Nonylphenol releases arachidonic acid in rat Sertoli cells via activation of PKA and PLA2.

In brief: The endocrine disruptor, nonylphenol (NP) increases 20:4n-6 release in Sertoli cells via PKA/cPLA2 activation. Our data show that lipid metabolism could be a target of NP-induced abnormal reproductive outcomes. Abstract: Nonylphenol (NP), an endocrine-disrupting chemical, is an environmental contaminant, and many notorious effects on male fertility have been reported in animal models and wild-type species. Here, we evaluated the effects of NP in follicle-stimulating hormone (FSH) signal transduction pathways and lipid metabolism using an in vitro model of rat Sertoli cell (SC) primary culture. Results show that an acute (1 h) SC exposure to NP (10 µM) increased the intra- and extra-cellular concentrations of free fatty acids (FFAs), mainly arachidonic acid (20:4n-6). Phosphatidylinositol seemed to be the major phospholipid source of this 20:4n-6 release by activation of the protein kinase A (PKA)/cytoplasmic phospholipase A2 (cPLA2) pathway. NP also increased diacylglycerols (DAG) levels and the expression (mRNA) of cyclooxygenase 2 (Cox2) and prostaglandin E2 (PGE2) levels. It is noteworthy that accumulation of lipid droplets took place after 24 h NP exposition, which was prevented by both a PKA inhibitor and a PLA2 inhibitor. Like FSH, NP triggers the release of 20:4n-6, which is a substrate for PGE2 synthesis via PKA/PLA2 activation. In addition, NP induces the formation of DAG, which could be required as a cofactor of the PKC-mediated activation of the COX2 inflammatory pathway. Our findings suggest that NP alters lipid homeostasis in SCs by inducing the activation of pro-inflammatory pathways that may trigger adverse effects in testis physiology over time. Concomitantly, the SC enhances the acylation of surplus FFAs (including 20:4n-6) in neutral lipids as a protective mechanism to shield itself from lipotoxicity and pro-inflammatory signals.

Hypothalamic-pituitary-gonadal axis response to photoperiod changes in female guanacos (Lama guanicoe).

The guanaco, a wild South American camelid, is renowned for its remarkable resilience to extreme conditions. Despite this, little is known about how reproductive hormones in female camelids are influenced during their seasonal breeding period, which occurs during long photoperiod. To explore this, the study investigated the response of the hypothalamic-pituitary-gonadal axis in female guanacos during short days (10L:14D; July) and long days (16L:8D; December) in the Mediterranean ecosystem (33°38'28″S, 70°34'27″W). Blood samples from 14 adult animals were collected, and measurements of melatonin, 17ß-estradiol, FSH, and LH concentrations were taken. The results showed that melatonin concentration was lower (P < 0.05) during long days than short days, whereas 17ß-estradiol, FSH, and LH concentrations were higher (P < 0.05) during long days compared to short days. Furthermore, the study detected the expression of the melatonin receptor 1A and kisspeptin in the hypothalamus and pituitary, suggesting that the pineal gland of female guanacos is sensitive to seasonal changes in day length. These findings also indicate a seasonal variation in the concentration of reproductive hormones, likely linked to the distinct modulation of the hypothalamic-pituitary-gonadal axis of female guanacos during short and long days.

The effect of photoperiod and melatonin on plasma prolactin concentrations in female guanaco (Lama guanicoe) in captivity.

The present study examined the effects of different photoperiods and melatonin treatment on plasma prolactin concentrations in guanacos, a South American camelid, in captivity. Fourteen adult female guanacos, not gestating or lactating and isolated from males, were studied. The control group was exposed to natural daylight, during short days (N = 7, 10L:14D) and long days (N = 7, 16L:8D). The treatment group (N = 7, 10L:14D) received melatonin implants every 23 days for 6 weeks during long days. Blood samples were taken at intervals of 1 week for 3 weeks, starting the third week of treatment. Prolactin concentrations were measured using competitive ELISA. Plasma concentrations of prolactin in non-lactating female guanacos have seasonal changes, with a higher concentration (p < .001) in short days (3.50 ± 2.24 ng/ml) than long days (1.10 ± 0.91 ng/ml). Melatonin treatment significantly decreases (p < .05) plasma concentrations of prolactin on the 21st day after the treatment. These findings are the first report of an endogenous circannual rhythm of plasma prolactin concentration and the action of melatonin treatment on prolactin secretion in this wild camelid.

Combined proteomic and miRNome analyses of mouse testis exposed to an endocrine disruptors chemicals mixture reveals altered toxicological pathways involved in male infertility.

