Cortactin and phagocytosis in isolated Sertoli cells

BACKGROUND: Cortactin, an actin binding protein, has been associated with Sertoli cell ectoplasmic specializations in vivo, based on its immunolocalization around the heads of elongated spermatids, but not previously identified in isolated Sertoli cells. In an in vitro model of Sertoli cell-spermatid binding, cortactin was identified around debris and dead germ cells. Based on this observation, we hypothesized that this actin binding protein may be associated with a non-junction-related physiological function, such as phagocytosis. The purpose of this study was to identify the presence and distribution of cortactin in isolated rat Sertoli cells active in phagocytic activity following the addition of 0.8 μm latex beads. RESULTS: Sertoli cell monocultures were incubated with or without follicle stimulating hormone (FSH; 0.1 μg/ml) in the presence or absence of cytochalasin D (2 μM), as an actin disrupter. Cortactin was identified by standard immunostaining with anti-cortactin, clone 4F11 (Upstate) after incubation times of 15 min, 2 hr, and 24 hr with or without beads. Cells exposed to no hormone and no beads appeared to have a ubiquitous distribution of cortactin throughout the cytoplasm. In the presence of cytochalasin D, cortactin immunostaining was punctate and distributed in a pattern similar to that reported for actin in cells exposed to cytochalasin D. Sertoli cells not exposed to FSH, but activated with beads, did not show cortactin immunostaining around the phagocytized beads at any of the time periods. FSH exposure did not alter the distribution of cortactin within Sertoli cells, even when phagocytic activity was upregulated by the presence of beads. CONCLUSION: Results of this study suggest cortactin is not associated with peripheralized actin at junctional or phagocytic sites. Further studies are necessary to clarify the role of cortactin in Sertoli cells

Nuclear Targeting of IGF-1 Receptor in Orbital Fibroblasts from Graves' Disease: Apparent Role of ADAM17

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with 125I IGF-1, 125I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis

Role of β-Catenin in Post-Meiotic Male Germ Cell Differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation

Microinjection Manipulation Resulted in the Increased Apoptosis of Spermatocytes in Testes from Intracytoplasmic Sperm Injection (ICSI) Derived Mice

The invention of intracytoplasmic sperm injection (ICSI) has possibly been the most important development in reproductive medicine, one that has given hope to thousands of infertile couples worldwide. However, concerns remain regarding the safety of this method since it is a more invasive procedure than in vitro fertilization (IVF), since a spermatozoon is injected into the oocyte cytoplasm. Using mice derived from IVF technology as a control, we assessed the influence of invasive microinjection in the process of transferring sperm into oocyte cytoplasm in ICSI procedure on the development and physiologic function of resultant offspring. Our results demonstrated that mice produced from ICSI and IVF had no significant difference in phenotypic indices including body weight, forelimb physiology, and learning and memory ability. However, increased spermatocyte apoptosis was observed in the testis of adult ICSI mice, when compared with IVF mice. And, decreased testis weight and marked damage of spermatogenic epithelia were found in aged ICSI mice. Furthermore, proteomic analysis verified that most of the differentiated proteins in testes between adult ICSI and IVF mice were those involved in regulation of apoptosis pathways. Our results demonstrated that the microinjection manipulation used in the ICSI procedure might pose potential risks to the fertility of male offspring. The changed expression of a series of proteins relating to apoptosis or proliferation might contribute to it. Further studies are necessary to better understand all the risks of ICSI

Preferences across the Menstrual Cycle for Masculinity and Symmetry in Photographs of Male Faces and Bodies

Background: Previous studies have shown that women increase their preference for masculinity during the fertile phase of the menstrual cycle. Evidence for a similar preference shift for symmetry is equivocal. These studies have required participants to choose between subtle variations in computer-generated stimuli, and preferences for more natural stimuli have not been investigated. Methodology/Principal Findings: Our study employed photographs of individual males to investigate women’s preferences for face and body masculinity and symmetry across the menstrual cycle. We collected attractiveness ratings from 25 normally cycling women at high- and low-fertility days of the menstrual cycle. Attractiveness ratings made by these women were correlated with independent ratings of masculinity and symmetry provided by different sets of raters. We found no evidence for any cyclic shift in female preferences. Correlations between attractiveness and masculinity, and attractiveness and symmetry did not differ significantly between high- and low-fertility test sessions. Furthermore, there was no significant difference between high- and low-fertility ratings of attractiveness. Conclusions: These results suggest that a menstrual cycle shift in visual preferences for masculinity and symmetry may be too subtle to influence responses to real faces and bodies, and subsequent mate-choice decisions

