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Seminoma and embryonal carcinoma (EC), two typical types of testicular germ cell tumors (TGCTs), present significant differences in growth behavior, expression characteristics, differentiation potential, clinical features, therapy, and prognosis. The purpose of this study was to compare the distinctive or preference metabolic pathways between seminoma and EC. The Cancer Genome Atlas revealed that many genes encoding metabolic enzymes could distinguish between seminoma and EC. Using well-characterized cell line models for seminoma (Tcam-2 cells) and EC (NT2 cells), we characterized their metabolite profiles using ultraperformance liquid chromatography coupled to Q-TOF mass spectrometry (UPLC/Q-TOF MS). In general, the integrated results from transcriptome and metabolite profiling revealed that seminoma and EC exhibited distinctive characteristics in the metabolisms of amino acids, glucose, fatty acids, sphingolipids, nucleotides, and drugs. Notably, an attenuation of citric acid cycle/mitochondrial oxidative phosphorylation and sphingolipid biosynthesis as well as an increase in arachidonic acid metabolism and (very) long-chain fatty acid abundance occurred in seminoma as compared with EC. Our study suggests histologic subtype-dependent metabolic reprogramming in TGCTs and will lead to a better understanding of the metabolic signatures and biology of TGCT subtypes.

Routine semen parameters have limited clinical diagnostic value for predicting male infertility. The aim of this study was to investigate the association between sperm DNA fragmentation index (DFI) and semen quality, and between DFI and clinical pregnancy rate of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

ABSTRACTMammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.

ABSTRACTIncreases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pHi) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pHiwith advancing age in CD1 mice. Furthermore, during the in vitrooocyte maturation process, the pHiwas maintained at a high level, up to ∼7.6, in 12-month-old mice. Normal pHiis necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pHimight be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitroalkalinization treatment of young oocytes, we observed that the increased pHicaused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pHiexhibited substantially the increase in chromosome misalignment.

Three new steroidal saponins, parisyunnanosides G–I (1–3), one new C21steroidal glycoside, parisyunnanoside J (4), and three known compounds, padelaoside B (5), pinnatasterone (6), and 20-hydroxyecdyson (7), were isolated from the rhizomes of Paris polyphyllaSmith var. yunnanensis. Compounds 1and 3have unique trisdesmoside structures that include a C-21 β-d-galactopyranose moiety. All compounds were evaluated for their cytotoxicity against human CCRF leukemia cells.

Abstract: We have analyzed a possible role of mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) in the regulation of FSH-induced tissue type plasminogen activator (tPA) production in granulosa cells (GCs) prepared from DES-treated immature rats; Treatment of the cells in the presence of FSH with MAPK inhibitors, such as UO126 or SB203580, significantly decreased the FSH-induced tPA production, suggesting that multiple signaling pathways may be involved in FSH-regulated tPA expression. We further examined possible signaling action involved in FSH-activated ERK1/2 and p38 MAPK on tPA production, and observed that FSH receptor occupancy led to both ERK1/2 and p38 MAPK phosphorylation. Such action might be through a protein kinase A-dependent pathway because the observed activation was destroyed by the addition of its specific inhibitor H89 to the culture. The inhibition of ERK1/2 and p38 MAPK activation by their specific inhibitors remarkably reduced FSH-induced tPA mRNA and its protein production. We further examined whether AP-1 located in the tPA promoter is involved in FSH-regulated tPA production, and demonstrated that FSH significantly stimulated AP-1 expression, whereas inclusion of H89, UO126, or SB20358 in the culture significantly decreased FSH-induced AP-1 expression. In summary, FSH-induced ERK1/2 and p38 MAPK activation is capable of regulating tPA production in cultured primary GCs, and that the transcript factor AP-1 may be important in the regulation of FSH-induced tPA expression.

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (Te) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (He) alone group and by Day 8 in the Tealone group but peaked at Day 3 in He+ Tegroup. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P< 0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling..

