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.     

Reproductive changes during the early stages of chronic renal insufficiency in the male rat

To examine the reproductive abnormalities during the early stages of chronic renal insufficiency, male rats were studied 1, 2, or 4 weeks after a 2-stage 5/6 nephrectomy or sham surgery. Serum creatinine and urea nitrogen were elevated 3 to 4-fold in nephrectomized rats at all times. Absolute testicular weight was not different between groups but relative testicular weight was higher in nephrectomized animals because of their lower body weight. Absolute prostate and seminal vesicle weights were depressed nephrectomized rats at 4 weeks. Serum luteinizing hormone (LH) was depressed by nephrectomy at all times whereas follicle-stimulating hormone (FSH) was elevated at 4 weeks. Testosterone and androstenedione in serum and testicular interstitial fluid were depressed by nephrectomy at all times. Pituitary LH and FSH were depressed in nephrectomized rats by 2 weeks. These data indicate that some, but not all, of the reproductive defects that accompany chronic renal insufficiency are already present 1 week after a large reduction in renal mass and that defects in both pituitary and testicular function probably occur.     

PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES

The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous.     

Evidence for a major role of inhibin in the feedback control of FSH in the male rat

Adult rats (16-18/group) received a single intratesticular injection of 25, 100 or 400 microliters 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 studied in Paraplast-embedded 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 less than 0.001) and testicular (r = -0.66, P less than 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 microliters 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.     

Feedback control of FSH secretion in the male rat

Site of feedback control of FSH secretion in the male rat was studied by measuring changes in serum LH, FSH and hypothalamic LH-RH by radioimmunoassay in rats after castration and after 500 rad X-irradiation to the testis. The rise in serum LH and FSH in castrated animals was associated with a significant fall in hypothalamic LH-RH 16 and 24 days after castration. Serum FSH rose significantly after X-irradiation without a significant change in serum LH or hypothalamic LH-RH content up to 30 days after irradiation. When pituitary halves from X-irradiated animals were incubated in vitro in the presence or absence of synthetic LH-RH, there was a significant rise in FSH (but not LH) released in the incubation medium in the absence of added LH-RH. The response of the pituitaries to LH-RH was, however, not different between control and irradiated rats. It is concluded that the testicular FSH-inhibitory substance acts predominantly at the pituitary gland on the LH-RH independent release of FSH.     

Effect of neonatal androgenization on the testosterone feedback sensitivity in adult rats in two lighting conditions

Neonatally androgenized and intact adult male Wistar rats received daily, during 1 week, either testosterone propionate or sesame oil injections in periodic or constant light. Serum and pituitary gonadotropins and hypothalamic LHRH were measured. In periodic light, neonatal androgenization did not change the serum concentration or pituitary contents of gonadotropins, or the hypothalamic content of LHRH. Testosterone injections decreased serum concentration and pituitary content of gonadotropin of intact rats but failed to decrease the pituitary gonadotropin content of neonatally androgenized rats. In constant light, serum FSH was decreased in neonatally androgenized rats. Testosterone injections decreased both serum LH and FSH concentrations of intact rats but only serum LH of androgenized rats. Pituitary gonadotropin and hypothalamic LHRH contents remained unchanged. We conclude that neonatal androgenization renders the male rat hypothalamo-pituitary axis more resistant to changes of testosterone concentration in adulthood. Constant light did not sensitize the neonatally androgenized rats to testosterone, but on the contrary, testosterone injections were less effective in constant than in periodical light.     

Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.     

Androgen secretion and characteristics of testicular HCG binding in cryptorchid rats

Response of the cryptorchid testis to gonadotrophic stimulation was assessed by comparison of the androgen production capability in vivo and in vitro with that of the normal scrotal testis. Serum androgen concentrations in cryptorchid rats were similar to those in normal rats, and the incremental increase 60 min after 50 i.u. hCG (i.v.) was about 7-fold for both groups. Basal and hCG-stimulated androgen production in vitro was higher for abdominal testes (557 and 3286 ng/pair) than for scrotal tests (157 and 504 ng/pair). Specific binding of hCG by testicular homogenates was slightly higher (P < 0.05) for cryptorchid testes when expressed per unit weight, but Scatchard analysis indicated that although hCG binding affinities did not differ (Ka = 2 x 10(10) M-1), hCG binding capacity of cryptorchid testes was only 75 ng, compared to 219 ng for scrotal testes. These data indicate that a discrepancy exists between androgen production in vivo and in vitro by cryptorchid testes and that normal serum androgen concentrations are maintained in the presence of decreased numbers of testicular LH/hCG receptors.     