The increase in male idiopathic infertility has been associated with daily exposure to endocrine disruptors chemicals (EDCs). Nevertheless, the mechanisms of action in relation to dysregulating proteins and regulatory microRNAs are unknown. We combined proteomic and miRNome analyses of mouse testis chronically exposed to low doses of a define mixture of EDCs [phthalates: bis(2-ethylhexyl), dibutyl and benzyl-butyl; 4-nonylphenol and 4-tert-octylphenol], administered in the drinking water from conception until adulthood (post-natal Day 60/75) and compared them with no-exposed control mice. We analysed fertility parameters and global changes in the patterns of mice testis proteome by 2D electrophoresis/mass spectrometry, along with bioinformatic analyses of dysregulated microRNAs, and their association with published data in human infertile patients. We detected a decrease in the potential fertility of exposed mice associated with changes in the expression of 18 proteins (10 up-regulated, 8 down-regulated). Functional analysis showed that 89% were involved in cell death. Furthermore, we found a group of 23 microRNAs/isomiRs (down-regulated) correlated with six of the up-regulated target proteins (DIABLO, PGAM1, RTRAF, EIF4E, IVD and CNDP2). Regarding this, PGAM1 up-regulation was validated by Western blot and mainly detected in Sertoli cells. Some of these microRNA/protein dysregulations were reported in human testis with spermatogenic failure. Overall, a chronic exposure to EDCs mixture in human males could potentially lead to spermatogenic failure through changes in microRNA expression, which could post-transcriptionally dysregulate mRNA targets that encode proteins participating in cell death in testicular cells. Finally, these microRNA/protein dysregulations need to be validated with other EDCs mixtures and concentrations.

The xenoestrogens biphenol-A and nonylphenol differentially regulate metalloprotease-mediated shedding of EGFR ligands.

The xenoestrogens bisphenol-A (BPA) and nonylphenol (NP) are endocrine disruptors used in the plastic polymer industry to manufacture different products for human use. Previous studies have suggested a role of these compounds in the shedding of signaling molecules, such as tumor necrosis factor α (TNF-α). The aim of this work was to evaluate the effect of BPA and NP on the sheddase ADAM17 and its newly discovered regulators iRhom1 and iRhom2 in the release of EGFR-ligands. We report that BPA and NP can stimulate the release of the ADAM17-substrates HB-EGF and TGF-α. In cells lacking ADAM17 (Adam17-/- mEFs) BPA-stimulated release of HB-EGF, but not TGF-α, was strongly reduced, whereas NP-stimulated shedding of HB-EGF and TGF-α was completely abolished. Inactivation of both ADAM17 and the related ADAM10 (Adam10/17-/- mEFs) completely prevented the release of these substrates. In the absence of iRhom1, BPA- or NP-stimulated release of HB-EGF or TGF-α was comparable to wild-type control mEFs, conversely the BPA-induced release of HB-EGF was abolished in iRhom2-/- mEFs. The defect in shedding of HB-EGF in iRhom2-/- mEF cells could be rescued by overexpressing iRhom2. Interestingly, the NP-stimulated release of HB-EGF was not affected by the absence of iRhom2, suggesting that NP could potentially activate both ADAM10 and ADAM17. We tested this hypothesis using betacellulin (BTC), an EGFR-ligand that is a substrate for ADAM10. We found that NP, but not BPA stimulated the release of BTC in Adam17-/- , iRhom2-/- , or iRhom1/2-/- , but not in Adam10/17-/- cells. Taken together, our results suggest that BPA and NP stimulate the release of EGFR-ligands by differentially activating ADAM17 or ADAM10. The identification of specific effects of these endocrine disruptors on ADAM10 and ADAM17 will help to provide a better understanding of their roles in cell signaling and proinflammatory processes, and provide new potential targets for treatment of reproductive or inflammatory diseases such as asthma or breast cancer that are promoted by xenoestrogens.

Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Bisphenol-A and Nonylphenol Induce Apoptosis in Reproductive Tract Cancer Cell Lines by the Activation of ADAM17.

