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PubMed1994

Cadherins and cadherin-associated molecules in the developing and maturing rat testis.

The calcium-dependent class of cell adhesion molecules known as cadherins mediate homotypic cell interactions in most epithelia. We have now investigated the expression and distribution of cadherins and cadherin-associated molecules in the developing and maturing rat testis. E-Cadherin was not detected in the seminiferous tubule at any time in development or in the adult. In contrast, Leydig cells expressed E-cadherin between day 15 of gestation and postnatal day 3. alpha- and beta-catenins were expressed throughout the developing testis, but were particularly prominent in Leydig cells. In the maturing testis, alpha-catenin and plakoglobin became progressively more restricted to the basal part of the seminiferous epithelium and by 23 days exhibited a pattern characteristic of the Sertoli cell junctional complex. beta-Catenin recruitment to the Sertoli cell junctional complex was not complete until 60 days. alpha-Catenin and plakoglobin were not present at sites of Sertoli cell-germ cell contacts. Northern blot analysis of testicular RNA showed three mRNA species hybridizing with N-cadherin cDNA. A pan-cadherin antibody specific for a region of the highly conserved C-terminal of all cadherins stained sites of Sertoli-spermatocyte and Sertoli-round spermatid contact in the adult rat seminiferous epithelium, but did not stain the Sertoli cell tight junctional complex. Western blots of testicular extracts indicated that the molecule(s) recognized by these antibodies had an approximate molecular mass of 120 kilodalton, typical of members of the cadherin family. Therefore, although Sertoli cells do not express E-cadherin, another member(s) of the cadherin family is present in the testis, but may not be directly involved in tight junction dynamics as in other cells. Instead, cadherin-mediated adhesion is likely to be involved in Sertoli cell-germ cell interactions. As catenins are not present at these sites, our results suggest a catenin-independent role of cadherins in germ cell adhesion to Sertoli cells.

Aging↗

PubMed1996

Induction of lipid peroxidation during steroidogenesis in the rat testis.

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. The cytochrome P450 enzymes of the steroidogenic pathway are known to produce free radicals. The present study was conducted to elucidate in vivo the gonadotropin regulation of free radical-mediated lipid peroxidation and the antioxidative defense system in the rat testis. GnRH antagonist (Org 30276; 1 mg/kg BW) and testosterone [40-mm SILASTIC brand (Dow-Corning) capsules] treatments were used to suppress serum gonadotropin levels. As expected, serum LH decreased to a very low level, whereas serum FSH decreased only slightly. Testosterone treatment for 8 days decreased the levels of the peroxide-metabolizing enzymes, catalase, glutathione peroxidase (GSH-Px), and glutathione transferase (-44%, -24%, and -31%, respectively; P < 0.01 for all). These changes predominately reflect the interstitial tissue, in which catalase and GSH-Px activities were much higher than in the seminiferous tubules. Testicular CuZn or Mn superoxide dismutase activities, which were high in the seminiferous tubules, were not affected by gonadotropin suppression. The total peroxyl radical-trapping capacity of the testis, or its components, vitamin E and ubiquinol 9, were not affected either. Lipid peroxidation was decreased after 8-day treatment, as detected by diminished formation of conjugated dienes and fluorescent chromolipids (-30% and -19%, respectively; P < 0.05 for both). Similar results of decreasing catalase and GSH-Px activities were found after gonadotropin suppression with GnRH antagonist treatment for 2 days or testosterone treatment for 5 days. Substitution with hCG, alone or in combination with recombinant human FSH, reversed the changes in enzyme activities, whereas FSH alone had no effect. After 5-day testosterone treatment, catalase messenger RNA expression was studied by Northern hybridization, and it was observed to parallel the changes in enzyme activity. The site of free radical production was studied by separating interstitial tissue and seminiferous tubules 5 h after hCG injection. GSH-Px was induced by hCG only in the interstitial tissue (+28%; P< 0.01), supporting the hypothesis of free radical production during steroidogenesis. Aminoglutethimide, an inhibitor of the P450 cholesterol side-chain cleavage enzyme, induced extensive lipid peroxidation in the testis. Presumably, aminoglutethimide leads to leakage of free radicals from the P450 enzyme when substrate oxygenation is prevented. In conclusion, the present study suggests that physiological LH action in the rat testis causes lipid peroxidation and maintains high activities of peroxide-metabolizing enzymes in the interstitial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoglutethimide↗

PubMed1996

Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis.