Abstract: Our previous studies have demonstrated that tissue type plasminogen activator (tPA) might be involved in matrix degradation of blood-testis barrier in rat. In this study, we have further investigated the effect of testosterone (T) on tPA production in rat Sertoli cells. Our results showed that Sertoli cells isolated from rat testes at various ages in vitro secreted tPA in an age-dependent manner. The tPA activity was detected on day 20 after birth, and reached maximum on day 60. The Sertoli cells isolated from the testes on day 20 were then cultured in the presence or absence of testosterone, FSH, and forskolin, the tPA activities were upregulated by T, FSH and forskolin. Addition of H89 or U0126, both inhibited the testosterone-, FSH-, and forskolin-induced tPA expression. It is suggested that FSH- and testosterone-stimulated tPA expression in Sertoli cells may be via PKA and ERK signal transduction. Furthermore, we have observed that testosterone stimulated tPA secretion at all the stages of spermatogenesis (II–VI, VII–VIII, IX–XII and XIII–I), the highest stimulation of tPA activity was observed at stages VII–VIII. This study further suggests that testosterone-induced tPA activity in the Sertoli cells might be related to the function of blood-testis barrier opening and/or closing.

Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43°C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.

In the present study, we started out to test whether the follicle-stimulating hormone (FSH)-activated p38 MAPK signaling cascade was involved in the regulation of steroidogenesis in granulosa cells (GCs). GCs were prepared from the ovaries of DES-treated immature rats and cultured in serum-free medium. Treatment of GCs with FSH (50 ng/ml) induced the phosphorylation of p38 MAPK rapidly with the phosphorylation being observed within 5 min and reaching the highest level at 30 min. Such activation was protein kinase A-dependent as indicated by the results using specific inhibitors. FSH stimulated the production of progesterone and estradiol as well as the expression of the steroidogenic acute regulatory protein (StAR) in a time-dependent manner, with a maximum level being observed in the production of progesterone and StAR at 48 h. Moreover, the potent p38 MAPK inhibitor SB203580 (20 μM) augmented FSH-induced progesterone and StAR production, while reduced FSH-induced estradiol production at the same time (P<0.01). RT-PCR data showed that inclusion of SB203580 in the media enhanced FSH-stimulated StAR mRNA production, while decreased the FSH-stimulated P450arom mRNA expression (P<0.05). Immunocytochemical studies showed that FSH treatment together with the inhibition of p38 MAPK activity resulted in a higher expression of StAR in mitochondria than FSH treatment alone. FSH also significantly up-regulated the protein level of LRH-1, a member of the orphan receptor family that activates the expression of P450arom in ovaries and testes. p38 MAPK inactivation down-regulated the basal and FSH-induced LRH-1 expression significantly. The intra-cellular level of DAX-1, another orphan receptor that inhibits StAR expression, also decreased upon p38 MAPK being inactivated. For the first time, the present study suggests that FSH-activated p38 MAPK signal pathway regulates progesterone and estrogen production in GCs differentially, and that the transcription factors LRH-1 and DAX-1 might play important roles in the process.

Successful embryo implantation involves complex interactions between the embryo and the uterus and is critical in establishing pregnancy. Proprotein convertase (PC) 6 (PC6) is one of the PC endoproteases regulating protein function through posttranslational activation of precursor proteins, including growth and differentiation factors. Here we show that PC6 protein is induced in the uterine stromal cells specifically at the site of embryo attachment during early pregnancy in mice. In vivo blocking of uterine production of PC6 protein using morpholino antisense oligonucleotides in mice resulted in total inhibition of implantation, revealing a vital role for PC6 in modulating the uterus for embryo implantation. Studies in primates (rhesus monkey and human) showed a dramatic upregulation of endometrial PC6 during the phase of uterine receptivity and at implantation, particularly during a critical uterine cell differentiation process termed decidualization. Thus, the current studies have demonstrated that PC6 is an essential molecule in modulating uterine function to support the establishment of embryo implantation. Interestingly, PC6 is one of the PCs identified to be important in processing the coat protein of HIV; inhibition of PCs has been suggested to be an effective approach to reduce HIV transmission. We therefore propose the novel concept that PC6 could be a potential nonhormonal target in the female reproductive tract for dual protection for women, both in preventing pregnancy and reducing HIV infection.