Correlation of the level of β-citryl-l-glutamic acid with spermatogenesis in rat testes

β-Citryl-l-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.     

Transport of IgG across the blood-luminal barrier of the male reproductive tract of the rat and the effect of estradiol administration on reabsorption of fluid and IgG by the epididymal ducts

In rats immunized systemically with tetanus toxoid the concentration of specific anti-tetanus-toxoid-specific IgG in fluid from the rete testis and cauda epididymidis were respectively 0.6% and 1.4% the concentration in blood serum. The extratesticular duct system reabsorbed 97% of the IgG and 99% of the fluid leaving the rete, but estradiol administration affected the site of reabsorption. In untreated rats, the ductuli efferentes reabsorbed 94% of the IgG and 96% of the fluid leaving the rete, whereas estradiol-treated rats reabsorbed 83% of the IgG and 86% of the fluid, and the ductus epididymidis fully compensated for these different effects of estradiol on the ductuli efferentes. The concentrations of IgG in secretions of the seminal vesicles and prostate gland were lower (0.1% and 0.3% respectively of the titers in blood serum) than in fluids from the extratesticular ducts, and were not affected by the administration of estradiol. RT-PCR showed that Fcgrt (neonatal Fc receptor, also known as FcRn) is expressed in the reproductive ducts, where IgG is probably transported across epithelium, being particularly strong in the ductuli efferentes (where most IgG was reabsorbed) and distal caput epididymidis. It is concluded that IgG enters the rete testis and is concentrated only 2.5-fold along the extratesticular duct system, unlike spermatozoa, which are concentrated 95-fold. Further, the ductus epididymidis can recognize and compensate for changes in function of the ductuli efferentes.     

Estrogen receptor in the ductuli efferentes, epididymis, and testis of rhesus and cynomolgus macaques

We obtained the testes, ductuli efferentes, and epididymides from adult rhesus and cynomolgus macaques and examined these tissues for estrogen receptors (ER) with immunocytochemistry (ICC) and a sucrose gradient assay. Both techniques employed monoclonal antibodies prepared against ER, and both showed that high concentrations of ER were present OFFy in the ductuli efferentes. Moreover, all specific staining was confined to the nuclei of the nonciliated, absorptive epithelial cells. The quantity of salt-extractable ER in the ductuli efferentes (834 +/- 161 [SEM] fmol/mg DNA [n = 8]) did not differ significantly from the amounts measured with the identical assay in oviducts and endometrium of estrogenized female macaques. Testes and epididymides of macaques had no specific staining by ICC and barely detectable amounts by biochemical analysis (7 +/- 4 [n = 3], 8 +/- 2 [n = 5], 33 +/- 16 [n = 3], and 6 +/- 3 [n = 8] fmol/mg DNA for testis and caput, corpus, and cauda epididymis, respectively). The functional significance of the high levels of ER in the ductuli efferentes of macaques remains to be determined.     

Regulation of infant and developing rat testicular gonadotropin and prolactin receptors and steroidogenesis by treatments with human chorionic gonadotropin, gonadotropin-releasing hormone analogs, bromocriptine, prolactin, and estrogen