Endogenous TRH-like products were analyzed in rat testis using a TRH enzyme immunoassay coupled to molecular sieve filtration and HPLC identification. Of the three immunoreactive peptides detected, the two major forms exhibited the same chromatographic properties as synthetic TRH and pGlu-Phe-Pro-NH2. These peptides accounted respectively for 33% and 54% of the total TRH immunoreactivity in the testis. In rat serum, HPLC analysis showed the presence of only one immunoreactive peak with a retention time similar to that of authentic TRH. Ethylene dimethanesulfonate treatment of adult rat was used to assess the effect of Leydig cell destruction on the TRH immunoreactivity content. The concentration of the three TRH immunoreactive peptides fell gradually after treatment and reached a minimum at day 21, where a marked decrease (98%) was observed. At day 41, the regeneration of Leydig cells was achieved, as shown by histochemistry and measurements of serum testosterone and testicular weight. However, no restoration of the TRH immunoreactive content was achieved, the three TRH-related peptides being only detectable in trace amount. On the other hand, no significant change in hypothalamic levels of TRH was observed at any treatment time, indicating that hypothalamic TRH biosynthesis was not influenced by testosterone. By using the indirect immunofluorescence technique, TRH immunoreactive cells were found in the interstitial space of the testis. These results suggest that Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis. A paracrine, or autocrine role for these products is suggested.

Animals↗

PubMed1996

All-trans-4-oxo-retinoic acid: a potent inducer of in vivo proliferation of growth-arrested A spermatogonia in the vitamin A-deficient mouse testis.

Vitamin A deficiency leads to an arrest of spermatogenesis and a loss of advanced germ cells in male mice. In the present study, the effects of several retinoids and carotenoids on these mouse testis were investigated. First, the proliferative activity of the growth-arrested A spermatogonia in vitamin A-deficient (VAD) mice testis was determined, 20, 24, or 28 h after administration of 0.5 mg all-trans-retinoic acid (RA). The bromodeoxy-uridine (BrdU) labeling index of A spermatogonia in control VAD testis was 5 +/- 1% (n = 4, mean +/- SD). When RA was injected (ip), the highest labeling index was found 24 h after RA administration; 49 +/- 5%. When various concentrations of RA, all-trans-4-oxo-retinoic acid (4-oxo-RA) or all-trans-retinol acetate (ROAc), ranging from 0.13-1 mg, were injected, the labeling index of A spermatogonia always increased in comparison with the VAD situation. A maximum index at 24 h was found when 0.5 mg 4-oxo-RA was injected: 56 +/- 3%. This labeling index was even higher than those after injection of RA or ROAc, 49 +/- 5% and 34 +/- 6% respectively. The increase of the BrdU labeling index was dose dependent. After an initial increase of the labeling indices with increasing retinoid doses, the labeling indices decreased at a higher concentration. This decrease is likely due to a concentration dependent timeshift of the optimum of BrdU labeling to shorter time intervals after retinoid administration because a labeling index of 66 +/- 1% was found 20 h after injection of 1 mg RA. At 24 h, this labeling index was halved: 33 +/- 2%. These indices show that the degree of synchronization of spermatogenesis is also dependent on the retinoid dose. When the dimers of RA and 4-oxo-RA, respectively beta-carotene (beta C) and canthaxanthin, were given, 24 h after administration BrdU-labeling indices comparable with the VAD value were found. Repeated injection of beta C twice a week did induce a reinitiation of spermatogenesis, but compared with RA, the activity of beta C was lower and delayed. It is concluded that 4-oxo-RA is active in adult mammals in vivo. It is at least as potent as RA in the induction of the differentiation and subsequent proliferation of growth-arrested A spermatogonia in VAD mice testis. Furthermore, the degree of synchronization of spermatogenesis is influenced by the retinoid dose. Finally, carotenoids were shown to act in the induction of spermatogonial cell proliferation too but with a lower and delayed activity.

