Hormonal regulation of DNA polymerase beta activity and expression in rat adrenal glands and testes

We investigated whether DNA polymerase beta activity and expression in rat adrenal glands and testes are controlled by the cAMP dependent protein kinase (A-kinase) phosphorylation system in addition to anterior pituitary hormones. DNA polymerase beta mRNA expression in rat testes was decreased by hypophysectomy and recovered with administration of gonadotropic hormone, suggesting that this enzyme is controlled at the mRNA level by this pituitary hormone. In addition, DNA polymerase beta activity in the adrenal glands and testes and the amount of mRNA in the testes increased when cAMP was administered to the normal rat. This activity was decreased by administration of the cyclic AMP-dependent protein kinase inhibitor, H(8). Moreover, when alkaline phosphatase was added to the assay system in vitro, a decrease in DNA polymerase beta activity was observed. These findings indicate that changes in the activity and expression of DNA polymerase beta are mediated via cAMP and the A-kinase system, and that phosphorylation of this enzyme is also involved in this expression.     

Hormonal regulation of DNA polymerase-beta activity in the rat thyroid gland

Using hypophysectomized rats, it has been shown that DNA polymerase-beta activity in the adrenal gland and testis is largely influenced by pituitary trophic hormones. Sucrose gradient centrifugation of thyroid extracts revealed three peaks of DNA polymerase-beta activity sedimenting at 3.3S, 7.3S and 12S. Of these, hypophysectomy induced a decrease in the 3.3S DNA polymerase-beta, whereas other molecular forms were affected only slightly. DNA polymerase-alpha and -gamma activities were unaffected by hypophysectomy. These changes in DNA polymerase-beta caused by hypophysectomy were reversed by daily i.p. injection of TSH. Furthermore, stimulation of the thyroid by excess TSH induced by the administration of 1-methyl-2-mercaptoimidazole resulted in an increase of all forms of thyroid DNA polymerase-beta. These results show that the level of DNA polymerase is relatively constant after hypophysectomy but that DNA polymerase-beta in the rat thyroid gland is also modulated by TSH mainly through the change of activity of the polymerase-beta which sediments at 3.3S.     

Pituitary regulation of Leydig cell function in the adult male rat.

The effects of hypophysectomy on serum testosterone, 125I-labelled hCG binding to testicular membranes and on testicular responsiveness were studied in adult rats. Serum testosterone decreased rapidly over the first 6 h after hypophysectomy. LH receptors were determined (pmol/testis) by measuring the specific binding of 125I-labelled hCG in membrane preparations of testes of rats hypophysectomized 1, 2, 3, 6, 9, or 15 days earlier. Hypophysectomy did not result in a decrease in 125I-labelled hCG binding on day 1 but this had decreased to 40% of that in intact controls by day 2. A gradual decline was found between days 2 and 6 at which time hCG binding had decreased to 15%. No further decrease occurred between days 6 and 15. Scatchard analysis indicated that the decline in hCG binding was due to a decreaffinity. FSH, testosterone, dihydrotestosterone, and oestradiol were unable to prevent the decline in hCG binding. Although serum testosterone, testicular testosterone content, and 125I-labelled hCG binding decreased rapidly after hypophysectomy, testicular responsiveness to LH was biphasic. The intraperitoneal administration of 25 microgram LH 2 h before decapitation increased testosterone in the circulation to a greater extent extent in animals hypophysectomized for 1 day than in intact controls while hCG binding affinities and capacities had not changed. Two or three days after hypophysectomy testicular responsiveness to LH was similar to that of intact controls even though hCG binding in hypophysectomized animals had decreased to 40 and 28% of intact controls respectively. It is concluded that (1) the testis is dependent on anterior pituitary hormones for maintenance of testicular LH receptors and testosterone secretion, (2) FSH, testosterone, dihydrotestosterone, or oestradiol cannot prevent the decline in testicular LH receptors resulting from hypophysectomy, and (3) steroidogenic capacity of the testis persists significantly longer than the hCG binding capacity of the testis.     

The effect of testosterone propionate on the concentration of testicular and epididymal androgen binding activity in the hypophysectomised rat.

