Serum FSH-suppressing activity of human recombinant inhibin A in male and female rats.

After a single i.v. injection of purified human recombinant inhibin A (hr-inhibin) or bovine follicular fluid (bFF) to 3-day castrated 35-day-old male rats, serum FSH concentrations fell (P less than 0.05) between 4 and 8 h, returning to control concentrations by 16-24 h. Administration of graded doses of hr-inhibin (0.625-10 micrograms/100 g body wt) and bFF (31.3-250 microliters/100 g body wt) resulted in a parallel dose-related suppression of serum FSH with a maximum suppression 50% of controls. Similar experiments in 2-day ovariectomized 85-day-old female rats also showed a dose-related suppression with a maximum suppression approximately 30% of controls. Serum LH concentrations remained unchanged in all studies with male or female rats. The biological activity of hr-inhibin in vivo was determined for male and female rats in terms of a standard bFF preparation defined by an in-vitro bioassay based on the suppression of FSH content in rat pituitary cells in culture. In males hr-inhibin exhibited a biopotency of 407 (159:1050; fiducial limits) U/micrograms protein and in females the biopotency was 358 (226:565) U/micrograms protein. These potencies are lower than that measured in the in-vitro bioassay (1120 (1040:1210) U/micrograms protein) and differences between in-vivo and in-vitro systems were attributed to the use of bFF rather than a purified human inhibin preparation as standard. These results indicate that hr-inhibin behaves similarly in vivo to bFF. Furthermore, based on the large working range and relatively good precision, the female rat system provides a good basis for an inhibin in-vivo bioassay method.

The effects of exogenously administered testosterone on spermatogenesis in intact and hypophysectomized rats.

The effects of exogenously administered testosterone on the maintenance of spermatogenesis in intact and hypophysectomized rats were examined. Adult male rats were given Silastic implants containing testosterone in lengths ranging from 0.5-20 cm, and their effects on daily sperm production (DSP); serum FSH, LH, and testosterone; and interstitial fluid testosterone were determined in intact and hypophysectomized rats over a 13-week period. In intact rats, DSP levels were suppressed to 4-30% of control values at lower testosterone doses (2- to 6-cm implants), while DSP levels were partially maintained (65-93%) at higher doses (greater than or equal to 8 cm implants). Under these conditions LH levels were suppressed while FSH levels were reduced to 30-60% of control values. A steep testosterone-induced change in DSP levels was observed in both intact (2- to 3-fold) and hypophysectomized (18-fold) animals over a narrow testosterone dose range (implant lengths, 6-8 cm). Interstitial fluid testosterone levels associated with this change in DSP levels range from 6-8% of control values, while serum testosterone levels were elevated 1- to 2-fold above control values. A comparison of the testosterone implant lengths that caused a 50% change in DSP levels after 7 weeks of treatment was similar in intact and hypophysectomized rats. At high testosterone doses (greater than or equal to 10-cm implants), maximal DSP levels decreased 10% by 7 weeks and 35% by 13 weeks. DSP and serum testosterone levels were highly correlated (r = 0.54-0.83), while DSP and interstitial fluid testosterone showed a less correlation (r = 0.36-0.70) under the various experimental conditions examined. In conclusion, the testosterone-induced maintenance of DSP in rats is associated with several dose-related responses or events that appear to be differentially regulated. At low testosterone concentrations, DSP levels are partially maintained in intact rats but are totally suppressed in hypophysectomised rats, suggesting that a pituitary factor, probably FSH, has a potentiating effect at these testosterone concentrations. At intermediate testosterone doses resulting in the testosterone-induced maintenance of DSP, the similarity of dose-response curves (ED50 and maximum response) in intact and hypophysectomised rats would suggest that pituitary hormones are not required for this aspect of the process. The observation that the maintenance of DSP by testosterone under some experimental conditions is poorly correlated with interstitial fluid testosterone levels raises questions as to the mechanisms by which testosterone acts to stimulate spermatogenesis.

Inhibin secretion is influenced by Leydig cells: evidence from studies using the cytotoxin ethane dimethane sulphonate (EDS).

Adult male rats given a single intraperitoneal injection of the Leydig cell cytotoxin ethane dimethane sulphonate (EDS) show a significant decrease in testosterone from 7 to 14 days, and elevation of serum FSH and LH levels commencing 7 days after treatment, returning to normal at 28 days for LH and 49 days for FSH. A significant rise in serum inhibin levels was seen at day 14 after EDS treatment with levels returning to normal at day 49. In a second series of experiments, silastic implants of testosterone, either 2.5 cm or 22.5 cm in length, were introduced subcutaneously into adult male rats which were treated with EDS 10 days later. Both doses of testosterone suppressed basal LH levels but did not significantly change FSH levels. The rise in FSH and LH levels seen in normal rats after EDS treatment did not occur in either group of testosterone-implanted rats. However, serum inhibin levels rose significantly in both groups after EDS treatment, suggesting that the rise in serum inhibin levels was not due to stimulation arising from the increase in FSH levels after EDS treatment. The data suggest that the rise in serum inhibin levels after EDS treatment is linked to destruction of the Leydig cells through mechanisms that require further investigation.