Endocrine-disruptor chemicals (EDCs), such as bisphenol A (BPA) and nonylphenol (NP), have been widely studied due to their negative effects on human and wildlife reproduction. Exposure to BPA or NP is related to cell death, hormonal deregulation, and cancer onset. Our previous studies showed that both compounds induce A Disintegrin And Metalloprotease 17 (ADAM17) activation. Here, we show that BPA and NP induce apoptosis in prostate and ovary cancer cell lines, in a process dependent on ADAM17 activation. ADAM17 knockdown completely prevented apoptosis as well as the shedding of ADAM17 substrates. Both compounds were found to induce an increase in intracellular calcium (Ca2+) only in Ca2+-containing medium, with the NP-treated cells response being more robust than those treated with BPA. Additionally, using a phosphorylated protein microarray, we found that both compounds stimulate common intracellular pathways related to cell growth, differentiation, survival, and apoptosis. These results suggest that BPA and NP could induce apoptosis through ADAM17 by activating different intracellular signaling pathways that may converge in different cellular responses, one of which is apoptosis. These results confirm the capacity of these compounds to induce cell apoptosis in cancer cell lines and uncover ADAM17 as a key regulator of this process in response to EDCs.

Effects of ascorbic acid on spermatogenesis and sperm parameters in diabetic rats.

Diabetes mellitus in human and animal models has been correlated with low sperm count, testicular abnormalities, high levels of germ cell death, and oxidative stress. In this study, we focus on three questions: (1) Is germ cell apoptosis stage-specific in diabetic male rats? (2) Could ascorbic acid (AA) reverse oxidative and histological damage and restore testicular dysfunction? (3) Could AA treatment restore fertility parameters in diabetic rats? Adult Sprague-Dawley rats were divided into four groups: control, diabetic, control plus AA, and diabetic plus AA. Seminiferous tubules underwent severe histological damage, together with a change in frequency of some stages of the seminiferous cycle, and germ cell apoptosis was increased in a stage-dependent manner in diabetic rats. We found a significant decrease in testosterone and higher levels of lipid peroxidation in diabetic rats when compared with controls. A major finding was that AA reversed the histological damage and peroxidation levels to control levels in diabetic rats, but testosterone levels remained unchanged. The pregnancy rate was decreased in females that mated with diabetic rats and those treated with AA, but the litter size was only reduced in the second case. Interestingly, spermatozoa from diabetic and AA-treated rats showed reduced motility and hyperactivation, but only diabetic rats had higher levels of apoptosis when compared with controls. These results suggest that treatment with AA reverses testicular damage in diabetic rats but is insufficient to restore testosterone levels, sperm motility, and fertility in a rat model.

Mitotic Inheritance of mRNA Facilitates Translational Activation of the Osteogenic-Lineage Commitment Factor Runx2 in Progeny of Osteoblastic Cells.

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that "bookmark" the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis.

Involvement of a P2X7 Receptor in the Acrosome Reaction Induced by ATP in Rat Spermatozoa.

The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non-physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), Suramin, and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS]). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue-G (BB-G) and 17-ß-oestradiol, two blockers of P2X7 receptors. Moreover, the ATP analog 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2X7 receptor. Here, we show the presence of P2X7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2X7 receptor, which may be functional during in vivo fertilization.

In vitro fertilization (IVF) in mammals: epigenetic and developmental alterations. Scientific and bioethical implications for IVF in humans.

The advent of in vitro fertilization (IVF) in animals and humans implies an extraordinary change in the environment where the beginning of a new organism takes place. In mammals fertilization occurs in the maternal oviduct, where there are unique conditions for guaranteeing the encounter of the gametes and the first stages of development of the embryo and thus its future. During this period a major epigenetic reprogramming takes place that is crucial for the normal fate of the embryo. This epigenetic reprogramming is very vulnerable to changes in environmental conditions such as the ones implied in IVF, including in vitro culture, nutrition, light, temperature, oxygen tension, embryo-maternal signaling, and the general absence of protection against foreign elements that could affect the stability of this process. The objective of this review is to update the impact of the various conditions inherent in the use of IVF on the epigenetic profile and outcomes of mammalian embryos, including superovulation, IVF technique, embryo culture and manipulation and absence of embryo-maternal signaling. It also covers the possible transgenerational inheritance of the epigenetic alterations associated with assisted reproductive technologies (ART), including its phenotypic consequences as is in the case of the large offspring syndrome (LOS). Finally, the important scientific and bioethical implications of the results found in animals are discussed in terms of the ART in humans.

Spermatozoa from mice deficient in Niemann-Pick disease type C2 (NPC2) protein have defective cholesterol content and reduced in vitro fertilising ability.