Animals↗

PubMed1999

Cloning of thyrotropin-releasing hormone precursor and receptor in rat thymus, adrenal gland, and testis.

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Adrenal Glands↗

PubMed1999

Hepatocyte growth factor and c-MET are expressed in rat prepuberal testis.

The hepatocyte growth factor (HGF) receptor (c-MET) is present in different mammalian tissues and transduces multiple biological effects. The HGF is known to regulate many fundamental cellular functions, such as cell growth, movement and differentiation, and is involved in embryonal morphogenesis. We have studied HGF and c-MET expression in prepuberal rat testis. c-MET gene expression was found in total testis and in homogeneous cell populations, as demonstrated by Northern blotting. In the seminiferous tubules, c-MET gene was only expressed in the myoid cells. In these cells, c-MET was detectable and constantly expressed for at least six days of culture. The interstitial tissue was also c-MET positive. The protein encoded by the MET proto-oncogene was detected in myoid cells, and HGF administration to these cells induced morphological changes in the cells. HGF expression was not detected by Northern blotting using RNA extracted from total testis. By contrast, when homogenous cell populations were used, HGF expression was detectable and exclusively localized in myoid cells. Myoid cell-conditioned medium was able to induce scattering of canine kidney epithelial (MDCK) cells, and the scatter effect of a 3-days conditioned medium was evident even after 7-fold dilution of the medium. Our findings demonstrate that HGF and its receptor are present in rat prepuberal testis. The coexpression of factor and receptor in the myoid cells suggests a new role for HGF as autocrine regulator of myoid cell function and, possibly, as regulator of mammalian testicular function.

Animals↗

PubMed2000

Localization of 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase isoform expression in the developing mouse testis--androstenedione is the major androgen secreted by fetal/neonatal leydig cells.

The final step in the biosynthesis of testosterone is reduction of androstenedione by the enzyme 17beta-hydroxysteroid dehydrogenase/ 17-ketosteroid reductase (17betaHSD/17KSR). In this study, we have examined expression of the four known reductive isoforms of 17betaHSD/ 17KSR (types 1, 3, 5, and 7) in the developing mouse testis and have determined changes in the localization of isoform expression and testosterone secretion during development. Using RT-PCR isoforms 1, 3, and 7 were shown to be expressed in the seminiferous tubules of neonatal testis, whereas isoforms 3 and 7 were expressed in the interstitial tissue of the adult testis. The type 7 isoform is unlikely to be involved in androgen synthesis and further study concentrated on the type 3 isoform. Developmentally, isoform type 3 was expressed in the seminiferous tubules up to day 10, showed little or no expression on day 20 and from day 30 was confined to the interstitial tissue. In situ hybridization confirmed that the type 3 isoform was expressed only in the seminiferous tubules in fetal testes and in the interstitial tissue in adult testes. In accordance with the localization of enzyme messenger RNA expression 17-ketosteroid reductase enzyme activity was very low in isolated interstitial tissue from neonatal testes while interstitial tissue from adult testes showed high activity. Seminiferous tubules from both neonatal and adult testes showed high levels of enzyme activity. The major androgen secreted by the interstitial tissue of prepubertal animals was androstenedione up to day 20 while 5alpha-androstanediol and/or testosterone were the major androgens secreted from day 30 onwards. These results show that fetal Leydig cells do not express significant levels of a reductive isoform of 17betaHSD/ 17KSR and that androstenedione is the major androgen secreted by these cells. Production of testosterone up until puberty is dependent upon 17betaHSD/17KSR activity in the seminiferous tubules--a "two cell" requirement for testosterone synthesis. Expression of the 17betaHSD/17KSR type 3 isoform (the main reductive isoform in the testis) declines in the seminiferous tubules before puberty but then reappears in the developing adult Leydig cell population.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Maternal exposure to octylphenol suppresses ovine fetal follicle-stimulating hormone secretion, testis size, and sertoli cell number.