The present review is a summary of mechanisms of spermatogenesis in primates with emphasis on anti-spermatogenesis of testosterone (T), gossypol, and “testicular heat stress” for development of male contraception. Both FSH and testosterone stimulate all phases of spermatogenesis. FSH is capable of amplifying the population of the differentiated spermatogonia (B1, B2, B3 and B4) and controls the spermatogonia production rate, and, in synergy with testosterone, regulating spermatogenesis in adult monkeys. Pituitary FSH beta gene expression is governed by a feedback of Beta inhibin, which is a major component of the testicular negative feedback signals. Beta inhibin secreted by Sertoli cells is in turn inhibited by testosterone from Leydig cells under the control of LH. Disturbance of the normal interaction of pituitary FSH with Sertoli cell Beta inhibin is responsible for azoospermia or oligozoospermia induced by exogenous T. Three possible regimens of T, gossypol and “heat stress” have been suggested for male contraception. They act on different sites and stages of spermatogenesis in testis or sperm activity in epididymis. Apoptosis induced by testosterone occurs mainly at stages VII–VIII of spermatogenesis while that by testicular “heat stress” mostly occurs at stages I–IV and X–XII. Low dose of gossypol mainly influences the sperm activity in the epididymis although it also acts on testicular spermatids.

Abstract: We have examined the expression of MMP-2, TIMP-1, and TIMP-3 mRNA at the implantation site of rhesus monkeys during early stage of pregnancy using in situ hybridization and Northern blot analysis. The results indicate that MMP-2 mRNA was mainly localized in the chorinoic villi and epithelial plaque, suggesting that MMP-2 may be involved in the process of epithelial plaque and trophoblast invasion. TIMP-3 was specifically expressed in the cells around the spiral arteries and maternal-fetal interface. Therefore, TIMP-3 may be the main inhibitor that restricts the trophoblast invasion. The TIMP-1 mRNA was detected in trophoblast villi and maternal decidua; however, its distribution was not cell-specific, suggesting a general role in the protection of trophoblast villi and maternal decidua from proteolysis by the MMPs secreted by themselves.

Abstract: We investigated the possible role of Hsp70-2 in germ cell apoptosis induced by heat stress in monkey unilateral cryptorchid testis. The study focused on in situ analysis of the testicular cell DNA fragmentation and on the possible relationship between Hsp70-2 expression and germ cell apoptosis. The TUNEL result showed that most of the germ cells were labeled in the cryptorchid testis on d 5 after induction of cryptorchidism; that with most of the apoptotic germ cells depleted, only a few germ cells were labeled on d 10; and that almost no apoptotic signal was observed in the cryptorchid testis on d 15 and thereafter. This indicates that the increasing germ cell degeneration in cryptorchid testis may take the form of apoptosis. Using in situ hybridization, immunohistochemistry, and Northern blot, we examined the changes of Hsp70-2 expression in the monkey cryptorchid testis. The level of Hsp70-2 mRNA decreased slightly, while the expression of HSP70-2 protein was almost unchanged at the early stage of germ cell apoptosis in the cryptorchid testis on d 5 and dropped dramatically along with the loss of apoptotic germ cells in the cryptorchid testis on d 10 after operation. It is therefore suggested that Hsp70-2 might not take part in inhibiting the apoptosis of germ cells at the early stage during operation-induced cryptorchid testis, and that Hsp70-2 gene does not belong to the immediate early related gene responsible for germ cell apoptosis induced by heat stress.

Whereas the linkage of infertility to cryptorchidism, the failure of the testis to descend into the scrotum at birth, has been well documented, the detailed molecular mechanism remains unclear. Here we report that the testicular orphan receptor-2 (TR2) expression, which modulates many signal pathways, was completely repressed in the surgery-induced cryptorchidism of the rhesus monkey. Further studies link TR2 repression to the induction of p53 and results suggest that induced p53 could repress TR2 expression via the p53→p21→CDK→Rb→E2F signal pathway. In return, TR2 could also control the expression of p53 and Rb through the regulation of human papillomavirus 16 E6/E7 genes. Together, our data suggest a feedback control mechanism between TR2 and p53/Rb tumor suppressors, which might play important roles in male infertility associated with cryptorchidism.

The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type‐1 (PAI‐1) and type‐2 (PAI‐2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI‐1 and PAI‐2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI‐1 and PAI‐2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI‐2 was noted in villous trophoblast whereas tPA and PAI‐1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI‐1 and PAI‐2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI‐1 and PAI‐2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI‐1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI‐2 appears mainly to play a role in degradation of trophoblast cell‐associated extracellular matrix, and thus may be of greatest importance during early stages of placentation.