The cholesterol content of the sperm membrane is regulated during both maturation in the epididymis and capacitation in the female tract, two processes required for the spermatozoa to acquire their fertilising ability. Because Niemann-Pick disease, type C2 (NPC2) protein is one of the most abundant components of the epididymal fluid and contains a functional cholesterol-binding site that can transfer cholesterol between membranes, it has been suggested for years that NPC2 could be involved in the regulation of cholesterol levels in spermatozoa during epididymal maturation. In the present study, western blot and immunohistochemistry analyses demonstrated significant levels of NPC2 in the mouse epididymal epithelium. Epididymal spermatozoa obtained from NPC2(-/-) mice were morphologically normal and had normal motility parameters, but had a reduced cholesterol content compared with that of wild-type (WT) spermatozoa, as determined by both biochemical and by flow cytometry analyses. These results suggest that NPC2 could be involved in regulating cholesterol levels in spermatozoa during epididymal maturation. To understand the relevance of epididymal NPC2 for sperm function, the ability of spermatozoa to undergo events influenced by epididymal maturation, such as capacitation and fertilisation, were compared between WT and NPC2(-/-) mice. Capacitated NPC2(-/-) spermatozoa exhibited defective tyrosine phosphorylation patterns and a reduced ability to fertilise cumulus-oocyte complexes compared with WT spermatozoa, supporting the relevance of mouse epididymal NPC2 for male fertility.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation.

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Differential effects of temperature on reactive oxygen/nitrogen species production in rat pachytene spermatocytes and round spermatids.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide and nitric oxide are produced by testis and spermatogenic cells in response to heat stress. However, the magnitude and mechanisms of this production in spermatogenic cells have not been described. In this work, we evaluated ROS/RNS production, its pharmacology, mitochondrial oxidative metabolism, membrane potential and antioxidant capacity at different temperatures in isolated rat pachytene spermatocytes and round spermatids. Our results showed an increment in ROS/RNS production by pachytene spermatocytes when increasing the temperature to 40 °C. Instead, ROS/RNS production by round spermatids did not change at temperatures higher than 33 °C. ROS/RNS production was sensitive to NADPH oxidase inhibitor diphenylene iodonium or the mitochondrial complex I inhibitor rotenone. No additive effects were observed for these two compounds. Our results suggest an important mitochondrial ROS/RNS production in spermatogenic cells. Oligomycin-insensitive oxygen consumption (uncoupled oxygen consumption) increased with temperature and was significantly larger in round spermatids than pachytene spermatocytes, indicating a likely round spermatid mitochondrial uncoupling at high temperatures. A similar conclusion can be reached by measuring the mitochondrial membrane potential using rhodamine 123 fluorescence in permeabilized cells or JC-1 fluorescence in intact cells. The antioxidant capacity was higher in round spermatids than pachytene spermatocytes at 40 °C. Our results strongly suggest that at high temperatures (40 °C) pachytene spermatocytes are more susceptible to oxidative stress, but round spermatids are more protected because of a temperature-induced mitochondrial uncoupling together with a larger antioxidant capacity.

Human globozoospermia-related genes and their role in acrosome biogenesis.

The mammalian acrosome is a secretory vesicle attached to the sperm nucleus whose fusion with the overlying plasma membrane is required to achieve fertilization. Acrosome biogenesis starts during meiosis, but it lasts through the entire process of haploid cell differentiation (spermiogenesis). Acrosome biogenesis is a stepwise process that involves membrane traffic from the Golgi apparatus, but it also seems that the lysosome/endosome system participates in this process. Defective sperm head morphology is accompanied by defective acrosome shape and function, and patients with these characteristics are infertile or subfertile. The most extreme case of acrosome biogenesis failure is globozoospermia syndrome, which is primarily characterized by the presence of round-headed spermatozoa without acrosomes with cytoskeleton defects around the nucleus and infertility. Several genes participating in acrosome biogenesis have been uncovered using genetic deletions in mice, but only a few of them have been found to be deleted or modified in patients with globozoospermia. Understanding acrosome biogenesis is crucial to uncovering the molecular basis of male infertility and developing new diagnostic tools and assisted reproductive technologies that may help infertile patients through more effective treatment techniques. This article is categorized under: Reproductive System Diseases > Environmental Factors Infectious Diseases > Stem Cells and Development Reproductive System Diseases > Molecular and Cellular Physiology.

Histomorphological changes between short and long days in ovary guanacos (Lama guanicoe).

This is the first morpho-histological comparison of guanaco ovaries between reproductive (long-days) and non-reproductive (short-days) seasons, and oestrogen receptor-alpha (ERα) and beta (ERß) detection. Different stages of follicle development were found in the cortical area, but no corpus luteum was detected. The size and frequency of antral follicles and large atretic follicles were higher in long-day ovaries than short-days, consistent with ovarian activity in this season. Differential expression of ERα and ERß was observed in follicles at different stages of development between short and long days. These data reveal histological and molecular differences between reproductive and non-reproductive seasons of guanaco ovaries.