Effects of gonadotropin-releasing hormone pulse-frequency modulation on luteinizing hormone, follicle-stimulating hormone and testosterone secretion in hypothalamo/pituitary-disconnected rams.

The effects of changes in pulse frequency of exogenously infused gonadotropin-releasing hormone (GnRH) were investigated in 6 adult surgically hypothalamo/pituitary-disconnected (HPD) gonadal-intact rams. Ten-minute sampling in 16 normal animals prior to HPD showed endogenous luteinizing hormone (LH) pulses occurring every 2.3 h with a mean pulse amplitude of 1.11 +/- 0.06 (SEM) ng/ml. Mean testosterone and follicle-stimulating hormone (FSH) concentrations were 3.0 +/- 0.14 ng/ml and 0.85 +/- 0.10 ng/ml, respectively. Before HPD, increasing single doses of GnRH (50-500 ng) elicited a dose-dependent rise of LH, 50 ng producing a response of similar amplitude to those of spontaneous LH pulses. The effects of varying the pulse frequency of a 100-ng GnRH dose weekly was investigated in 6 HPD animals; the pulse intervals explored were those at 1, 2, and 4 h. The pulsatile GnRH treatment was commenced 2-6 days after HPD when plasma testosterone concentrations were in the castrate range (less than 0.5 ng/ml) in all animals. Pulsatile LH and testosterone secretion was reestablished in all animals in the first 7 days by 2-h GnRH pulses, but the maximal pulse amplitudes of both hormones were only 50 and 62%, respectively, of endogenous pulses in the pre-HPD state. The plasma FSH pattern was nonpulsatile and FSH concentrations gradually increased in the first 7 days, although not to the pre-HPD range. Increasing GnRH pulse frequency from 2- to 1-hour immediately increased the LH baseline and pulse amplitude. As testosterone concentrations increased, the LH responses declined in a reciprocal fashion between Days 2 and 7. FSH concentration decreased gradually over the 7 days at the 1-h pulse frequency. Slowing the GnRH pulse to a 4-h frequency produced a progressive fall in testosterone concentrations, even though LH baselines were unchanged and LH pulse amplitudes increased transiently. FSH concentrations were unaltered during the 4-h regime. These results show that 1) the pulsatile pattern of LH and testosterone secretion in HPD rams can be reestablished by exogenous GnRH, 2) the magnitude of LH, FSH, and testosterone secretion were not fully restored to pre-HPD levels by the GnRH dose of 100 ng per pulse, and 3) changes in GnRH pulse frequency alone can influence both gonadotropin and testosterone secretion in the HPD model.

Effects of ethane dimethane sulphonate (EDS) on seminiferous tubule function in rats.

The effects of a single injection of ethane dimethane sulphonate (EDS) on aspects of seminiferous tubule function were assessed over a period of 49 days. Ethane dimethane sulphonate, which is known to cause destruction of Leydig cells, reduced the levels of testosterone in both serum and testicular interstitial fluid for 21 days, after which recovery occurred. The low testosterone levels were associated with elevated serum levels of LH and FSH. Daily sperm production was decreased from 14 to 42 days post-EDS but returned to control levels at 49 days. The production of seminiferous tubule fluid, measured after unilateral efferent duct ligation, decreased significantly at 7 and 14 days but then recovered. The testicular content of androgen binding protein (ABP) was decreased from 14 to 28 days but returned to normal thereafter. These results demonstrate significant effects on seminiferous tubule function, which may be due to the decrease in testosterone or be associated with a direct effect of EDS.

Ultrastructure and possible function of giant crystalloids in the Sertoli cell of the juvenile and adult koala (Phascolarctos cinereus).

Sertoli cells of the juvenile and adult koala testis exhibit a unique morphology due to their large nuclei and in particular, a remarkable abundance of large cytoplasmic crystalloid inclusions. Numerous crystalloid subunits in immature Sertoli cells are aggregated into distinct clusters where by assembly and union, they form large slender crystalloids consisting of an ordered substructure of filaments and tubules. Adult Sertoli cells contain large numbers of basally-positioned crystalloids up to 60 micron in length and the observations suggest a possible mechanism for their growth from collections of tubules assembled together within membrane-bound inclusions. The trunk and adluminal cytoplasm of the adult Sertoli cell also contains crystalloids, usually single, positioned between germ cells or their excess residual cytoplasm. Following sperm release, crystalloids are not shed from the seminiferous epithelium but are retained within the apical Sertoli cell cytoplasm. Although their subsequent fate could not be determined crystalloids did not show evidence of breakdown or phagocytosis by the Sertoli cell, suggesting that they may be reutilized and possibly function to stabilize the association between Sertoli cell cytoplasm and the developing germ cells.