Expression of both mRNA and protein of the steroidogenic acute regulatory protein (StAR), in correlation with progesterone (P) production and LH receptor (LHR) mRNA expression, was studied in the corpora lutea (CL) of gonadotropin-induced-pseudopregnant and pregnant rats at various stages of CL development. Immature female rats, 21–22 days old, were injected s.c. with 20 IU eCG to stimulate follicle growth and then with 20 IU hCG 48 h later to induce ovulation. The ovaries were removed at various stages of CL development; either CL were isolated and snap frozen for total RNA analysis, or whole ovaries were fixed in Bouin's fluid for paraffin sectioning. The results of in situ hybridization, immunohistochemistry, and Northern blotting showed that the increase in StAR mRNA and protein expression was well correlated with the increase in serum P concentration. StAR expression was restricted to the luteal cells or theca cells in antral follicles. Both StAR mRNA and protein in the CL of pseudopregnant rats increased steadily on Day 1 and Day 4, reached highest levels on Day 4, and then dropped sharply on Day 8 when luteolysis takes place. LHR mRNA content was high on Day 1 but dropped significantly on Day 2. LHR mRNA increased to high levels on Day 4 and 8 and then declined on Day 12. StAR mRNA and protein levels in the CL of pregnant rats were high during early luteal development (Day 2, 4), increased even further on Day 9, and decreased on Day 13 when luteolysis takes place. It is therefore suggested that the expression of StAR coincides well with the capacity of P production in the CL and that StAR expression can be used as a functional “marker” of CL development.To study the possible effect of cytokines on StAR expression, pseudopregnant rats on Day 5 were injected s.c. with 10 IU hCG plus 20 μg prolactin (PRL), with or without 500 IU tumor necrosis factor α (TNFα) 30 min later. TNFα significantly inhibited hCG/PRL-induced StAR and LHR mRNA expression at 1 and 3 h post-TNFα. It is suggested that the luteolytic effect of TNFα may be mediated by its direct inhibition on StAR expression or by an indirect decrease in LHR expression.

In mammalian ovaries, luteinizing hormone (LH) induces androgen biosynthesis by theca interna cells, whereas both follicle-stimulating hormone (FSH) and LH stimulate aromatase activity by granulosa cells. The joint action of these two types of cell and pituitary hormones forms the basis of a 2-cell, 2-gonadotropin hypothesis for biosynthesis of estrogen. We tested the synergism between these cell types using antisera against the estrogen precursors progesterone and testosterone. Ovaries were obtained from immature rats two days after a single injection of pregnant mare serum gonadotropin (PMSG). Granulosa cells were obtained by puncturing the follicles, and ovarian remnants were dissociated with collagenase to derive cells from the theca interstitium. Granulosa and theca interstitial cells were cultured alone or in combination for 20 h. Granulosa cells secreted negligible amounts of estrogen and testosterone, but contained high aromatase activity. Treatment with both FSH and human chorionic gonadotropin (hCG) increased production of progesterone in granulosa cells. In contrast, theca interstitial cells had negligible aromatase activity. Treatment with hCG, but not FSH, increased androgen production by theca interstitium; the hCG action was further augmented by the inclusion of exogenous progesterone. Furthermore, co-culture of gonadotropin-treated granulosa and theca interstitial cells resulted in substantial increases in estrogen production, indicating synergism between the two types of cell. The increases in estrogen production in the co-cultures were accompanied by decreases in progesterone content. To test the possibility that progesterone released by granulosa cells may serve as a substrate for production of androgen in theca cells, specific antiserum was used to adsorb progesterone present in the medium. Addition of the progesterone antibody inhibited gonadotropin-stimulated production of testosterone (39% decrease) and estrogen (64% decrease) by the combined cell cultures. The inhibitory effect on estrogen production could be reversed by the addition of progesterone (10−6M) or testosterone (10−6M) but not by the addition of a synthetic progestin, R5020. Since testosterone released from theca cells may serve as the substrate for aromatases in granulosa cells, specific testosterone antiserum was also used. Production of estrogens by the combined cell cultures was inhibited (78%) by the testosterone antiserum, but the inhibitory effect was completely reversed by exogenous testosterone or progesterone. Thus, synergistic interactions between granulosa and theca interstitial cells are important in effecting maximal estrogen biosynthesis. Progesterone produced by granulosa cells is utilized by the theca interstitium to form androgens that are further aromatized into estrogens by granulosa cells.