The effect of testosterone propionate on the level of androgen binding activity in both testis and epididymis was investigated by both a dextran coated charcoal (DCC) method and by equilibrium polyacrylamide gel electrophoresis (PAGE) which measures specifically androgen binding protein.There was a decrease in androgen binding activity in both testis and epididymis after hypophysectomy. Testosterone propionate treatment (1 mg/day), begun at one day posthypophysectomy, maintained androgen binding activity above the level in testes from hypophysectomised rats for 30 days (PAGE) or 60 days (DCC) and 60 days in epididymides (PAGE). The ability of testosterone propionate treatment (250, 500 and 750 μg/day) to restore androgen binding activity in testis and epididymis of chronically hypophysectomised rats (30 days) was tested using a 3-day treatment period. These doses restored testicular androgen binding activity to 60, 36 and 75% of the levels in control animals, respectively. The degree of restoration of androgen binding activity in epididymis in this time interval was inferior to that in the testis at all doses. Prolonged treatment with testosterone propionate (1 mg/day for 10, 30 and 60 days) resulted in complete restoration of androgen binding activity in testis, expressed per mg protein (PAGE and DCC), and over 50% restoration in the epididymis (PAGE).It was concluded that testosterone propionate is able to maintain and restore testicular and epididymal androgen binding activity and, in particular, androgen binding protein, after hypophysectomy and that this hormone may, in the short term, influence testicular rather than epididymal levels of binding activity.     

Effect of long term deprivation of luteinizing hormone on Leydig cell volume, Leydig cell number, and steroidogenic capacity of the rat testis

Leydig cells atrophy, losing cytoplasmic volume and the capacity for testosterone secretion, within 1-2 weeks of LH deprivation. We investigated the effects of long term (0-16 weeks) LH deprivation on the volume of an average Leydig cell, the volume of Leydig cells per testis, the number of Leydig cells per testis, and testosterone secretion by in vitro perfused testes. Endogenous LH was suppressed in adult rats by testosterone/estradiol-filled (TE) Silastic implants. The presence of Leydig cells in testes was verified by 1) morphological examination using light and electron microscopy, 2) histochemical localization of 3 beta-hydroxysteroid dehydrogenase activity (3 beta HSD), and 3) conversion of pregnenolone to progesterone by in vitro perfused testes. Marked quantitative differences existed in Leydig cell morphology among control and treated rats. The volume of an average Leydig cell and the total volume of Leydig cells per testis decreased (P less than 0.01) rapidly and progressively after TE implantation. At 16 weeks, the average Leydig cell lost 90% of its cytoplasmic volume and 65% of its nuclear volume. Analysis of variance failed to detect a significant decline in Leydig cell number per testis, despite a 16% reduction from the value in control rats (22.2 +/- 1.5 x 10(6)) in rats treated for 16 weeks (18.7 +/- 1.5 x 10(6)). After TE implantation, LH-stimulated testosterone secretion by in vitro perfused testes diminished (P less than 0.01) rapidly to 5% of the control values at 1 week and less than 0.3% of the control value from 4-16 weeks. In contrast, 25% of 3 beta HSD activity was retained (P less than 0.01 vs. controls) at 16 weeks, based on the rate of pregnenolone conversion to progesterone. Moreover, testes of treated rats secreted progesterone at a rate twice that of controls, when the steroid secretion rates were expressed per volume of Leydig cell cytoplasm. Loss of the testosterone-secreting capacity of testes after LH withdrawal was associated with a loss in the volume, but not a significant loss in the number, of Leydig cells. Thus, LH was required to maintain the differentiated structure and function of Leydig cells, but was not required to maintain the overwhelming majority of Leydig cells in the adult rat testis through 16 weeks. Moreover, at least one steroidogenic enzyme, 3 beta HSD, was retained by Leydig cells after long term LH deprivation.     