Cytostatic Factor Proteins Are Present in Male Meiotic Cells and β-Nerve Growth Factor Increases Mos Levels in Rat Late Spermatocytes

Background: In co-cultures of pachytene spermatocytes with Sertoli cells, beta-NGF regulates the second meiotic division by blocking secondary spermatocytes in metaphase (metaphase II), and thereby lowers round spermatid formation. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of cytostatic factor (CSF) in their cytoplasm. By analogy, we hypothesized the presence of CSF in male germ cells. Methodology/Principal Findings: We show here, that Mos, Emi2, cyclin E and Cdk2, the four proteins of CSF, and their respective mRNAs, are present in male rat meiotic cells; this was assessed by using Western blotting, immunocytochemistry and reverse transcriptase PCR. We measured the relative cellular levels of Mos, Emi2, Cyclin E and Cdk2 in the meiotic cells by flow cytometry and found that the four proteins increased throughout the first meiotic prophase, reaching their highest levels in middle to late pachytene spermatocytes, then decreased following the meiotic divisions. In co-cultures of pachytene spermatocytes with Sertoli cells, beta-NGF increased the number of metaphases II, while enhancing Mos and Emi2 levels in middle to late pachytene spermatocytes, pachytene spermatocytes in division and secondary spermatocytes. Conclusion/Significance: Our results suggest that CSF is not restricted to the oocyte. In addition, they reinforce the view that NGF, by enhancing Mos in late spermatocytes, is one of the intra-testicular factors which adjusts the number of round spermatids that can be supported by Sertoli cells

Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes

Maintaining the integrity of sperm DNA is vital to reproduction and male fertility. Sperm contain a number of molecules and pathways for the repair of base excision, base mismatches and DNA strand breaks. The presence of Poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme, and its homologues has recently been shown in male germ cells, specifically during stage VII of spermatogenesis. High PARP expression has been reported in mature spermatozoa and in proven fertile men. Whenever there are strand breaks in sperm DNA due to oxidative stress, chromatin remodeling or cell death, PARP is activated. However, the cleavage of PARP by caspase-3 inactivates it and inhibits PARP's DNA-repairing abilities. Therefore, cleaved PARP (cPARP) may be considered a marker of apoptosis. The presence of higher levels of cPARP in sperm of infertile men adds a new proof for the correlation between apoptosis and male infertility. This review describes the possible biological significance of PARP in mammalian cells with the focus on male reproduction. The review elaborates on the role played by PARP during spermatogenesis, sperm maturation in ejaculated spermatozoa and the potential role of PARP as new marker of sperm damage. PARP could provide new strategies to preserve fertility in cancer patients subjected to genotoxic stresses and may be a key to better male reproductive health

Germ Cell-Specific Targeting of DICER or DGCR8 Reveals a Novel Role for Endo-siRNAs in the Progression of Mammalian Spermatogenesis and Male Fertility

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process

Additive Effects of Nicotine and High-Fat Diet on Hepatocellular Apoptosis in Mice: Involvement of Caspase 2 and Inducible Nitric Oxide Synthase-Mediated Intrinsic Pathway Signaling

Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to nonalcoholic fatty liver disease (NAFLD). The health risk associated with smoking is exaggerated by obesity and is the leading causes of morbidity and mortality worldwide. We recently demonstrated that combined treatment with nicotine and a high-fat diet (HFD) triggers greater oxidative stress, activates hepatocellular apoptosis, and exacerbates HFD-induced hepatic steatosis. Given that hepatocellular apoptosis plays a pivotal role in the pathogenesis of NAFLD, using this model of exacerbated hepatic steatosis, we elucidated the signal transduction pathways involved in HFD plus nicotine-induced liver cell death. Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice daily IP injections of 0.75 mg/kg BW of nicotine or saline for 10 weeks. High-resolution light microscopy revealed markedly higher lipid accumulation in hepatocytes from mice received HFD plus nicotine, compared to mice on HFD alone. Addition of nicotine to HFD further resulted in an increase in the incidence of hepatocellular apoptosis and was associated with activation of caspase 2, induction of inducible nitric oxide synthase (iNOS), and perturbation of the BAX/BCL-2 ratio. Together, our data indicate the involvement of caspase 2 and iNOS-mediated apoptotic signaling in nicotine plus HFD-induced hepatocellular apoptosis. Targeting the caspase 2-mediated death pathway may have a protective role in development and progression of NAFLD