Increased expression of ERp57 in rat oocytes during meiotic maturation is associated with sperm-egg fusion.

Oocyte meiotic maturation is a developmental transition that starts during germinal-vesicle breakdown and ends at the arrest in metaphase of meiosis II. This transition is associated with changes to both the proteins that are synthesized and the abundance/distribution of post-translational modifications that are crucial for subsequent fertilization and embryogenesis. Here, we isolated and cultured rat oocytes in vitro during both metaphase of meiosis I (MI) and meiosis II (MII) stages, respectively, and then compared their proteomic profiles by high-resolution, two-dimensional gel electrophoresis (2DE) followed by mass spectrometry. We found that the expression of five proteins was up-regulated while six proteins were down-regulated when comparing MI to MII oocytes. The expression of ERp57, an endoplasmic reticulum chaperone, underwent a dramatic increase between MI and MII oocytes, and became concentrated in a dome-shaped area of the cell surface within the microvillar region. A similar profile was observed during spermatogenesis, and sperm ERp57 eventually localized to the head and flagellum surfaces, finally ending in the equatorial region of acrosome-reacted sperm. Given the localization pattern, we tested and found that a polyclonal antiserum created against recombinant rat ERp57 significantly inhibited spermatozoa from penetrating zona pellucida-free oocytes without affecting either sperm motility or the acrosome reaction. These results indicate that ERp57 expression on oocytes, and possibly sperm, plays an important physiological role during sperm-egg fusion.

Palmitoyl-protein thioesterase 1 (PPT1): an obesity-induced rat testicular marker of reduced fertility.

Male obesity may lead to declines in testosterone levels, reproductive hormonal profile, and semen quantity. To assess the effects of obesity on spermatogenesis, Sprague-Dawley rats fed a high-fat diet served as a model of induced obesity. The litter sizes for females mated to obese males were significantly lower as compared to females mated with normal-diet-fed controls. Their serum high-density lipoprotein, low-density lipoprotein, cholesterol, and estradiol levels increased in obese males, but testosterone and follicle-stimulating hormone levels decreased. Testicular morphology disruptions included Sertoli-cell atrophy, disrupted tight junctions, and mitochondrial degeneration in spermatogenic cells. To further investigate the molecular mechanisms leading to high-fat-diet-induced changes, we employed testicular proteomic analysis on rats fed both types of diet. Three spots were up-regulated in rats fed a high-fat diet whereas two others were downregulated. One of the upregulated spots was palmitoyl-protein thioesterase 1 (PPT1), a lipoprotein metabolizing related enzyme localized to Sertoli cells. In a Sertoli-cell line cultured in a high-fat supplemented medium, PPT1 abundance was accompanied by increases in the endocytic vesicle-associated protein, clathrin, and decreases in the tight junctional proteins, ZO-1 and occludin. In conclusion, declines in rat male fertility induced by a high-fat diet are associated with an altered testicular protein expression pattern as well as disruption of testicular Sertoli-cell and spermatogenic-cell morphology. PPT1 expression may provide a testicular marker of reduced fertility in obese males, as increases in its expression may be detrimental to Sertoli-cell function during spermatogenesis.

In vitro and in vivo differentiation of induced pluripotent stem cells into male germ cells.

The introduction of induced pluripotent stem cell (iPSC) lines has been a breakthrough in the field of stem cell research. However, the extent of pluripotency among those cell lines tends to be variable due to their different epigenetic signatures. Mouse iPS cell line 4.1 has been established via retroviral transfer of human transcription factors Oct4, Sox2, Klf4, and c-Myc; the germline competence of this line has not been determined. In the present study, we induced the differentiation of miPS-4.1 cells into male germ cells, in vivo and in vitro. In the in vitro model, the behavior of miPS-4.1 cells was identical to that of differentiating mouse embryonic stem cells (ESCs). We obtained primordial germ cell-like cells (PGC-LC) that were positive for alkaline phosphatase (AP) activity. In continuous culture, these cells expressed pluripotent marker Oct4 and male germline markers C-kit and MVH. For our in vivo model, miPS-4.1 cells were co-transplanted with neonatal testicular cell suspension. We observed ectopically reconstituted seminiferous tubule structures, in which the miPS-4.1 cells were homing and developing. In conclusion, we successfully induced the differentiation of miPS-4.1 cells into male germ cells, albeit their epigenetic characteristics. Our study provides a system to examine the mechanisms of male germ cell development and might help to supply an effective treatment for male infertility in the future.