Regulation of testicular proopiomelanocortin gene expression

The POMC gene is expressed in testicular Leydig cells, but its mRNA is about 150-200 nucleotides shorter in these cells than in the pituitary. For this reason this testicular mRNA has been termed POMC-like mRNA. The purpose of the present study was to define the ontogeny and regulation of POMC gene expression in rat testis. The level of POMC-like mRNA was very low in the testes of 15- and 20-day-old animals. A dramatic increase in mRNA concentration was observed between 20 and 25 days of age, and maximal levels were detected at 40 days. The ontogeny of testicular POMC gene expression correlates with the reported increases in Leydig cell numbers, immunostainable beta-endorphin in Leydig cells, and testicular LH receptors during development. Since these observations suggested that the expression of testicular POMC gene might be influenced by LH, we studied the effect of hypophysectomy and hCG treatment on testicular POMC-like mRNA. Total contents of testicular RNA and POMC-like mRNA decreased in parallel with the decline of testicular weight after hypophysectomy. Administration of hCG to rats 6 days after hypophysectomy prevented the regression of testes and the decrease in testicular POMC-like mRNA content. An increase in the total amount of testicular POMC-like mRNA was observed relative to that in hypophysectomized controls after 8 days of hCG injection. Similar results were obtained when hCG was administered to rats 13 days after hypophysectomy. The effect of glucocorticoid deprivation on testicular POMC-like mRNA was also studied. The POMC-like mRNA concentration did not increase in testis as it did in the anterior pituitary after adrenalectomy, suggesting that glucocorticoids are not a primary regulator of POMC-like mRNA in the testis. In summary, the ontogeny of expression of testicular POMC gene correlates closely with the maturation pattern of Leydig cells; and the expression of testicular POMC gene is regulated by gonadotropins and not by glucocorticoids. We conclude that the regulation of POMC-like mRNA in the testis is different from that in the pituitary.     

The effects of testicular denervation on spermatogenesis in the Sprague-Dawley rat

In the rat, regression of spermatogenesis during the chronic stages of spinal cord injury (SCI) occurs in the presence of normal function of the pituitary-testis hormone axis, thus suggesting that nonendocrine mechanisms might be involved. The current study examined whether disruption of neural input to the testis contributes to the cascade that leads to the regression of spermatogenesis. Four weeks after denervation of the superior spermatic nerve (SSN), testis weight was 25% lower (p < 0.01) than that of the contralateral sham-operated testis. Defects in spermatogenesis including phagocytosis of mature spermatids, vacuolization of spermatid nuclei, delayed spermiation and incomplete cellular associations were observed in >60% of the tubules. In the remaining 30-40% of tubules, the seminiferous epithelium was severely regressed. While cutting the inferior spermatic nerve (ISN) alone did not affect spermatogenesis significantly, it enhanced the effect of SSN denervation on both spermatogenesis and testis weight (p < 0.01). Spermatogenesis was totally regressed in the SSN/ISN-denervated testes. At this time, quantitatively normal spermatogonial proliferation was maintained in SSN- or ISN-denervated testes. Twelve weeks after surgery, regression of the seminiferous epithelium characterized by absence of proliferating spermatogonia, while undifferentiating spermatogonia were present, was observed in all SSN-denervated testes. At this time, regression of the seminiferous epithelia also occurred in >30% of the tubules in ISN-denervated testes. At both times, serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were normal and >60% of normal testicular testosterone concentrations were maintained in the denervated testes. These results indicate that disruption of neural input to the testis is not a cause for the decrease in spermatogonial proliferation during the acute phase of SCI, but may contribute to the chronic effects of SCI on spermatogenesis.     

Identification of insulin-like growth factor-I and its receptors in the rat testis

As part of a study of the testicular production and action of insulin-like growth factor-I (IGF-I), adult rat testes were extracted with acidified methanol, yielding an immunoreactive IGF-I fraction corresponding in size to human IGF-I. The mean IGF-I content (+/- SEM) of testes weighing approximately 1.1 g was 51.5 +/- 5.6 ng/testis, and was not due to serum contamination. After a 3-day fast testicular IGF-I decreased by 80%, whereas serum IGF-I levels declined by 90%. Testicular homogenates and isolated Leydig cells were shown to contain specific IGF-I receptors, Ka = 2 X 10(9) M-1, with 10% IGF-II cross-reactivity. The concentration of these receptors was 2 pmol binding sites per testis, or 3.3 fmol per 10(6) Leydig cells. However IGF-I at 250 ng/ml had no effect on basal or hCG-stimulated testosterone production by isolated Leydig cells, measured over 3 h. Although an effect of IGF-I over longer incubation periods cannot be excluded, it is also possible that testicular IGF-I has a mitogenic role, rather than acting on differentiated testicular functions.     