Infant (5-day-old) male rats were treated with hormonal regimens to alter their exposure to gonadotropins, prolactin (Prl), and estrogen, and the response of testicular endocrine functions was measured. Human chorionic gonadotropin (hCG) or a potent gonadotropin-releasing hormone agonist analog (GnRH-A) resulted in a short-lived decrease of testicular receptors (R) for luteinizing hormone (LH), but no deleterious effects were found on testicular capacity to produce testosterone (T), which is a typical response of the adult testis. Only GnRH-A, through probable direct testicular action, induced a relative blockade of C21 steroid side-chain cleavage that was observed in vitro upon hCG stimulation. Human chorionic gonadotropin treatment, but not GnRH-A treatment, increased testicular Prl-R. GnRH antagonist analog (GnRH-Ant) treatment did not affect testicular LH-R, but decreased Prl-R and testicular T production. Decrease of serum Prl by bromocriptine had no effect on testicular LH-R or Prl-R, but slightly decreased T production in vitro. Ovine Prl increased binding sites for LH/hCG. The postnatal rats were insensitive to negative effects of diethylstilbestrol when monitored by testis weight, T, and LH-R. In conclusion, the responses to changes in the hormonal environment differed greatly between infant and adult testes. Mainly positive effects of elevated gonadotropin and Prl levels were seen on infant rat Leydig cell functions. Likewise, decreased tropic hormone levels, and exposure to estrogen, were ineffective in bringing about the inhibitory actions seen in the adult.     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

Correlation of the level of β-citryl-l-glutamic acid with spermatogenesis in rat testes

β-Citryl-l-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.     

Response of adult male rats to LH-RH after neonatal immunization with antiserum to LH-RH

Male rats were passively immunized at Day 5 of age with LH-RH antiserum. Their response to LH-RH at 100 days of age was examined. Serum FSH and LH concentrations increased more than in control rats, although basal plasma levels were similar. The pituitary content of LH, but not FSH, was significantly increased, and hypothalamic content of LH-RH was similar in both groups. The testes and seminal vesicles were smaller in experimental animals than in controls. It is suggested that transient blockade of LH-RH secretion during the neonatal period produces abnormalities in pituitary-testicular functon in the adult rat.     

Effects of x-ray irradiation on the subsequent gonadotropin secretion in normal and neonatally estrogenized female rats.

Female rats were irradiated with 190R of X-rays at 10 days of age and sacrificed 4, 7 or 12 months later. Their ovaries were histologically examined and serum levels and pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. Both serum levels and pituitary contents of LH and FSH rose significantly 4 and 7 months after irradiation, although the ovaries were markedly reduced in weight. On the contrary, 12 months after irradiation, the ovaries increased in weight and consisted mostly of polyhedral, hyperplastic interstitial cell masses, and both LH and FSH in the serum and pituitary were reduced to normal levels. These characteristic changes in the ovarian weight and histological appearance could not be observed in the similarly irradiated animals which were received daily injections of estrone for the first 30 days of postnatal life, i.e., daily injections of 50 mug for the first 10 days, 100 mug for the middle 10 days and 200 mug for the last 10 days. Serum LH levels of the estrogenized irradiated rats at 7 or 12 months of age did not elevate although those of FSH were significantly higher than the non-irradiated intact levels. From these results, a rise in the blood levels of LH and the FSH may be attributed to the increase in weight and the histological changes in the ovaries of the irradiated female rats, and the elevation of only FSh level may not result in the abnormal growth of the irradiated ovaries.     

Human chorionic gonadotropin increases the concentration of macrophages in neonatal rat testis

The purpose of these studies was to determine whether treatment of newborn rats with exogenous FSH or hCG would alter the concentration or size of testicular macrophages. Animals were injected once daily with various doses of FSH, hCG, or vehicle for 8-10 days beginning the day following birth. After immunohistochemical labeling of the macrophages with a monoclonal antibody specific for rat macrophages, the concentration and size of macrophages were determined by use of a point-counting method. Body weight, testis weight, and serum levels of testosterone and FSH were also measured. It was found that hCG significantly increased the concentration of macrophages within the interstitium but did not affect the size of the cells. Both testicular weight and serum testosterone concentrations increased after hCG treatment. Although FSH increased the weight of the testis, neither the size nor concentration of macrophages was altered. These results raise the possibility that the number of macrophages within the interstitial compartment of the normally maturing rat testis is under the control of LH.     