Endoplasmic reticulum protein 29 (ERp29), a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse.

BACKGROUND: Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process. METHODS: Laser scanning confocal microscopy (LSCM) and Western blot analysis were employed to probe for ERp29 protein in BALB/c mouse epididymal and acrosome-reacted spermatozoa. We prepared rabbit polyclonal antibodies against mouse recombinant ERp29 (rERp29) to characterize: 1) fertilization rate (FR); 2) fertilization index (FI); 3) sperm motility and 4) acrosome reaction (AR). RESULTS: Confocal microscopy indicated that ERp29 was partially localized at the sperm head of the epididymal caput as well as over the whole head and part of the principal piece of the tail region from the epididymal cauda. However, when the acrosome reacted, ERp29 remained in the equatorial and post-acrosomal regions of the sperm head, which is the initial site of sperm-oocyte membrane fusion. Such localization changes were confirmed based on the results of Western blot analysis. Furthermore, the antibodies against mouse rERp29 inhibited the spermatozoa from penetrating into the zona pellucida (ZP)-free oocytes. The functional blocking antibodies reduced both mouse sperm-oocyte FR and FI at concentrations of 100 and 200 micro g/ml compared with pre-immunized rabbit IgG or with anti-mouse recombinant bactericidal/permeability-increasing protein (BPI, a sperm surface protein unrelated to sperm-oocyte fusion) antibodies (100 micro g/ml), but they had no effect on sperm motility and AR. CONCLUSION: This study demonstrates that ERp29 on mouse spermatozoa membrane changes during epididymal transit and AR. Accordingly, in mice this protein may be one of the important factors involved in sperm fertilization by facilitating sperm-oocyte membrane fusion.

Expression of the retinoic acid-metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development.

AIM: To study the expression pattern of the retinoic acid metabolizing enzymes RALDH2 and CYP26b1 during mouse postnatal testis development at both mRNA and protein levels. METHODS: Real-time polymerase chain reaction and Western blot analysis were performed to determine the relative quantity of RALDH2 and CYP26b1 at both mRNA and protein levels at postnatal day 1, 5, 10, 20, and in adult mice (70 days testes). Testicular localization of RALDH2 and CYP26b1 during mouse postnatal development was examined using immunohistochemistry assay. RESULTS: Aldh1a2 transcripts and its protein RALDH2 began to increase at postnatal day 10, and remained at a high level through postnatal day 20 to adulthood. Cyp26b1 transcripts and CYP26b1 protein did not change significantly during mouse postnatal testis development. RALDH2 was undetectable in the postnatal 1, 5 and 10 day testes using immunohistochemistry assay. At postnatal day 20 it was detected in pachytene spermatocytes. Robust expression of RALDH2 was restricted in round spermatids in the adult mouse testis. In the developing and adult testis, CYP26b1 protein was confined to the peritubular myoepithelial cells. CONCLUSION: Our results indicate that following birth, the level of retinoic acid in the seminiferous tubules might begin to increase at postnatal day 10, and maintain a high level through postnatal day 20 to adulthood.

[Estrogens affect Sertoli cells and the blood-testis barrier in pubertal rats].

OBJECTIVE: To investigate the correlation of exogenous estrogens with the expression of FasL in Sertoli cells and the blood-testis barrier during the differentiation and maturation period of Sertoli cells, and to discuss the related factors that influence the blood-testis barrier of pubertal rats. METHODS: Super-physiological doses of exogenous estrogenic compounds (diethylstilbestrol and estradiol) were administered to pubertal Sprague-Dawley rats in vitro and in vivo, the FasL expression in the Sertoli cells of the rats detected by immunohistochemistry and Western blot, and the changes in the blood-testis barrier observed with the electron microscope. RESULTS: After the exposure to exogenous estrogens, the FasL expression was markedly up-regulated in the immature Sertoli cells (P < 0.05) as well as in the Sertoli cell membrane and the blood-testis barrier of the epithelium. The tracer lanthanum passed through the blood-testis barrier and reached the whole layer of the epithelium at 18 days. CONCLUSION: Super-physiological dose of exogenous estrogens can change the expression and distribution of FasL in immature Sertoli cells and affect the structure of the blood-testis barrier.