Prolactin binding by testes of unilaterally cryptorchid rats: the effect of hCG, testosterone, prolactin and orchiopexy

The effect of unilateral cryptorchidism on prolactin binding to the testes was studied in the rat. Cryptorchidism was rendered surgically for 3 weeks and 3, 6 and 9 weeks later prolactin binding was measured in testicular homogenates. Prolactin binding to the cryptorchid testes decreased significantly at 3 weeks with a further decrease at 6 and 9 weeks. Binding by the contralateral testes decreased at 3 weeks and increased at 6 and 9 weeks. To examine the possible mechanism of these changes one group of rats was treated for 5 weeks with testosterone and another with hCG. Testosterone treatment resulted in a significant fall in prolactin binding to normal, cryptorchid and contralateral testes. hCG also produced a slight but significant reduction in prolactin binding. To study the effect of surgical relocation of the testes into the scrotum, orchiopexy was performed in another group of rats. Orchiopexy increased prolactin binding only if performed 3 weeks after cryptorchidism. At 6 and 9 weeks after cryptorchidism orchiopexy did not increase prolactin binding. Treatment of cryptorchid rats with prolactin for 5 weeks induced an increase in prolactin binding to control, cryptorchid and contralateral testes. It is concluded that testicular atrophy follows upon placement of a testis within the peritoneal cavity. This atrophy lowers the total amount of prolactin binding and increases binding to the contralateral testes. Intratesticular concentration of testosterone may play a major role in the decrease of prolactin binding. Orchiopexy improves prolactin binding only if performed before 6 weeks of age. Administration of prolactin augments prolactin binding to the testes, irrespective of their location.     

Temporal gene expression is restored concomitantly with germ cells in the experimentally regressed rat testis.

The present study was designed to examine the effect of hypophysectomy and subsequent testosterone administration on germ cell numbers and germ cell- and Sertoli cell-specific mRNA levels in adult rats. Rats were hypophysectomized and 4 weeks later received 24-cm testosterone-containing polydimethylsiloxane (PDS) implants. Sham-hypophysectomized rats received an empty PDS implant. At 0 and 3 days, and at 1, 2, 4, and 8 weeks, rats were killed. One testis from each rat (n = 4/group) was used to prepare total RNA; the other testis was used to enumerate stage VII-VIII germ cells. cDNA probes for germ cell and Sertoli cell products were used to monitor germ cell- and Sertoli cell-specific mRNAs on Northern blots. Four weeks after hypophysectomy (0 days), preleptotene and pachytene spermatocytes and round and elongating spermatids were reduced in number to 54%, 12%, 1%, and 0%, respectively, of the control values. Testosterone administration caused a time-dependent increase in germ cell numbers; after 8 weeks of testosterone treatment, preleptotene and pachytene spermatocytes and round and elongating spermatids were 75%, 79%, 74%, and 22%, respectively, of control values. Lactate dehydrogenase-C, phosphoglycerate kinase-2, protamine-1, and sulfated glycoprotein-2 mRNA levels (on a per micrograms RNA basis) were 34%, 34%, less than 1%, and 580% of control values, respectively, 4 weeks after hypophysectomy and 79%, 87%, 61%, and 192% of control values, respectively, after 8 weeks of testosterone treatment. Pachytene spermatocyte and round spermatid numbers increased, while Sertoli cell sulfated glycoprotein-2 mRNA levels decreased, with respect to 4 week hypophysectomy values, as early as 3 days after implantation of testosterone capsules. In contrast, germ cell (lactate dehydrogenase-C, phosphoglycerate kinase-2, and protamine-1) mRNA levels increased to the greatest extent between 1-4 weeks after the start of testosterone treatment and, after a short lag period, reflected increases in germ cell type and number. The results indicate that cell-specific mRNAs appear concomitantly with germ cell reappearance in a time-dependent manner in the testes of testosterone-treated hypophysectomized adult rats.     