A hormone response element, CNTFRα-NBRE (5′-AAAGGTCA-3′) has been identified in the fifth intron of the α component of ciliary neurotrophic factor receptor gene (CNTFR-I5) for the human TR3 orphan receptor (TR3). A specific binding between in vitro expressed TR3 and CNTFRα-NBRE was demonstrated by using electrophoretic mobility shift assay. A reporter gene assay using chloramphenicol acetyltransferase (CAT) showed that CNTFR-I5 has an enhancer activity that could be induced by TR3 in a dose-dependent manner. This induction was significantly reduced in the absence of CNTFRα-NBRE. Together, these results indicate CNTFRα-NBRE is sufficient to mediate TR3 action in inducing the enhancer activity of CNTFR-I5. Our finding may, therefore, suggest CNTFRα is a target gene regulated by TR3 and expand the role of TR3 in the nervous system.

The present study was designed to investigate the effect of prolactin (PRL) on plasminogen activator inhibitor-I (PAI-I) and tissue type plasminogen activator (tPA) gene expression in eCG-primed granulosa cells in vitro. At 46 h after the hormone treatment, ovaries were removed, and granulosa cells were prepared for culture. Cells were incubated for various times in serum-free medium in the presence or absence of LH and PRL alone or in combination. tPA and PAI-I activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA from granulosa cells was prepared using the NP-40 method and was assayed for PAI-I and tPA mRNA levels. We demonstrated the following. 1) PRL increased PAI-I mRNA production in cultured granulosa cells. Inclusion of LH with PRL had a synergistic effect on increasing PAI-I mRNA levels. After 48-h culture, 3-fold increases in PAI-I mRNA levels were seen with LH in combination with PRL as compared with PRL alone. The synergistic increase in PAI-I mRNA levels occurred in a dose- and time-dependent manner. 2) The increase in PAI-I mRNA synthesis by PRL alone, or by PRL in combination with LH, was well correlated with the changes in PAI-I activity and antigen levels in the conditioned media. 3) PRL in the culture also dramatically decreased LH-induced tPA mRNA and activity in a dose- and time-dependent fashion. The decrease in the tPA activity by PRL was also correlated with an increase in the amount of PA-PAI-I complexes in the cell-conditioned media. 4) In situ hybridization of tPA and PAI-I mRNAs in the cultured granulosa cells also showed that PRL was capable of enhancing PAI-I mRNA while diminishing tPA mRNA production induced by LH. This suggests that the dose- and time-dependent decrease in the gonadotropin-induced tPA activity in the culture by the presence of PRL may be due to decreasing tPA mRNA synthesis on one hand and to neutralization of the tPA activity by the increased PAI-I activity on the other.

Two types of plasminogen activator (tissue-type, tPA; urokinase-type, uPA) have been demonstrated in ovarian granulosa cells, but only tPA activity was found in denuded oocytes. Immature rats were treated subcutaneously with 20 IU pregnant mare’s serum gonadotropin (PMSG) to stimulate follicle maturation, followed 2 days later by an injection of 10 IU human chorionic gonadotropin (hCG) to induce ovulation. Cellular plasminogen activator activities were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by a fibrin-overlay technique. Cumulus-oocyte complexes from rats before and after PMSG treatment contained low amounts of tPA, but not uPA, activity. After hCG treatment, tPA activity showed a time-dependent increase, reaching a maximum at 24 h after injection. At 12 and 24 h after hCG treatment, uPA activity was also detected. The appearance of high molecular weight lysis zones further suggested the formation of plasminogen activator-inhibitor complexes. Morphological analysis indicated that the increases in oocyte tPA activity were correlated with the extent of cumulus cell expansion and dispersion. In denuded oocytes, tPA activity also progressively increased during the periovulatory period to a maximum at 24 h after hCG treatment. In contrast, neither uPA activity nor activator-inhibitor complex was detected. Secretion of the proteases was measured in the conditioned media of cumulus-oocyte complexes cultured for 24 h in vitro. Substantial increases in tPA release were found in complexes obtained at 8 and 12 h after hCG injection, with lower secretion from complexes obtained at 24 h after hCG treatment. To examine hormonal responsiveness, cumulus-oocyte complexes were incubated with follicle-stimulating hormone (FSH) or hCG for 24 h, and the total (medium plus cell) tPA activity was determined. Tissue PA activity was markedly increased by both FSH and hCG in cumulus-oocyte complexes obtained from PMSG-treated, but not control, rats. After in vivo hCG treatment, the elevated basal tPA activity was not affected b y gonadotropins. Thus, gonadotropin stimulation of ovulation is associated with increased celluar content and secretion of tPA activity by the cumulus-oocyte complexes. The observed periovulatory changes in protease activity may be related tofollicle rupture or cumulus cell expansion and dispersion.