Age-dependent expression of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testis and epididymis.

AIM: To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic (Cres) gene in mouse testis and epididymis during postnatal development. METHODS: The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. RESULTS: (1) In both the testis and epididymis, Cres mRNA was first detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. (2) In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. (3) The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. CONCLUSION: The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.

Anthraquinones sensitize tumor cells to arsenic cytotoxicity in vitro and in vivo via reactive oxygen species-mediated dual regulation of apoptosis.

Cellular oxidation/reduction state affects the cytotoxicity of a number of chemotherapeutic agents, including arsenic trioxide. Reactive oxygen species (ROS), the major intracellular oxidants, may be a determinant of cellular susceptibility to arsenic. Our previous studies showed that a naphthoquinone and an anthraquinone (emodin) displayed the capability of producing ROS and facilitating arsenic cytotoxicity in both leukemia and solid tumor cell lines. We therefore attempted to test emodin and several other kinds of anthraquinone derivatives on EC/CUHK1, a cell line derived from esophageal carcinoma, and on a nude mouse model, with regard to their effects and mechanisms. Results showed that anthraquinones could produce ROS and sensitize tumor cells to arsenic both in vivo and in vitro. The combination of emodin and arsenic promoted the major apoptotic signaling events, i.e., the collapse of the mitochondrial transmembrane potential, the release of cytochrome c, and the activation of caspases 9 and 3. Meanwhile a combination of emodin and arsenic suppressed the activation of transcription factor NF-kappaB and downregulated the expression of a NF-kappaB-specific antiapoptotic protein, survivin. These two aspects could be antagonized by the antioxidant N-acetyl-L-cysteine. Therefore anthraquinones exert their effects via a ROS-mediated dual regulation, i.e., the enhancement of proapoptosis and the simultaneous inhibition of antiapoptosis. In vivo study showed that emodin made the EC/CUHK1 cell-derived tumors more sensitive to arsenic trioxide with no additional systemic toxicity and side effects. Taken together, these results suggest an innovative and safe chemotherapeutic strategy that uses natural anthraquinone derivatives as ROS generators to increase the susceptibility of tumor cells to cytotoxic therapeutic agents.

Mechanisms of glucocorticoid-induced Leydig cell apoptosis.

The high levels of corticosterone (CORT) that are typically achieved during stress induce apoptotic death of Leydig cells. The intracellular mechanisms by which CORT acts on Leydig cells to induce apoptosis are unknown, and the present study tested for mediation by Fas ligand (FasL), a member of the tumor necrosis factor ligand family, in association with caspase activation. In addition, another apoptotic pathway involving in the participation of mitochondria was studied by evaluation of mitochondrial membrane potential (DeltaPsi) loss and generation of reactive oxygen species (ROS), which are early apoptotic events in many cell types. Rat Leydig cells were isolated from adrenalectomized rats on day 90 postpartum at 3, 6, 12, 24 and 48 h after the start of CORT administration (at a dose of 5 mg total/100 g body weight per day intraperitoneally in two daily injections starting 3 days after surgery). Both FasL and Fas receptor protein levels, analyzed by Western blot and fluorescent immunohistochemistry, increased at 6 h after the start of CORT administration, peaking at 24 h and declining thereafter. Leydig cell caspase-3 activity was analyzed in vitro. Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nM CORT in vitro, and the abundance of the fragments was more pronounced at 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Western blotting analysis revealed that procaspase-3 was present only at low levels in untreated control Leydig cells, and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced, and the cleaved, active caspase-3 forms appeared and increased through 24 h. These results indicated that FasL/Fas and caspase were implicated in CORT-mediated Leydig cell apoptosis. Decreased DeltaPsi and increased ROS generation were also measurable in Leydig cells for up to 2 days following CORT administration in vitro. These data indicate that activation of the Fas system, cleavage of procaspase-3, loss of DeltaPsi and increased ROS generation are all implicated in the process of CORT-induced Leydig cell death.

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period.