Intratesticular factors and testosterone secretion: the role of luteinizing hormone in relation to changes during puberty and experimental cryptorchidism

Testicular interstitial fluid (IF) from the rat contains a nongonadotropic polypeptide factor (or factors) which can enhance human CG (hCG)-stimulated testosterone production by Percoll-purified Leydig cells in vitro. The potential importance of this factor has been investigated by measuring its effective levels in testicular IF from individual rats during sexual maturation or after the induction of unilateral cryptorchidism (UCD). In both situations, the effect of modulating LH drive to the testis was also assessed. In abdominal testes from UCD rats, levels of the IF factor were nearly doubled (P less than 0.001) when compared to levels in the contralateral scrotal testis; this was associated with more than an 85% reduction in IF testosterone levels. Further lowering of testosterone levels by the injection of an antiserum to LH beta, raised levels of the IF factor by 30-40% (P less than 0.01) in both scrotal and abdominal testes. Conversely, raising of testosterone levels by injection of hCG caused more than a 90% reduction (P less than 0.001) in levels of the IF factor in both scrotal and abdominal testes. During sexual maturation, levels of the IF factor doubled (P less than 0.01) between 30 and 40 days of age, remained high until 70 days of age, and then decreased to the lower levels found in adult rats. High pubertal levels of the IF factor were associated with the most rapid phase of testicular growth and with a steady increase in the IF levels of testosterone. Injection of pubertal or adult rats with antiserum to ovine LH (oLH) reduced testosterone levels by approximately 85% and doubled (P less than 0.01) levels of the IF factor(s) in both groups. It is concluded that levels of the IF-factor are influenced: by testosterone levels and/or LH drive and by the maturational and/or functional status of the testis. These results also suggest that changing levels of the IF factor may be of physiological significance during puberty.     

The role of luteinizing hormone in regulation of testicular inhibin alpha and beta-B subunit messenger RNAs in immature and adult animals

In vivo and in vitro evidence indicates that FSH is a primary regulator of testicular inhibin production. However, recent reports suggest that LH may also promote inhibin secretion in vivo. To investigate whether LH regulates inhibin subunit messenger RNA (mRNA) expression as well, we examined the effects of hypophysectomy and LH replacement on the testicular content of inhibin alpha and beta-B subunit mRNAs in sexually immature and adult rats. Twenty- and 60-day-old intact and hypophysectomized rats received saline or 0.25, 2.5, 25.0, or 250 micrograms ovine LH/100 g body wt sc, twice daily for 7 days beginning on the day after hypophysectomy (n = 5/group in three separate experiments). The inhibin subunit mRNA content of each testis was measured for statistical analysis by dot-blot hybridization, and experimental results were confirmed by northern analysis of poly(A) RNA from each sample. In immature animals, the testicular content of both inhibin subunit mRNAs was decreased after hypophysectomy; inhibin alpha and beta-B mRNA levels were decreased to 6.9 +/- 0.9% and 31.7 +/- 2.7% of intact control values, respectively. In adult animals, hypophysectomy resulted in a more modest decrease in inhibin alpha subunit mRNA per testis (44.9 +/- 3.1% of controls) but had no effect on beta-B subunit mRNA. Replacement of LH to immature hypophysectomized animals did not alter levels of mRNA for either inhibin subunit. However, in adults LH restored testicular inhibin alpha subunit mRNA content in testes of hypophysectomized animals to levels seen in intact, saline-treated control animals. LH replacement also slightly but consistently decreased testicular beta-B subunit mRNA content compared to levels seen in hypophysectomized, saline-treated rats. These results indicate that although the response of inhibin subunit mRNAs to pituitary input decreases as a function of sexual maturation, LH may play an important role in regulation of inhibin subunit mRNA expression in adult but not immature testes. The mechanism(s) of LH action on testicular inhibin subunit gene expression is currently unknown, but may involve either direct actions of LH on Leydig cell inhibin subunit mRNAs or indirect actions of interstitial cell factors on Sertoli cell inhibin subunit gene expression.     