We have tested the hypothesis that maternal exposure to octylphenol, a putative endocrine disrupting chemical, will suppress gonadotropin secretion with a concomitant decrease in testis size and Sertoli cell number during fetal life in the lamb. In Exp 1, pregnant ewes received a continuous iv infusion of diethylstilbestrol (DES; 50 microg/kg x day), octylphenol (1000 microg/kg x day), or vehicle (1:4, alcohol-saline) from days 110-115 of gestation. The fetuses were chronically catheterized in utero, and blood samples were collected every 8 h to monitor gonadotropin secretion. In Exp 2, pregnant ewes received twice weekly sc injections of DES (0.5 microg/kg x day), octylphenol (1000 microg/kg x day), or corn oil from day 70 of gestation to birth. The pituitary gland and testes were collected from the lambs at the end of the treatment period. In Exp 1, maternal exposure to octylphenol suppressed (P < 0.05) FSH concentrations without any effect (P > 0.05) on LH concentrations compared with those in control fetuses. In Exp 2, long-term maternal exposure to octylphenol or a 1000-fold lower dose of DES suppressed (P < 0.05) FSH, messenger RNA levels and the number of FSHbeta-immunopositive cells in the pituitary gland and reduced testis weight and the number of Sertoli cells in the testis compared with those in control lambs. We conclude that maternal exposure to octylphenol inhibits the secretion of FSH in the fetus with a concomitant decrease in testis size and Sertoli cell number at birth.

Animals↗

PubMed2002

The goitrogen 6-n-propyl-2-thiouracil (PTU) given during testis development increases Sertoli and germ cell numbers per cyst in fish: the tilapia (Oreochromis niloticus) model.

The main objectives of the present study were to investigate the effects of 6-n-propyl-2-thiouracil (PTU) on Sertoli cell proliferation, germ cell number, and testis size in Nile tilapias (Oreochromis niloticus). In this regard, young fish (approximately 1 g BW and approximately 3.5 cm total in length) were treated for a period of 40 d with different concentrations (100 and 150 ppm) of PTU. The animals were killed and analyzed on d 1, 30, 40, 98, and 208 after the beginning of the treatment. On d 30 and 40 the spermatogenic process was delayed in fish treated with PTU compared with the control group. Also at these periods, treated tilapia had decreased (P < 0.05) body weight and total length. On d 98 body weight and total length had recovered in PTU-treated fish and were similar (P > 0.05) to those of the controls. However, testis weight and gonadosomatic index (testis mass/body weight) were approximately 100% higher (P < 0.05) in treated tilapia. Similarly, the area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells per testis were significantly (P < 0.05) greater in treated fish. Nevertheless, nuclear volume and individual Leydig cell volume were significantly lower (P < 0.05) in tilapia receiving PTU treatment. Compared with controls, at 208 d all parameters analyzed presented the same trend as that observed at 98 d. In general, at 98 d the different PTU concentrations used during the treatment period induced similar effects. However, at 208 d the mean values observed for several parameters were significantly higher (P < 0.05) in fish exposed to 150 ppm. Probably due to the higher density of Sertoli cells per cyst in treated tilapia, these cells presented a smaller (P < 0.05) nucleolus and a trend to decrease its support capacity (efficiency). However, the meiotic index (germ cell loss during the two meiotic divisions) was similar (P > 0.05) in the three groups of fish investigated. Remarkably, the results found in tilapia were similar to those found for rats treated with PTU. This suggests strongly that the mechanisms of control of Sertoli cell and Leydig cell proliferation seem to be preserved during vertebrate evolution.

Animals↗

PubMed2004

Coexistence of intracellular and membrane-bound progesterone receptors in human testis.