Crystalloid formation in Leydig cells of rats (Rattus fuscipes). An ultrastructural and hormonal study.

The ultrastructure of Leydig cells in a seasonally breeding rodent, Rattus fuscipes, was studied in the breeding and non-breeding season and compared with Leydig cell morphology after suppression of gonadotrophin secretion induced by hypophysectomy or chronic administration of testosterone. Serum luteinizing hormone (LH) and testosterone (T) were measured and in-vitro T production by testes was assessed by stimulation with human chorionic gonadotrophin (hCG). In non-breeding wild-trapped rats and rats with experimental suppression of gonadotrophins, the Leydig cells were atrophied and exhibited variable amounts of cytoplasmic lipid and crystalloid inclusions, the latter commonly dominating the cytoplasmic area. Compared with fertile rats, serum LH and hCG-stimulated T production of experimentally regressed rats was significantly reduced, confirming structural features indicative of Leydig cell inactivity. Atrophy of Leydig cell nuclei was accompanied by the formation of unusual intranuclear vesicles sometimes containing small crystalloids. Ultrastructural analysis suggested transfer of the vesicles to the cytoplasm where their unification gave rise to much larger crystalloid bodies. Crystalloids occurred when serum LH was depressed and with either full (T treatment) or arrested spermatogenesis (hypophysectomy) suggesting that their formation is governed by pituitary function and is not dependent upon the degree of spermatogenic activity.

Effects of testosterone on testicular inhibin and fluid production in intact and hypophysectomized adult rats.

Rats were given s.c. implants of high (HT) or low (LT) doses of testosterone and 10 days later hypophysectomy or sham-operation was performed. The rats were killed after 50 days. Unilateral efferent duct ligation was performed 16 h before death to measure seminiferous tubule fluid production and the increment in testicular inhibin values (inhibin production). Inhibin levels in testis cytosols were measured by a pituitary cell culture bioassay. The LT implants maintained serum testosterone at control values and decreased testicular weight whereas HT implants raised serum testosterone 3-fold and maintained testicular weight at 75-85% of pretreatment levels. In intact rats, LT implants caused no change in testicular inhibin content but decreased inhibin production; no significant changes occurred with HT implants. After hypophysectomy both values were significantly suppressed and could not be maintained by HT or LT implants. However, the HT implants partly restored inhibin production despite their inability to influence testicular inhibin content. In contrast, tubule fluid production depended mainly on intratesticular testosterone levels and occurred normally in intact or hypophysectomized rats with HT but not LT implants. These results indicate that inhibin and seminiferous tubule fluid production, both functions of the Sertoli cell, are under different hormonal control. The maintenance of inhibin production by the testis requires the support of pituitary hormones, presumably FSH, while seminiferous tubule fluid production requires testosterone, presumably through LH stimulation of Leydig cells. These findings are consistent with the hypothesis that inhibin is produced in response to trophic stimulation by FSH.

Seasonally and experimentally induced changes in testicular function of the Australian bush rat (Rattus fuscipes).

Serum concentrations of LH, FSH and testosterone were measured monthly throughout the year in male bush rats. Testicular size and ultrastructure, LH/hCG, FSH and oestradiol receptors and the response of the pituitary to LHRH were also recorded. LH and FSH rose in parallel with an increase in testicular size after the winter solstice with peak gonadotrophin levels in the spring (September). The subsequent fall in LH and FSH levels was associated with a rise in serum testosterone which reached peak levels during summer (December and January). In February serum testosterone levels and testicular size declined in parallel, while the pituitary response to an LHRH injection was maximal during late summer. The number of LH/hCG, FSH and oestradiol receptors per testis were all greatly reduced in the regressed testes when compared to active testes. In a controlled environment of decreased lighting (shortened photoperiod), temperature and food quality, the testes of sexually active adult males regressed at any time of the year, the resultant testicular morphology and endocrine status being identical to that of wild rats in the non-breeding season. Full testicular regression was achieved only when the photoperiod, temperature and food quality were changed: experiments in which only one or two of these factors were altered failed to produce complete sexual regression.

Effects of the induction of experimental cryptorchidism and subsequent orchidopexy on testicular function in immature rats.