Testicular function in experimental uremia

A model for the study of testicular function in experimental uremia induced by subtotal nephrectomy in mature male rats is described. Glomerular filtration rate was reduced by 87%, resulting in stable uremia for up to 13 weeks with characteristic biochemical changes and reduction in weight of testes, prostate, seminal vesicles, and epididymis but not of heart, liver, spleen, or epididymal fat-pads in comparison with pair-fed and sham-operated, ad libitum-fed littermate controls. Basal serum LH, FSH, and testosterone were reduced and PRL levels increased in chronic uremic rats compared with pair-fed and sham-operated controls. Intratesticular and peripheral venous testosterone levels were reduced by 73-85%, whereas spermatic vein testosterone levels were reduced by 47% suggesting a reduction in blood flow to the uremic rat testis. Sensitivity of uremic Leydig cells to human CG stimulation was normal to increased both in vivo and in vitro without changes in LH receptor affinity or numbers. Fertility was markedly impaired in uremic rats but was restored to normal by either human CG or testosterone treatment. Spermatogenesis was minimally depressed after up to 3 months of uremia. These studies suggest that this experimental paradigm is a suitable model for studying the pathogenesis of early changes in uremic hypogonadism. The predominant early changes of uremic hypogonadism are due to central defects in regulation of pituitary LH secretion with consequent testicular and peripheral hypoandrogenism but initial preservation of spermatogenesis.     

Increase in DNA polymerase alpha activity associated with DNA synthesis due to FSH or oestrogen in ovaries of immature rats

DNA polymerase activities and DNA content of ovaries from immature intact rats (4-29 days after birth), hypophysectomized rats and hormone-treated hypophysectomized rats were measured. During normal ovarian growth DNA polymerase alpha activity and DNA content of ovaries increased. The polymerase activity decreased gradually after hypophysectomy without any alteration in the DNA content. Administration of ovine FSH (2 micrograms/day) or oestradiol-17 beta (1 mg/day) to hypophysectomized rats enhanced ovarian DNA content and DNA polymerase alpha activity, whereas DNA polymerase beta activity did not change significantly. These results suggest that DNA polymerase alpha participates in DNA synthesis in these ovaries. The specific activity of DNA polymerase alpha (the activity per microgram DNA) in the ovaries increased between 4 and 14 days after birth, and then remained almost constant; the specific activity declined gradually after hypophysectomy. Administration of FSH or oestradiol-17 beta but not of ovine LH, progesterone or testosterone to hypophysectomized rats restored the specific activity. Mixing experiments with different kinds of ovarian extracts suggested that no activators of DNA polymerase alpha were present in the extracts. These results suggest that FSH or oestrogen causes the induction of DNA polymerase alpha accompanied by DNA synthesis during cell proliferation in ovaries of immature rats.     

Effects of hypophysectomy and subsequent FSH and testosterone treatment on inhibin production by adult rat testes

The hormonal control of inhibin production by adult rat testes was investigated using an in-vitro inhibin bioassay validated for the measurement of inhibin activity in charcoal-treated rat testicular extracts. The effect of hypophysectomy examined at 16 h, 3, 7 and 42 days after surgery showed a decrease in testicular inhibin content and seminiferous tubule fluid production by 7 days and a decrease in inhibin production by 42 days. Serum FSH and LH were suppressed 3 days after surgery. In 30-day chronically hypophysectomized adult rats treated for 3 days with twice daily s.c. injections of (a) human FSH (hFSH, 22 i.u./rat per day), (b) testosterone (5 mg/rat per day), (c) hFSH + testosterone (same doses as a and b), or (d) human chorionic gonadotrophin (hCG, 12 i.u./rat per day), hFSH or hFSH and testosterone stimulated an increase in testicular inhibin content but not in inhibin production or tubule fluid production. Testosterone and hCG had no effect on these parameters. It is concluded that in vivo, FSH alone stimulates an increase in testicular inhibin content. The failure to observe an increase in inhibin production in vivo is attributed to the suppression of seminiferous tubule fluid production under the same experimental conditions.     

Changes in testicular blood flow and testosterone production during aspermatogenesis after irradiation

Exposure of the testes of anaesthetized adult rats to 527 rads of gamma-irradiation caused testis weight to fall slowly at first and then more rapidly from 21 days afterwards, reaching a minimum at 52 days, when spermatogenesis was severely disrupted. The weights of the accessory organs and the concentrations of testosterone in peripheral blood were slightly reduced; the concentrations in blood from the testicular veins were lower than control at shorter intervals after irradiation, but at later times tended to be similar or greater than control. Testicular blood flow per testis followed testis weight closely, and as a result the production of testosterone by the smaller testes (calculated as the product of plasma flow and the veno-arterial difference in testosterone concentration) was markedly reduced especially when the rats had been stimulated with human chorionic gonadotrophin (hCG). Serum FSH and LH rose appreciably as testis weight fell but there was a proportionately greater rise in FSH than LH, in comparison with surgically castrated animals. Increased amounts of extratubular, extracellular fluid were found in the aspermatogenic testes, but injection of hCG still caused increases in capillary permeability and the amount of fluid in the testis. These results indicate that during aspermatogenesis following irradiation (as with heat and efferent duct ligation) the capacity of the testes to secrete testosterone is severely limited by decreased testicular blood flow, not by the ability of the Leydig cells to release testosterone into their immediate environment.     