Progesterone and progesterone receptors (PR) play a crucial role in female reproduction, but their roles in male reproductive physiology are largely unknown. Our previous studies demonstrated the presence of a specific membrane-bound PR in mature human spermatozoa that is known to regulate important sperm functions. The present study was undertaken to determine whether there exist PR in human testis and to investigate their molecular characteristics and expression profiles. PR mRNA and protein were detected in the spermatogenic cells, Sertoli cells, and occasionally the Leydig cells. PR protein was localized in nucleus and cytoplasm of spermatogonia, primary and secondary spermatocytes, and round spermatids in a stage-specific manner. Intense PR localization was observed in stages IV and V, whereas it was low at stages I, II, and III of spermatogenesis. RT-PCR studies revealed the presence of transcripts for PR in human testis and spermatogenic cells. In accordance with the reported molecular sizes of the known isoforms of PR, two mRNA transcripts of 3.8 and 2.8 kb for PR in adult human testis and spermatogenic cell RNA were detected by Northern blot hybridization. Western blot analysis of testicular and spermatogenic cell lysates revealed two bands of 120 and 90 kDa, corresponding to the conventional PR. In these tissue lysates, an additional band of approximately 55 kDa was detected that was also observed as a single band in sperm lysates, indicating that this smaller protein may correspond to the membrane-bound PR. The membrane-bound PR protein was demonstrated on the spermatogenic cells when probed with progesterone-bound fluorescein conjugate. The results of the present study demonstrate the existence of both intracellular PR-B and PR-A mRNA and protein in the spermatogenic cells of the human testis. A membrane-bound PR was also localized in these cells. The varying levels of intracellular PR during different stages of spermatogenesis and the presence of the membrane-bound PR imply the significance of progesterone in male reproductive events such as regulation of spermatogenesis.

Adult↗

PubMed2006

Testicular adrenal rest tumors in adult males with congenital adrenal hyperplasia: evaluation of pituitary-gonadal function before and after successful testis-sparing surgery in eight patients.

CONTEXT: In male patients with congenital adrenal hyperplasia (CAH), testicular adrenal rest tumors (TART) are frequently present. These tumors can interfere with testicular function. Intensifying glucocorticoid therapy does not always lead to tumor regression and improvement of testicular function. Recently, testis-sparing surgery was introduced for treatment of TART. OBJECTIVE: The aim of this study was to evaluate tumor volume, symptoms, and pituitary-gonadal function in male patients with CAH caused by 21-hydroxylase deficiency and bilateral TART before and after testis-sparing surgery. SETTING: This study was conducted at Radboud University Nijmegen Medical Centre in The Netherlands. PATIENTS: Eight adult male CAH patients with bilateral TART and infertility were included. INTERVENTIONS: Evaluation of testicular magnetic resonance imaging, symptoms, fasting serum concentrations of ACTH, LH, FSH, inhibin B, 17-OH progesterone, androstenedione, testosterone, and estrone, and semen analysis (six of eight patients) was performed before and 6 and 22 months after testis-sparing surgery. MAIN OUTCOME MEASURES: The main outcome measures were absence of residual tumor and improvement of symptoms and pituitary-gonadal function. RESULTS: Residual tumors were not found on any of the patients' magnetic resonance imaging after surgery. Two patients reported testicular pain and discomfort that disappeared after surgery. Parameters of pituitary-gonadal function did not improve after surgery: semen analysis showed azoospermia (five patients) or oligospermia (one patient) without improvement, and all patients had persistently low inhibin B concentrations. CONCLUSION: Testis-sparing surgery did not improve pituitary-gonadal function despite successful removal of the tumors. Further studies are needed to investigate whether surgery at an earlier stage in the natural history of TART can prevent permanent testicular damage.

17-alpha-Hydroxyprogesterone↗

PubMed1975

Studies on the human testis. VI. NADH-linked reactions of microsomal steroid 20alpha-and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase.

