Sertoli cells are important in determining the fate of spermatogenic cells by providing nutrition and structural support via cell junctions. In this study, we sought to examine the effect of 43 C warming on cell junctions in seminiferous epithelium and the expression of junction-associated molecules in Sertoli cells. Electron microscopy showed the appearance of large vacuoles between Sertoli and germ cells and adjacent Sertoli cells, leading to disruption of corresponding cell junctions 24 h after terminating the heat treatment. Using primary Sertoli cells isolated from pubertal monkey testes, we demonstrated that expression of adherens junction-associated molecules, such as N-cadherin and beta-catenin, and tight junction-associated molecule zonula occludens protein 1 was significantly reduced in 24-48 h after heat treatment. In contrast, intermediate filament vimentin expression was up-regulated in 6-48 h. Androgen receptor (AR) and Wilms' tumor gene 1 expression dramatically decreased after heat treatment. Both proteins completely disappeared immediately after terminating heat treatment and began to recover after 6 h. Treatment of the monkey Sertoli cells with an AR antagonist, flutamide, could mimic the heat-induced changes in the expression of junction-associated molecules in Sertoli cells. Furthermore, overexpression of AR in the Sertoli cells up-regulated the expression of N-cadherin, beta-catenin, and zonula occludens protein 1 and down-regulated vimentin expression. Their expression after heat treatment could be rescued by the AR overexpression. These results indicate that the decreased AR expression after heat treatment is involved in heat-induced cell junction disruption.
Specialized junctions, which occur at sites of Sertoli-Sertoli and Sertoli-germ cell contact of seminiferous epithelium, play pivotal roles in spermatogenesis. Slight increase in scrotal temperature can induce oligospermia or azoospermia via increasing germ cell apoptosis. In this study, we demonstrated that the expression of tight junction (TJ) components, such as occludin, claudin-3 and zonula occludens-1 (ZO-1), was reduced 24-48h after a single mild scrotal heat exposure (43°C for 30min), whereas mRNA levels of claudin-11 were increased. Moreover, the protein localization of occludin and ZO-1 was lost from the blood-testis barrier (BTB) site, whereas claudin-11 immunostaining became diffuse and cytoplasmic 2days following heat exposure. Electron microscopic analysis showed that 2days after the heat treatment, the intercellular space between the two adjacent Sertoli cells was expanded, coupled with defragmentation of actin bundles and the endoplasmic reticulum. In addition, the TJ permeability increased significantly 2days after the heat exposure and recovered approximately 10days later. Heat-induced reversible BTB disruption was associated with a transient induction of transforming growth factor (TGF)-β2, -3 and p38 mitogen-activated protein kinase activation. However, the TGF-β antagonist only partially prevented the heat-induced BTB disruption. In conclusion, the expression of TJ-associated molecules and BTB were reversibly perturbed after mild testicular hyperthermia, and the induction of TGF-β expression may be partially involved in heat-induced BTB damage.
Increasing evidence has shown that excess androgen may be a main cause of polycystic ovary syndrome (PCOS). However, the molecular mechanism of androgen action on the ovary is unclear. To investigate the possible impacts of androgen on early follicular development, neonatal mouse ovaries mainly containing primordial follicles were cultured with testosterone. We demonstrated that the number of primary follicles was increased after 10 d culture with testosterone treatment via phosphatidylinositol 3-kinase/Akt pathway. Androgen induced Forkhead box (Foxo)-3a activation, and translocation of Foxo3a protein from oocyte nuclei to cytoplasm, which might be a key step for primordial follicle activation. Interestingly, testosterone was also capable of down-regulating growth and differentiation factor-9 expression via its receptor. In summary, we infer that intraovarian excess androgen in PCOS might result in excess early follicles by inducing oocyte Foxo3a translocation and follicular arrest by down-regulating growth and differentiation factor-9 expression.
Notch signaling is an evolutionarily conserved pathway, which regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Notch signaling are expressed in mammalian ovaries, but the exact functions of this pathway in follicle development is still unclear. In this study, primary follicles were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). We found that the cultured follicles completely stopped developing after L-658,458 and DAPT treatment, most of the granulosa cells were detached, and the oocytes were also degenerated with condensed cytoplasma. Further studies demonstrated that the proliferation of granulosa cells was dependent on the Notch signaling. L-658,458 and DAPT treatment inhibited proliferation of in vitro cultured primary granulosa cells and decreased the expression of c-Myc. Lentivirus mediated overexpression of Notch intracellular domain 2, and c-Myc could promote the proliferation of granulosa cells and rescue the growth inhibition induced by L-658,458 and DAPT. In conclusion, Notch signaling is involved in follicular development by regulating granulosa cell proliferation.
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 intracellular 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.
Stem cell factor (SCF) is essential for the development of primordial follicles. One of its functions is to prevent oocytes from apoptosis. However, the underlying mechanism remains largely unknown. By using cultured ovaries that are rich in primordial follicles, the anti-apoptotic action of SCF and the potential signal transduction pathways were investigated. The apoptosis was evaluated by means of in situ 3'-end labeling. The expressions of proteins were analyzed with immunohistochemistry and Western blot. The data showed that SCF significantly prevented oocytes from apoptosis in the cultured organs. Addition of a specific pharmacological inhibitor of PI3K abolished the anti-apoptotic action of SCF while that of a MEK inhibitor did not. The phosphorylation of two mitogen activated protein kinases (MAPKs) (p42 and p44) and AKT, the respective substrates of MEK and PI3K, were enhanced by SCF treatment. Not surprisingly, the MAPK activation occurred only in theca cells. The expressions of apoptosis-related gene products, the Bcl-2 family proteins, in response to SCF treatment were also investigated. While SCF up-regulated the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, it did the opposite to the pro-apoptotic factor Bax. The PI3K inhibitor reversed the regulation of SCF on Bcl-xL and Bax but not on Bcl-2. Therefore, it seemed that SCF initiated an anti-apoptotic signal starting from its membrane receptor c-kit to Bcl-2 family members through PI3K/AKT and other signaling cascades in the oocytes of primordial follicles.
This proof-of-concept study demonstrates that transient testicular warming enhances and hastens the effect of T implant on the suppression of spermatogenesis in monkeys.
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 degrees 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.
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