, at least for the duration of one cycle.
R. K. Chandolia, G. F. Weinbauer, U. Fingscheidt, J. M. S. Bartlett and E. Nieschlag
Summary. The effects of combined treatment with an antagonist of gonadotrophinreleasing hormone (ANT) and the antiandrogen flutamide (FL) on spermatogenesis were studied in the presence and absence of exogenous follicle-stimulating hormone (FSH). After treatment for 2 weeks, the combination of ANT (RS 68439, 450–500 μg/kg per day, s.c.) with 10, 20 or 40 mg FL/day, s.c. was as effective as ANT plus the Leydig cell toxin ethane dimethane sulphonate (75 mg/kg per week, i.p.) in terms of reduction in weight of testes, epididymides and seminal vesicles. Thus, a daily dose of 10 mg FL/kg was sufficient to block the androgen action in the testes of ANT-treated rats. In a second experiment, rats received ANT and ANT + FL (10 mg/kg) alone or in combination with a highly purified human FSH preparation (5 or 10 iu, twice a day) for 2 weeks. FSH did not affect testosterone concentration or weight of epididymides and seminal vesicles, but ANT+FL markedly enhanced the ANT-induced reduction of testis weight, seminiferous tubule diameter and numbers of germ cells, as revealed by qualitative and quantitative analysis of testis histology. In the absence of FL, testis size and numbers of germ cells, including elongated spermatids, were increased by FSH. In the presence of FL, the effects of FSH were less pronounced with respect to the germ cells, in terms of both numbers of cells and the effective dose of FSH. Irrespective of treatment with FL, exogenous FSH increased the inhibin concentrations in serum, indicating that Sertoli cells remained responsive to FSH. From the present study it is concluded that (i) FL accelerates ANT-induced testicular involution, (ii) FSH has a role in adult spermatogenesis and (iii) the effects of FSH on advanced germ cells are influenced by androgens.
Keywords: FSH; spermatogenesis; GnRH antagonist; antiandrogen; rat
G. F. Weinbauer, J. M. S. Bartlett, U. Fingscheidt, C. G. Tsonis, D. M. de Kretser and E. Nieschlag
Summary. Adult rats (16–18/group) received a single intratesticular injection of 25, 100 or 400 μl glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1·5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studed in Paraplastembedded tissue stained with PAS and haematoxylin–eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = −0·70, P < 0·001) and testicular (r = −0·66, P < 0·001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0·48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 μl groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7–14%) of testicular testosterone and unaltered serum FSH concentrations.
These findings indicate that (i) inhibin is involved in the in-vivo regulation of FSH secretion in the adult male rat, (ii) testicular inhibin content, at least in part, may be regulated at the testicular level, and (iii) serum inhibin concentrations to some extent reflect the extent of damage to spermatogenesis and Sertoli cells.
Keywords: inhibin; testosterone; FSH; feedback; testis; rat