NADH-linked 20alpha- and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase activities were demonstrated in the microsomal fraction of the human testis. The microsomal 20alpha-hydroxysteroid dehydrogenase showed substrate affinity to pregnenolone and progesterone and not to 17alpha-hydroxyprogesterone and preferred NADH to NADPH as a hydrogen donor. In the presence of NADH, the optimal pH for the enzyme was 7.7 and the apparent Michaelis constants of the enzyme for progesterone and pregnenolone at 37 C and pH 7.4 were 6.9-7.1 X 10-6M and in the order of 10-5M, respectively, 17alpha, 20beta-Dihydroxypregn-4-en-3-one was the only significant metabolite produced from 17alpha-hydroxyprogesterone by microsomal fraction of the human testis in the presence of NADH. The apparent Michaelis constant of microsomal 20beta-hydroxysteroid dehydrogenase for 17alpha-hydroxyprogesterone in the presence of NADH was in the order of 10-5M at 37 C and pH 7.4. The microsomal 17alpha-hydroxylase catalyzed the metabolism of pregnenolone and progesterone at a similar rate in the presence of NADH. The optimal pH and the apparent Michaelis constant at 37 C and pH 7.4 of the NADH-linked reaction of 17alpha-hydroxylase for progesterone were 7.7 and 5.3-5.4 X 10-7M, resepctively. The NADH-linked enzyme activity for progesterone was competitively inhibited by both pregn-5-ene-3beta, 20alpha-diol (inhibition constant: 1.7 X 10-7M) and 20alpha-hydroxypregn-4-en-3 one (inhibition constant: 6.6 X 10-7M), and was resistant to poor oxygen supply during incubation. The results indicate that the microsomal 20alpha-hydroxysteroid dehydrogenase is a different enzyme from the one in the soluble fraction of the human testis and that microsomal 17alpha-hydroxylase in the human testis is activated by NADH as well as NADPH.

Binding, Competitive↗

PubMed1989

Testosterone synthesis and adenylate cyclase activity in the early human fetal testis appear to be independent of human chorionic gonadotropin control.

Androgen secretion by the fetal testis is essential for male phenotypic differentiation. In the human fetus testosterone formation is initiated soon after the differentiation of the testis (approximately 8 weeks of gestation), and the maximal testosterone content in fetal testes is achieved between 10 and 15 weeks of fetal life. The testosterone content of the fetal testis declines at the beginning of the third trimester and remains low until after birth. In an effort to understand the regulation of the onset of testosterone formation in the human fetal testis we measured adenylate cyclase activity in response to hCG stimulation in homogenates of fetal testes obtained from first and second trimester human abortuses. Basal adenylate cyclase activity was 50 pmol/mg protein.min at 10 weeks gestation, the peak activity was 137 pmol/mg protein.min at 12 weeks gestation, and activity declined thereafter to 8 pmol/mg protein.min by 16 weeks gestation. NaF-stimulated (0.6 mmol/L) and forskolin-stimulated (50 mumol/L) activities were 4- to 8-fold greater than basal adenylate cyclase activities. The maximal forskolin-stimulated activity occurred at 11 weeks (803 pmol/mg protein.min), and it fell to 35 pmol/mg protein.min by 17 weeks gestation. In contrast, hCG-stimulated (1 mumol/L) adenylate cyclase activity was only slightly greater than basal rates at all ages examined. In addition, hCG did not stimulate baseline testosterone formation in minces of testes obtained between 12 and 18 weeks of gestation. These findings suggest that the onset of testosterone formation in human fetal testes may be independent of gonadotropin control.

Adenylyl Cyclases↗

PubMed2003

No circadian rhythms in testis: Period1 expression is clock independent and developmentally regulated in the mouse.

Spermatogenesis is a process whereby haploid spermatozoa differentiate through meiosis from precursor stem cells. We examined the expression of circadian clock genes in the testis, to assess clock control over the timing of different developmental events. Clock genes are known to oscillate with circadian rhythmicity in the central clock structure, the suprachiasmatic nucleus of the hypothalamus, but also in peripheral tissues. Here we show that Per1 gene expression in the testis is constant over a 24-h period and that the Per1 transcript is expressed at a level higher than the peak values of the Per1 oscillations observed for other tissues. Bmal1, another clock gene whose expression oscillates in other tissues, also shows constant expression levels in the testis. In addition, the levels and phosphorylation state of the PER1 protein are not oscillating at all times of day. Strikingly, Per1 is restricted primarily to step 7 to 10 spermatids and thus appears to be developmentally regulated. The expression of the Clock transcript is also developmentally regulated, but it is found principally in spermatogonia and spermatocytes up until the time of the first meiotic division. Per1 expression is not altered in testes from Clock mutant mice, suggesting that CLOCK does not activate Per1 in male germ cells, in contrast to what it does in other mouse tissues. Taken together, our observations suggest that the testis, in contrast to all other peripheral tissues, lacks a functioning circadian clock.