Testis cords, embryonic precursors of the seminiferous tubules, are fundamental for testis structure and function. Delay or disruption of testis cord formation could result in gonadal dysgenesis. Although mechanisms regulating testis cord formation during sex determination have been well-studied, the genes and signaling pathways involving in testis cord maintenance after the cords have formed are not well characterized. It is now clear that the maintenance of cord structure is an active process. In this review, we summarize the recent findings regarding the regulation of testis cord integrity by a series of Sertoli cell transcription factors, including the WT1-SOX8/SOX9-beta-CATENIN-DHH network, GPR56, STIM1, and NR0B1 (also known as DAX1). In particularly, we emphasize the underappreciated role of peritubular myoid cells in testis cord maintenance and their cooperation with Sertoli cells. The regulation of the size, shape, and number of testis cords by Sertoli cell proliferation (e.g., SMAD4, GATA4, and TGF-beta signaling), Leydig cell products (e.g., ACTIVIN A), vascular development (a lesson learned from PDGF signaling), and available gonad space (as observed in Ift144mutant mice) is also addressed. Further efforts and new genetic models are needed to unveil the gene networks and underlying mechanisms regulating testis cord integrity and morphology after sex determination.

Evidence has suggested that tissue-plasminogen activator (tPA) produced by granulosa cells (GCs) plays a crucial role in degradation of the follicular wall during the process of ovulation in rat. By using a primary immature rat GCs, a study was conducted to analyze the possible molecular mechanism of FSH-induced tPA production in the cultured primary GCs. Possible role of mitogenactivated protein kinase (MAPKs) and activator protein 1(AP-1) was examined. GCs obtained from the ovaries of DES-treated immature rats were cultured in a serum-free medium. The tPA activity in the cultured GCs was assessed by colorimetric determination. We have demonstrated that FSH increased the expression of the intracellular tPA in GCs in a time-dependent manner, with a maximum stimulation at 100 ng/ml at 48h. The treatment with H89 (10 μM, PKA inhibitor), UO126 (10 μM, ERK1/2 inhibitor) or SB203580 (20 μM, p38 MAPK inhibitor) significantly inhibited the FSHinduced tPA production, suggesting that multiple signaling pathways, including cAMP/PKA, MEK/ERK1/2 and p38 MAPK cascades, may be involved in the regulation of FSH-induced tPA expression. We further investigated the effect of FSH-activated ERK1/2 and p38 MAPK on tPA production in the primary GCs. FSH receptor occupancy leads to phosphorylation/activation of ERK1/2 and p38 MAPK in a time-dependent manner. Phosphorylation of ERK1/2 and p38 MAPK induced by FSH was observed within 10 min. The highest level of ERK1/2 activation was at 20 min, while p38 MAPK was at 30 min. It was observed that such activation was protein kinase A-dependent as indicated by using its specific inhibitor H89. Inhibition of ERK1/2 activity by U0126 and inhibition of p38 MAPK activity by SB203580 remarkably reduced the FSH-induced tPA production. RT-PCR data also showed that inclusion of the inhibitors of ERK1/2 or SB203580 in the incubation media significantly decreased the FSHinduced tPA mRNA levels. Confocal immunocytochemical studies further confirmed the data obtained by Western blot, RT-PCR and colorimetric measurement. We further investigated expression of AP-1 element in the tPA promoter area. RT-PCR and EMSA data demonstrated that FSH significantly stimulated AP-1 expression, inclusion of H89, ERK1/2 or SB20358 in cultured GCs significantly decreased the FSH-induced AP-1 expression. Therefore, FSH-induced ERK1/2 and p38 MAPK activation is capable of regulating tPA production in the cultured primary GCs, and transcript factor AP-1 in the tPA promoter may be important in regulation of FSH-induced tPA expression. (platform)

