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Summary. Subcutaneous injections of testosterone propionate to adult male rats at a dose of 2·5 or 10 mg/kg body weight, 3 times per week for 7 weeks, resulted in a 75% reduction in serum LH and more than 50% reduction in intratesticular testosterone concentration, but serum FSH levels remained unchanged. The free -SH content, measured as iodo[14C]acetamide binding, increased by 70–100% in testicular sperm heads after suppression of testicular testosterone, and by 25–30% in caput epididymal sperm heads but was decreased by 70–80% in cauda epididymal sperm heads. These results demonstrate an alteration in the oxidative state of sperm nuclear basic proteins, suggesting incomplete nuclear maturation. These changes may be specific for the suppression of intratesticular testosterone, thus illustrating the androgen dependency of sperm head maturation. The contrast effects noted between the iodo[14C]iodoacetamide binding by the caput and the cauda epididymal sperm heads indicate that testosterone propionate treatment may affect the mechanisms regulating the oxidation of the sulphydryl residues in sperm heads during epididymal transit. This alteration may not directly relate to the tissue androgen concentrations.
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Introduction
In the last two decades, it has become increasingly evident that disturbances of the hypothalamic–pituitary–gonadal axis account for only a small percentage of the cases seen in male infertility clinics. In an attempt to clarify the pathophysiology of idiopathic male infertility and also for the development of new methods for male contraception, researchers have focused on local regulators of intratesticular events (Bartlett et al., 1989; Hamilton and Waites, 1989). The number of factors implicated in paracrine regulation has been steadily increasing and it is essential that the data available are critically examined. The object of this review is to pinpoint the factors of potential or proven physiological significance and to concentrate on the more recent advances made in the field.
The two major areas of activity within the testis centre on steroidogenesis and spermatogenesis. The intratesticular control of steroidogenesis has already been extensively reviewed (Sharpe, 1990) and this paper
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Summary. Out-of-season male rhesus monkeys were used to compare the effectiveness of pulsatile treatment with LH-RH and administration of LH-RH agonist on testicular function.
Treatment with LH-RH agonist (1·0 μg Hoe 766/day) for 11 weeks resulted in partial stimulation of pituitary and testicular functions. The pituitary LH response to the agonist increased during treatment. Testosterone levels were stimulated to within the normal range and 2 of the 4 treated monkeys produced ejaculates, but sperm counts were below normal.
Pulsatile treatment with LH-RH (100 ng every 96 min for 7 days alternating with LH-RH agonist treatment for 7 days) in 2 monkeys induced full testicular activity after 7 weeks. Ejaculations were induced at a time when the rhesus monkey is normally sexually inactive. Seminal characteristics at the end of treatment were similar to values in the normal breeding season. In samples collected from one monkey over a 24-h period before treatment there were no LH spikes and very low testosterone levels. During pulsatile LH-RH treatment distinct LH and testosterone spikes occurred, comparable to those in the breeding season.
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Summary. The in-vitro bioassay for LH, using mouse Leydig cells, has been modified for the direct measurement of serum LH in the male rhesus monkey. Validation of the assay demonstrated good reliability in terms of accuracy, precision and sensitivity (1·5 mi.u./ml). Basal LH concentrations in laboratory-maintained monkeys with and without anaesthesia were not significantly different from those in free-ranging, feral monkeys. LH-RH (50 μg i.v.) elicited a 30-fold increase in LH concentrations after 30 min. LH levels in castrated adult monkeys were approximately 50 times the normal levels. Intact and castrated juvenile males had only very low LH levels. LH from the serum of an adult male castrate was further characterized by Sephadex G100 column chromatography.
The in-vitro bioassay provides a preferable alternative to the heterologous radioimmunoassay method for the routine determination of LH in the rhesus monkey.
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Summary. Morphometric study revealed that, at 40 days after the start of vitamin A replacement, A1 spermatogonia and preleptotene spermatocytes appeared in more than 70% of the whole mounts of seminiferous tubules of vitamin A-deficient rats. By 42 days, the appearance of these cell types was reduced by 50%, and A2 and A3 spermatogonia were predominant. By 46 days, A1–A3 spermatogonia appeared in <30% of the tubular length while A4, intermediate and B spermatogonia became the major cell types in the basement compartment of seminiferous tubules. The predominance of spermatogonia noted at given times was corroborated by higher frequencies of tubular cross-sections of stages in which that particular type of spermatogonium resides. These results indicate that seminiferous tubules of vitamin A-replaced–vitamin A-deficient rats are 'enriched' for particular stages. Tracing the development of [3H]thymidine-labelled preleptotene spermatocytes revealed normal kinetics of germ cell differentiation in these animals. Furthermore, the spermatogonial proliferations in the vitamin A-replaced–vitamin A-deficient rats were quantitatively normal. We suggest that vitamin A replacement may result in temporal suppression of the differentiation of A2–B spermatogonia, leading to a stimulation or synchronization of certain groups of undifferentiating spermatogonia which undergo active proliferation simultaneously. These synchronized populations of spermatogonia continue to proliferate and differentiate, thus resulting in the stage-enrichments noted at later times.
Keywords: vitamin A deficiency; spermatogenesis; seminiferous epithelium; stage enrichment; rat
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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.
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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.
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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
Department of Obstetrics and Gynecology, Center for Reproductive Medicine and Andrology, Department of Obstetrics and Gynecology, amedes Hamburg, Campus Grosshadern LMU Munich, 81377 Munich, Germany
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Department of Obstetrics and Gynecology, Center for Reproductive Medicine and Andrology, Department of Obstetrics and Gynecology, amedes Hamburg, Campus Grosshadern LMU Munich, 81377 Munich, Germany
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Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved–thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2±4.5% (fresh) to 13.6±1.8 (DMSO), 9.5±1.7 (PrOH), or 6.8±1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2±2.5%) and primary follicles (28.1±5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2±3 and 5.4±2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.
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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.