AIM: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. METHODS: The indirect immunofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. RESULTS: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary spermatocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. CONCLUSION: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

Spatial and temporal expression of germ cell nuclear factor in murine epididymis.

AIM: To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period. METHODS: The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope. RESULTS: GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells. CONCLUSION: GCNF may play an important role in epididymal differentiation and development and in sperm maturation.

[Influence of Ureaplasma urealyticum infection on the sperm-egg binding associated molecule, sulfogalactosylglycerolipid].

OBJECTIVE: To study the influence of Ureaplasma urealyticum (Uu) infection on the sperm-egg binding associated molecule, sulfogalactosylglycerolipid (SGG). METHODS: Epididymal sperm was collected from adult mice. The sperm suspension was randomly divided into 4 groups: Uu group (coincubated with Uu suspension), medium group (coincubated with Uu medium), normal group and PRS group. The indirect immunofluorescence technique was used to localize SGG on the sperm membrane and to observe the influence of Uu on SGG. RESULTS: In the epididymal sperm, SGG was localized to the head plasma membrane overlaying the acrosomal region. The SGG-positive rate of the sperm coincubated with Uu medium was 82.0%, while that of the sperm coincubated with Uu suspension was reduced to 39.0% (P = 0.001). CONCLUSION: Uu can adhere to the sperm surface. SGG might be a membrane receptor on the sperm surface for Uu infection of the mammalian male genital tract. The blockage of SGG by Uu might be one of the molecular mechanisms correlative to male infertility induced by Uu infection.

Bactericidal/permeability-increasing protein originates in both the testis and the epididymis and localizes in mouse spermatozoa.

Bactericidal/permeability-increasing protein (BPI) is an endogenous antibiotic protein with activity against gram-negative bacteria. In the present study, we examined the expression of BPI in postnatal mouse testes and epididymides as well as the subcellular localization within epididymal spermatozoa. Our results showed that, BPI mRNA was expressed in testis and epididymis independently. Throughout the epididymis, the BPI protein level gradually decreased in the epididymal epithelium in a spatial manner, specialized within the cytoplasm of clear cells in the cauda part. We detected BPI proteins in intact acrosome, implying its testicular origin; on the other hand, after the acrosome reaction, BPI proteins were observed dispersed across the entire sperm head, especially enriched at the equatorial segment. Our findings suggested a dual origin of the BPI that generated both in the testis and epididymis, and associated with mouse spermatozoa. BPI protein might be involved in the dynamics modification of the sperm plasma membrane and also the fertilization process.

The role of Zn-alpha2 glycoprotein in sperm motility is mediated by changes in cyclic AMP.

Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-alpha2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the beta(3)-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (P<0.05 and P<0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.

Identification and characterization of ERp29 in rat spermatozoa during epididymal transit.

The mammalian epididymis is able to create sequential changes in the composition of luminal fluid throughout its length, wherein spermatozoa undergo morphological, biochemical, and physiological modifications. Subsequently, spermatozoa acquire the ability for fertilization upon reaching the epididymal cauda. In this study, protein variations in Sprague-Dawley rat spermatozoa along the caput and caudal regions of epididymis were investigated by high-resolution two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry. From total protein spots on the 2DE maps, 43 spots were shown to be significantly modified as sperm traverse the epididymis, and seven unambiguous proteins were identified from them. Finally, using indirect immunofluorescence, we demonstrated that localization of one of these seven proteins, the endoplasmic reticulum protein (ERp29) precursor, which was first reported in mammalian spermatozoa, was apparently up-regulated as the sperm underwent epididymal maturation and expressed mainly on caudal sperm. Western blot analysis also revealed that ERp29 precursor, from both whole spermatozoa and membrane proteins, increased significantly as the sperm underwent epididymal maturation. Furthermore, the results from immunofluorescence-stained epididymal frozen sections demonstrated that ERp29 was localized in cytoplasm of epididymal epithelia, and the fluorescence intensity was significantly higher in the caudal epididymis than in the caput. These clues indicated that the ERp29 precursor, perhaps related to secretory protein synthesis and absorbed by spermatozoa, may play a vital role in sperm maturation during the epididymal transit, particularly, in the sperm/organelle membrane.