Scrotal hypothermia is essential for normal spermatogenesis, and temporal heat stress causes a reversible disruption of the blood-testis barrier (BTB). Previous studies have shown that androgen receptor (AR) expression in primary monkey Sertoli cells (SCs) was dramatically reduced after temporary heat treatment. However, the mechanisms underlying the heat-induced reversible disruption of the BTB, including whether it is directly regulated by the AR, remain largely unknown. In this study, we demonstrated that the AR acts upstream to regulate the heat-induced reversible change in the BTB in mice. When the AR was overexpressed in SCs using an adenovirus, the heat stress-induced down-regulation of BTB-associated proteins (zonula occludens-1, N-cadherin, E-cadherin, alpha-catenin, and beta-catenin) was partially rescued. AR knockdown by RNA interference or treatment with flutamide (an AR antagonist) in SCs inhibited the recovery of BTB-associated protein expression after 43°C heat treatment for 30 min. The results of an in vivo AR antagonist injection experiment further showed that the recovery of BTB permeability induced by temporal heat stress was regulated by the AR. Furthermore, we observed that the colocalization and interactions of partitioning-defective protein (Par) 6-Par3-aPKC-Cdc42 polarity complex components were disrupted in both AR-knockdown and heat-induced SCs. AR overexpression in SCs prevented the disruption of these protein-protein interactions after heat treatment. AR knockdown or treatment with flutamide in SCs inhibited the restoration of these protein-protein interactions after heat treatment compared with heat treatment alone. Together, these results demonstrate that the AR plays a crucial role in the heat-induced reversible change in BTB via the Par polarity complex.

Wt1is specifically expressed in Sertoli cells in the developing testis. A previous study has demonstrated that Wt1plays a critical role in maintaining the integrity of testicular cords. However, the underlying mechanism is unclear. In this study, we found that the laminin-positive basal lamina lining the testicular cords was fragmented and completely absent in some areas of Wt1−/flox; Amh-Cre testes, indicating that the testicular cord disruption can be attributed to the breakdown of the basement membrane. To explore the molecular mechanism underlying this effect, we examined the expression of cell adhesion molecules (CAMs) and testicular cord basal lamina components by real-time RT-PCR, Western blotting, and immunostaining. Compared with control testes, the expression of CAMs (such as E-cadherin, N-cadherin, claudin11, occludin, beta-catenin, and ZO-1) was not obviously altered in Wt1−/flox; Amh-Cre testes. However, the mRNA level of Col4a1and Col4a2was significantly decreased in Wt1-deficient testes. Immunostaining assays further confirmed that the collagen IV protein levels were dramatically reduced in Wt1−/flox; Amh-Cre testes. Moreover, luciferase and point mutation analyses revealed that the Col4a1and Col4a2promoters were additively transactivated by WT1 and SOX9. Given this finding and previous results showing that SOX9 expression declines rapidly after Wt1deletion, we conclude that the loss of Wt1in Sertoli cells results in the downregulation of the important basal lamina component, which in turn causes the breakdown of the basal lamina and subsequent testicular cord disruption.

Our previous studies have demonstrated that tissue type plasminogen activator (tPA) is involved in matrix degradation of the blood-testis barrier in rat. In this study we have further investigated the effect of testosterone on expression and secretion of tPA and its inhibitor type-1 (PAI-1) in rat testes. tPA localized in the Sertoli cells, while PAI-1 was expressed in both Sertoli cells and spermatids of monkey testis. The rat Sertoli cells from the testes at various ages in vitro secreted tPA in an age dependent manner. No detectable level of tPA was secreted in the Sertoli cells isolated from the first day after birth, its activity was detected on day 20, and reached maximum on day 60. The Sertoli cells on day 20 were cultured in the presence of testosterone, FSH and forskolin, the tPA and PAI-1 activities were dose-dependently stimulated. To the culture addition of H89 and U0126, both inhibited the FSH-, forskolin- and testosterone-induced tPA and PAI-1 activity. It is suggested that FSHand testosterone-stimulated tPA and PAI-1 expression in Sertoli cells may be via PKA and ERK signal transduction. Furthermore, we found that testosterone stimulated the tPA secretion at all the stages, but the highest stimulation of tPA activity was observed at stages. This study further suggests that testosterone-induced tPA activity in the Sertoli cells might be related to the blood-testis barrier opening and/or closing, as well as to the spermiation. (poster)