Regulation of testicular inhibin subunit messenger ribonucleic acid levels in vivo: effects of hypophysectomy and selective follicle-stimulating hormone replacement

To examine the pretranslational regulation of inhibin subunits in the rat testis by FSH, we studied the effects of hypophysectomy with or without selective FSH replacement on testicular inhibin subunit mRNA levels in immature and adult animals. In the first experiment (Exp I), sexually immature (20-23 days old) intact and hypophysectomized male rats were killed 1, 3, and 7 days after surgery, and the testicular content of inhibin subunit mRNAs was determined by filter hybridization. A second group of immature, intact, or hypophysectomized rats was treated with saline or FSH for 7 days as follows: I) intact, saline; II) hypophysectomized, saline; III) hypophysectomized, FSH [0.05 microgram/100 g BW, sc, twice daily (BID)]; IV) hypophysectomized, FSH (0.50 microgram/100 g BW, sc, BID); V) hypophysectomized, FSH (5.0 micrograms/100 g BW, sc, BID); and VI) hypophysectomized, FSH (50.0 micrograms/100 g BW, sc, BID). In the second experiment (Exp II), adult (60 days old) intact or hypophysectomized animals were treated with saline, FSH, and/or testosterone for 7 days as follows: I) intact, saline; II) hypophysectomized; saline; III) hypophysectomized, 22-mm testosterone implant; IV) hypophysectomized, FSH (50.0 micrograms/100 g BW, sc, BID; and V) hypophysectomized, 22-mm testosterone implant plus FSH (50.0 micrograms/100 g BW, sc, BID. The effects of FSH and testosterone on testicular inhibin subunit mRNA levels were measured by filter hybridization. In Exp I, the level of inhibin alpha-subunit mRNA per testis was significantly lower in hypophysectomized rats than in intact controls at all time points after surgery. Replacement of FSH to hypophysectomized immature rats led to a dose-dependent increase in alpha-subunit mRNA per testis. However, hypophysectomy and FSH replacement had no significant effect on beta-B-subunit mRNA. In adult rats (Exp II), hypophysectomy significantly lowered and FSH replacement increased testicular inhibin alpha-subunit mRNA levels. Replacement of testosterone to adult animals, either alone or in combination with FSH, had no effect on expression of inhibin alpha-subunit mRNA. beta-B mRNA levels in adult testis were not significantly altered by any of the treatments. beta-A-Subunit mRNA levels were below the detection threshold of filter hybridization in both Exp I and II. Collectively, these data demonstrate that FSH regulates alpha- but not beta-B-subunit mRNA in the testis of both immature and adult rats in vivo. Differential regulation of inhibin subunits may provide a mechanism for creation and regulation of functional diversity of inhibin-related peptides in the testis.     

Regulation of Sertoli cell transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels during the restoration of spermatogenesis in the adult hypophysectomized rat