ARNTL Transcription Factors↗

PubMed2003

Pem homeobox gene promoter sequences that direct transcription in a Sertoli cell-specific, stage-specific, and androgen-dependent manner in the testis in vivo.

Although many genes are expressed selectively in Sertoli cells, regulatory sequences sufficient to drive Sertoli cell-specific expression in the postnatal and adult testis in vivo have not been identified. In the present study, we identified promoter sequences from the Pem homeobox gene that direct Sertoli cell-specific expression in an androgen-dependent and stage-specific manner. Immunohistochemical and RNA analysis demonstrated that 0.6-kb 5'-flanking sequence directed transgene expression specifically in the testis and the epididymis but not in any other tissues tested. In the adult testis, this promoter fragment targeted the transgene expression specifically to Sertoli cells during stages IV-VIII of the seminiferous epithelial cycle, thereby mimicking the expression pattern of the endogenous Pem gene. This promoter fragment also recapitulated Pem's normal postnatal expression pattern, as it directed transcript induction between d 6 and d 9 post partum. Deletion of 0.3 kb from the 5'-end of the transgene had no effect on androgen-dependent Sertoli-specific expression but altered stage-specific expression in adult testes and caused premature postnatal expression. Our results suggest that there are at least two regulatory regions in the Pem proximal promoter: one that directs androgen receptor-dependent expression specifically in Sertoli cells within the testis and another that confers stage-specific expression in neonates and adults by acting as a negative regulator. To our knowledge, this is the first identification of regulatory regions that direct faithful developmentally regulated gene expression in postnatal and adult Sertoli cells in vivo.

5' Flanking Region↗

PubMed2000

Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.

Aged↗

PubMed1988

Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX----XY chimaeric mouse testes.

The distribution of XX and XY cells in XX----XY chimaeric mouse testes was analysed by enzyme marker analysis of separated testicular tissues and by in situ DNA marker analysis of air-dried testicular cells and testis sections. XX cells contributed to the Leydig cells, the peritubular cells and the vascularized connective tissue of the tunica albuginea. The Sertoli cells, on the other hand, appeared to be exclusively XY. These results indicate that during the development of the testis, Sertoli cell differentiation is triggered by cell-autonomous activity of the Y chromosomal testis-determining gene Tdy. Subsequent steps in testis differentiation may be a consequence of Sertoli cell activity.

Animals↗

PubMed2003

Quantitative changes in yolk protein and other components in the ovary and testis of the sea urchin Pseudocentrotus depressus.

Both male and female sea urchins accumulate the major yolk protein (MYP; the most abundant yolk granule protein in sea urchin eggs) in the nutritive phagocytes of immature gonads before gametogenesis. In this study, quantitative changes in MYP as well as in other biochemical components in the ovary and testis were examined in the course of gametogenesis in Pseudocentrotus depressus. Before gametogenesis, both the ovary and testis contained large quantities of proteins, lipids and polysaccharides. MYP reached about 80% of total protein in both sexes. In the testis, MYP decreased rapidly as spermatogenesis proceeded, and the fully mature testis contained little MYP; the levels of lipids and polysaccharides also decreased. In contrast, the levels of nucleic acids and proteins other than MYP increased markedly. In the ovary, MYP decreased gradually as oogenesis proceeded, and the fully mature ovary contained less than half of the initial amount of MYP. Polysaccharides also decreased, whereas proteins other than MYP increased. These results, taken together with those from other studies, suggest that MYP serves as a protein reserve that accumulates before gametogenesis and is used as material for synthesizing new substances constituting gametes in both male and female sea urchins.

Animals↗