Cryptorchidism surgically induced in 14-day-old rats, was allowed to persist until 35 days when one group was killed to assess testicular function. In a second group the cryptorchid testis was returned to the scrotum surgically (orchidopexy) and subsequently killed at 130 days. A third group remained persistently cryptorchid to 130 days, while in a fourth group two sham operations were performed at 14 and 35 days. At 35 days, cryptorchidism resulted in a significant decline in testis weight due to suppressed spermatogenesis. Sertoli cell function as measured by seminiferous tubule fluid (TF) production after unilateral efferent duct ligation and androgen-binding protein (ABP) production was significantly depressed in the cryptorchid group. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were significantly elevated with cryptorchidism but serum testosterone levels were unchanged. Although morphometric measurements showed no change in Leydig cells cross-sectioned area, in vitro human chorionic gonadotropin (hCG)-stimulated testosterone production was significantly increased in the cryptorchid group at higher hCG doses. Similar changes were found in cryptorchid testes at 130 days except that Leydig cell cross-sectional area was now significantly increased. Orchidopexy at 35 days restored spermatogenesis and fertility during test mating was not impaired. TF production, ABP accumulation and serum FSH levels returned to normal following orchidopexy.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric studies on lipid inclusions in Sertoli cells during the spermatogenic cycle in the rat.

The volume and surface area of lipid inclusions often present in the cytoplasm of rat Sertoli cells was measured directly from semi-thin sections of perfusion-fixed testicular tissues using an image analyser linked to a light microscope. Sertoli cell nuclei were used as a reference for comparing any variations in the measured parameters of lipid inclusions during the rat spermatogenic cycle. Volume density of Sertoli cell lipid inclusions was assessed by morphometric analysis of Sertoli cells photographically reconstructed from electron micrographs. Maximum lipid content in Sertoli cells occurred during stages IX-XIV of the spermatogenic cycle, then declined at stages I-III and remained low from stages IV-VIII. The persistence and increase in number of many large Sertoli cell lipid inclusions beyond the stage where spermatid residual bodies are phagocytosed within the Sertoli cells (stage IX) suggests that the synthesis and lipolysis of Sertoli cell lipid inclusions represents an intrinsic functional cycle of the Sertoli cells. Stage-dependent variations in the lipid content of rat Sertoli cells offers morphological evidence that the metabolic duties of the Sertoli cells are synchronised with the spermatogenic cycle to provide local coordination of the proliferation and maturation of the germ cells.

Studies on seminiferous tubule fluid production in the adult rat: effect of hypophysectomy and treatment with FSH, LH and testosterone.

Seminiferous tubule fluid production in adult rats was studied using the technique of unilateral efferent duct ligation (EDL), the production rate representing the difference in testis weight over the time since ligation. Following EDL, fluid production increases linearly for 6 h and linearly at a slightly slower rate for a further 18 h with a sharp decrease thereafter. No differences in fluid production were noted for rats aged between 90-310 days. Forty-eight hours after hypophysectomy there was a significant fall (26%) in fluid production prior to any significant decrease in testis weight. Fluid production continued to decline with time after hypophysectomy eventually reaching a plateau 16-44 days later at levels approximately 15% of those found in control rats. Treatment of rats hypophysectomized 4 days earlier with ovine FSH for 3 days did not restore fluid production, but treatment with ovine LH, testosterone propionate (TP) or FSH together with TP for a similar duration all restored fluid production to normal. On the other hand, treatment of intact adult rats with ovine LH significantly increased fluid production but the effect of treatment with testosterone alone did not reach significance. The results indicate that in the adult rat, seminiferous tubule fluid production is controlled principally by testosterone secreted by the Leydig cell in response to LH stimulation.

Testosterone and 5alpha-reduced androgen secretion by rabbit testes-epididymides perfused in vitro.

Mass spectrometric determinations confirmed that testosterone, 5alpha-androstan-17betaol-3-one, 5alpha-androstan-3alpha, 17beta-diol and 5alpha-androstan-3beta, 17beta-diol were present in venous effluent of in vitro perfused rabbit testes-epididymides. Testosterone, dihydrotestosterone, 3alpha-androstanediol and 3beta-androstanediol were secreted at 3.1 plus or minus 0.9, 0.7 plus or minus 0.2, 0.4 plus or minus 0.1 and 0.6 plus or minus 0.1 mug/h when testes-epididymides were perfused with an artificial medium containing 2.5 ng/ml NIH-LH-S17, ovine. Surprisingly, testosterone constituted only 64% of the total mass of the four androgens secreted. These results probably reflect in vivo androgen secretion since hourly collections of spermatic vein blood from anesthetized rabbits contained 2.7 plus or minus 0.9, 0.8 plus or minus 0.3, 0.15 plus or minus .04 and 0.5 plus or minus 0.2 mug of testosterone, dihydrotestosterone, 3alpha-androstanediol and 3beta-androstanediol, respectively. Once again testosterone constituted only 64% of the total mass of the four androgens.