The purpose of this study was to determine whether the levels of sulfated glycoprotein-2 (SGP-2) and/or transferrin mRNA in Sertoli cells responds to testosterone in the adult rat testis and, if so, to distinguish between the effects of testosterone and those of changing populations of germ cells. To this end we have examined changes in the steady state levels of these two mRNAs in relationship to changes in intratesticular testosterone concentration and germ cell numbers after treatment of adult hypophysectomized rats with testosterone. Hypophysectomy for 4 weeks caused intratesticular testosterone concentrations to become reduced from 50 to 3 ng/ml and caused significant reductions in the numbers of testicular spermatozoa (undetectable), round spermatids (nearly undetectable), and pachytene spermatocytes (12% of normal). Intratesticular testosterone concentrations rose to 30 ng/ml within 3 days after implantation of testosterone-containing polydimethylsiloxane capsules into the hypophysectomized rats. Three days after the initiation of testosterone treatment, increases were seen in the numbers of round spermatids (10% of normal) and pachytene spermatocytes (29% of normal). The major increases in the numbers of these cells and of spermatozoa occurred between days 14-56 of testosterone treatment, with pachytene spermatocytes reaching a maximum of 75% of the control level by day 28, and round spermatids and spermatozoa reaching 75% and 21% of the control levels, respectively, by 56 days. The steady state levels of SGP-2 mRNA were not affected by hypophysectomy, testosterone administration, or subsequent increases in germ cell numbers. In contrast, transferrin mRNA levels were reduced by hypophysectomy. As found for SGP-2, transferrin mRNA levels were unresponsive to increased testosterone concentration, but, unlike SGP-2 mRNA, transferrin mRNA increased as germ cells were restored after testosterone administration. These results suggest that SGP-2 mRNA is not regulated by testosterone or germ cells, but that the level of transferrin mRNA is influenced by the interaction of Sertoli and germ cells in the adult rat testis.     

Endogenous estrogens inhibit mouse fetal Leydig cell development via estrogen receptor alpha

It is now accepted that estrogens play a role in male fertility and that exposure to exogenous estrogens during fetal/neonatal life can lead to reproductive disorders in the male. However, the estrogen receptor (ER)-mediated processes involved in the regulation of male reproduction during fetal and neonatal development are still largely unclear. We previously reported that ER beta deficiency affects gametogenesis in mice but changes neither the number nor the differentiated functions of fetal Leydig cells. We show here that ER alpha-deficient mice (ER alpha-/-) display higher levels of testicular testosterone secretion than wild-type mice from fetal d 13.5 onwards. This results from higher levels of steroidogenic activity per fetal Leydig cell, as indicated by the hypertrophy of these cells and the higher levels of mRNA for StAR, P450c17 and P450scc in the testis, for a similar number of Leydig cells. Because LH is not produced on fetal d 13.5 and because no change in plasma LH concentration was observed in 2-d-old ER alpha-deficient mice, LH is probably not involved in the effects of estrogens on testicular steroidogenesis in fetal and early neonatal Leydig cells. Furthermore, inactivation of ER beta did not change the effect of ER alpha inactivation on steroidogenesis. Lastly, in an organ culture system, 1 mum diethylstilbestrol decreased the testosterone secretion of wild-type fetal and neonatal testes but not of ER alpha-/- testes. Thus, this study shows that endogenous estrogens physiologically inhibit steroidogenesis via ER alpha by acting directly on the testis early in fetal and neonatal development.     

Nuclear accumulation of androgens in perfused rat accessory sex organs and testes.

The uptake of androgens into the nuclei of caput epididymis, ventral prostate, seminal vesicle and testis was studied by recirculating physiological and pharmacological concentrations of [3H]testosterone in an artificial medium through the lower half (hemicorpus) of castrated or hypophysectomized rats. The accumulation of dihydrotestosterone in accessory sex organ nuclei was saturable, inhibited by perfusion of excess testosterone or cyproterone acetate, and associated with binding to 3S salt-extractable molecules. In castrated preparations the mean saturation levels (pmol/mg DNA) were different in the three organs: seminal vesicle, 2.8; ventral prostate, 1.8; caput epididymis, 0.9. The saturation level was significantly lower in ventral prostate of hypophysectomized rats (1.2) treated with testosterone to regenerate the accessory sex organs. Testosterone was the major nuclear androgen in the testes of mature hypophysectomized preparations perfused with testosterone. Although there was a large amount of nonspecific accumulation, testosterone binding to 3S molecules was shown by sucrose gradient centrifugation. Binding of dihydrotestosterone to 3S molecules in testicular nuclei was also demonstrated. The ratio of dihydrotestosterone to testosterone was different in immature and mature testicular nuclei and was altered by treatments known to affect testicular 5 alpha-reductase activity. The results suggest that in rat accessory sex organs and immature testis the major active androgen is dihydrotestosterone, whereas in mature testis it is testosterone. The shift in the predominant nuclear androgen in the testis from dihydrotestosterone to testosterone is most simply explained by the maturational change in 5 alpha-reductase activity.