Etoposide induces apoptosis and upregulation of TACE/ADAM17 and ADAM10 in an in vitro male germ cell line model.

Etoposide is a widely used anticancer drug in the treatment of different tumors. Etoposide is known to activate a wide range of intracellular signals, which may in turn induce cellular responses other than apoptosis. ADAM10 and TACE/ADAM17 belong to a family of transmembrane extracellular metalloproteinases involved in paracrine/juxtacrine regulation of many signaling pathways. The aim of this work was to evaluate if etoposide induces upregulation of ADAM10 or TACE/ADAM17 in two cell lines (GC-1 and GC-2) derived from male germ cells. Results showed that etoposide induced apoptosis in a dose-response manner in both GC-1 and GC-2 cells. Apoptosis started to increase 6h after etoposide addition in GC-2 cells, whereas the same was observed 18h after addition to the GC-1 cells. Protein and mRNA levels of ADAM10 and TACE/ADAM17 increased 18h after etoposide was removed from the GC-1 cells. In GC-2 cells, the protein levels of both proteins increased 12h after etoposide was removed. ADAM10 mRNA increased after 3h and then steadily decreased up to 12h after removal, whereas TACE/ADAM17 mRNA decreased after etoposide removal. Finally, apoptosis was prevented in GC-1 and GC-2 cells by the addition of pharmacological inhibitors of ADAM10 and TACE/ADAM17 to the culture medium of etoposide-treated cells. Our results show for the first time that etoposide upregulates ADAM10 and TACE/ADAM17 mRNA and protein levels. In addition, we also show that ADAM10 and TACE/ADAM17 have a role in etoposide-induced apoptosis.

Involvement of TACE/ADAM17 and ADAM10 in etoposide-induced apoptosis of germ cells in rat spermatogenesis.

Germ cell apoptosis is important to regulate sperm production in the mammalian testis, but the molecular mechanisms underlying apoptosis are still poorly understood. We have recently shown that in vitro, etoposide induces upregulation of TACE/ADAM17 and ADAM10, two membrane-bound extracellular metalloproteases. Here we show that in vivo these enzymes are involved in etoposide-, but not in heat shock-, induced apoptosis in rat spermatogenesis. Germ cell apoptosis induced by DNA damage was associated with an increase in protein levels and cell surface localization of TACE/ADAM17 and ADAM10. On the contrary, apoptosis of germ cells induced by heat stress, another cell death stimulus, did not change levels or localization of these proteins. Pharmacological in vivo inhibition of TACE/ADAM17 and ADAM10 prevents etoposide-induced germ cell apoptosis. Finally, Gleevec (STI571) a pharmacological inhibitor of p73, a master gene controlling apoptosis induced by etoposide, prevented the increase of TACE/ADAM17 levels. Our results strongly suggest that TACE/ADAM17 participates in in vivo apoptosis of male germ cells induced by DNA damage.

Apoptosis, necrosis and autophagy are influenced by metabolic energy sources in cultured rat spermatocytes.

Apoptosis, necrosis and autophagy are mechanistically related processes that control tissue homeostasis and cell survival. In the testis, germ cell death is important for controlling sperm output, but it is unknown whether or not germ cells can switch from apoptosis to necrosis, as has been reported in other tissues. Furthermore, autophagy has not been reported in spermatogenesis. Spermatocytes (meiotic cells) and spermatids (haploid cells) use lactate rather than glucose as their primary substrate for producing ATP. The metabolism of glucose, but not lactate, reduces ATP levels and increases intracellular [H(+)] and [Ca(2+)], both of which are associated with apoptosis and/or necrosis in somatic cells. In this work, we evaluated whether different energy sources, such as lactate or glucose, can influence spermatocyte death type and/or survival in primary cultures. Spermatocytes cultured for 12 h without an energy source died by necrosis, while spermatocytes cultured with 5 mM glucose showed a significant increase in apoptosis, as evidenced by caspase activity, TUNEL assay and phosphatidylserine exposure. Apoptosis was not observed in spermatocytes cultured with 5 mM lactate or deoxyglucose. Autophagy markers, such as LC3-II and autophagosomes, were detected after 12 h of culture, regardless the culture conditions. These results suggest that the availability of glucose and/or lactate affect the type of death or the survival of primary spermatocytes, where glucose can induce apoptosis, while lactate is a protective factor.

Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers.

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.