Pituitary and gonadal function during physical exercise in the male rat

The effects of training and acute exercise on serum testosterone, luteinizing hormone (LH) and corticosterone levels and on testicular endocrine function in male rats were studied. In the first part of the study, the rats were trained progressively on a treadmill, over 8 weeks. Training did not change the basal levels of serum testosterone, LH and corticosterone, or the testicular concentrations of testosterone and its precursors progesterone and androstenedione. The levels of testicular LH (30.3 +/- 2.6 ng/g wet wt, mean +/- SEM) and lactogen (150 +/- 14 pg/g) receptors were unchanged after training. However, the capacity of testicular interstitial cell suspensions to produce cAMP and testosterone increased by 20-30% during in vitro gonadotropin stimulation. In the second part, the trained and untrained control animals underwent acute exhaustive exercise. Serum testosterone levels decreased by 74 and 42% in trained and untrained rats, respectively (P less than 0.02), and corticosterone rose by 182% in trained and 146% in untrained rats (P less than 0.01), whereas the LH level was unchanged. Testicular levels of testosterone and its precursors decreased, with the exception of unchanged androstenedione, in trained rats; the cAMP concentration was unchanged. In both trained and untrained rats, acute exercise decreased the capacity of interstitial cell suspensions to produce cAMP, whereas there were no consistent effects on testosterone production. Acute exercise had no effect on LH or lactogen receptors in testis tissue. In conclusion, training had no effect on serum or testicular androgen concentrations, but increased Leydig cell capacity to produce testosterone and cAMP. Acute exercise decreased serum and testicular testosterone concentrations without affecting serum LH. A direct inhibitory effect of the increased serum corticosterone level on the hypothalamic-pituitary level and/or testis may be the explanation for this finding.     

Stimulatory effect of follicle-stimulating hormone on rat Leydig cells

Summary The effects of FSH on the testicular interstitial tissue of immature hypophysectomized rats were studied by comparing morphological changes in Leydig cells with quantitative changes in interstitial tissue histology using morphometric analysis. Three groups of rats received subcutaneous injections of 0.5 ml saline vehicle or 10 g rFSH or 20 ng oLH (equivalent to the amount of LH known to contaminate the FSH), twice daily for 7 days. Administration of FSH significantly increased testis weight and stimulated more advanced spermatogenesis compared to saline or LH. Morphometric analysis of testes of LH-treated rats showed a small but significant increase in total interstitial cell volume compared to saline treatment. FSH caused much greater increases in the total volume of interstitial tissue and interstitial cells than either saline or LH and significantly increased the total volume of interstitial fluid by comparison with the other groups. FSH but not saline or LH treatment resulted in a striking hypertrophy of Leydig cells, to produce cells ultrastructurally identical to Leydig cells from adults. Since the target tissue of FSH is the seminiferous epithelium, the observed effects on Leydig cells by FSH treatment suggest that the secretion of factors by the seminiferous tubules may mediate the maturation of Leydig cells.     

Nutritional influences on sexual maturation in the rat

The effect of altered nutrition on sexual maturation may depend in part on the nature and timing of the dietary change. The data are conflicting as to whether rats undernourished before weaning but normally fed after weaning have delayed puberty, but such undernourished rats clearly weigh less at vaginal opening than do normally fed animals. Altered nutrition after weaning can change the timing of puberty, and in such cases the body weight at puberty of the animals given the modified diet is frequently abnormal. The factors regulating the age and weight at puberty of rats fed altered diets seem to include the degree of underfeeding, as reflected in the growth rate, and the composition of the diet. Undernourished immature male rats have low serum testosterone secondary to gonadotropin deficiency. Basal luteinizing hormone (LH) in these animals is either low or "inappropriately normal" relative to their hypoandrogenic state (low serum testosterone and sexual accessory gland weights), and serum LH increases after luteinizing hormone-releasing hormone (LHRH) or castration are normal or minimally reduced. Serum follicle-stimulating hormone (FSH) in undernourished rats is subnormal basally and after administration of LHRH, but not after castration, which suggests that the low basal serum FSH is due to inhibition of FSH output by a testicular factor. Spermatogenesis may be unaltered by dietary changes severe enough to cause hypoandrogenism, although very severe under-nutrition will impair sperm production.