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.

SUMMARY 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. Mol. Reprod. Dev . 81: 315–325, 2014. © 2014 Wiley Periodicals, Inc.

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.

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.

Objective To investigate the biological origin of characteristic autofluorescence in normal gastric epithelial cells and different degree differentiated gastric cancer cells. Methods The behaviors of autofluorescence at wavelength 500-530 nm between gastric cancer cells and normal gastric epithelial cells were compared by laser confocal fluorescence microscopy. The site of autofluorescence was examined after stained by Rodamine 123. Alteration of autofluorescence intensity was assessed after the addition of oxidative respiratory chain blocking agent carbonylcyanide-mchlorophenylhydrazone (CCCP). Results Autofluorescence at wavelength 500-530 nm mainly came from mitochondria. The autofluorescence intensity of all the different degree differentiated gastric cancer cells was weaker than that of the normal gastric epithelial cells. The autofluorescence intensity increased with the addition of CCCP. Conclusions The weakening of autofluorescence at wavelength 500-530nm might help the diagnosis of gastric cancer. The origin of the autofluorescence was probably the redox coenzyme(reduced nicotinamide adenine dinucleotide,NADH) in mitochondria.

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.

Objective To clone rat ERp57 gene and express the recombinant protein;then,to generate anti-rERp57 antibodies and localize its distribution on rat spermatozoa and oocytes.Methods Rat ERp57 cDNA was amplified from total RNA of SD rat testis by RT-PCR and PCR product was subcloned into prokaryotic expression vector pET-28a(+). Then,this recombinant plasmid was transformed into E.coli.BL21 and recombinant ERp57 was expressed in host cells by IPTG induction,and purified by Ni-NTA resin under denature condition.Finally,rabbit anti-rat recombinant ERp57 polyclonal antibodies were generated,and the levels of ERp57 both in rat spermatozoa and oocytes were detected by Western blot and its distribution was localized by indirect immunofluorescence analysis.Results Rat ERp57 gene, protein and anti-rat rERp57 were successfully cloned,expressed and generated respectively.ERp57 existed in both rat mature spermatozoa and oocytes.Its distribution was mainly localized on the inner part of sperm head and appeared to be translocated to equatorial region after acrosome reaction.For oocytes,however,it was almost localized on the whole cell surface.Conclusion These results suggested that the distribution and expression of ERp57 in both rat spermatozoa and oocytes might be associated with the sperm-oocyte fusion.

The aim of this study is to investigate the effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats. Ninety-eight healthy male rats were divided into four groups: three exposure groups and a control group. Carbendazim was administered orally to male rats at 0, 20, 100 and 200 mg/kg for 80 days prior to mating. Each male was cohabited with an unexposed female for a maximum of 5 days. In 100 and 200 mg/kg groups, the mating index was relatively increased, the fertility index was decreased, and the testis weight, the sperm counts and motility were also decreased. The levels of luteinizing hormone (LH) showed a decreasing tendency and there was a statistical difference between the 200 mg/kg group and the control group. There were no obvious effects on the levels of follicle stimulating hormone (FSH) and testosterone (T). Histopathological evaluation showed atrophic seminiferous tubules, decreased germ cells, and increased sloughing of germ cells. Flow cytometric analysis of the testicular tissue revealed that carbendazim inhibited meiotic transformation and interfered with the spermatogenic process. These results suggest that carbendazim has adverse effects on spermatogenesis, resulting in reduced fertility in male rats.

Objective:To investigate the possible functions of zonadhesin D4 domain in the process of fertilization.Methods:Two cDNA fragments coding for VWD subdomain and the rest C-termianl fragment of D4 domain were cloned and expressed in E.coli.Antibodies against recombinant D4 domain were raised in New Zealand white rabbits immunized with recombinant fragments.The expression of this protein on mouse sperm and the specificity of the polyclonal antibodies were determined by indirect immunofluorescent staining and Western blot analysis.After treated sperm with these antibodies,sperm-zona pellucida binding assay and in vitro fertilization were carried out in BALB/c mice.Results:Recombinant D4 domain was successfully cloned and expressed.The expression of the protein was limited on the anterior head of mouse sperm and the antibodies against it were specific.The treatment of sperm with polyclonal antibodies did not significantly decrease the average number of sperm bound to zona pellucida.Meanwhile,the percentage of fertilized eggs was not decreased significantly in comparison to control rabbit IgG.Conclusion:The results suggest that polyclonal antibodies against recombinant zonadhesin D4 domain may not inhibit in vitro fertilization of mouse.

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.

目的:分析胃良恶性黏膜的固有荧光差异.方法:收集胃癌手术标本和胃低级别上皮内瘤变黏膜标本制成冰冻切片,在激光共聚焦显微镜下观察并测定荧光强度.结果:固有荧光主要位于胃壁黏膜层和黏膜下层.与正常胃黏膜相比,在胃癌和低级别上皮内瘤变组织中平均绿红荧光强度比较低(1.57±0.69,2.06±0.51 vs 3.75±1.41,P<0.01,0.05).同一患者的正常胃黏膜固有荧光强度明显高于胃腺癌胃黏膜(P＜0.05),而与低级别上皮内瘤变胃黏膜之间无显著差异.结论:胃癌黏膜绿色固有荧光强度变化可作为恶性病变的标志.

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.

Objective: To identify and localize ERp29 in spermatozoa from BALB/c mouse epididymal caput and cauda. Methods: ERp29 was identified by Western Blot, and localized by indirect immunofluorescence employed laser scanning confocal microscopy. Results: ERp29 was present in BALB/c mouse spermatozoa from epididymal caput and cauda, which was mostly localized on the partial region of sperm head from epididymal caput; however, it was localized on the whole sperm head and tail region (principal piece) from cauda. Additionally, the spermatozoa from epidiymal cauda contained more ERp29 protein compared with that in spermatozoa from the caput. Conclusion: ERp29 might play an important role in mouse sperm maturation during epididymal transit.

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.

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.

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.

Objective: To identify and localize AAT in human semen, especially in spermatozoa. Methods: AAT was identified by Western Blotting, and localized by indirect immunofluorescence studies using laser scanning confocal microscope. Results: AAT was present in human semen and spermatozoa, which was mostly localized on the head region of spermatozoa, in particular on the equatorial plate. Conclusion: This research will help further investigation of the possible relationship between AAT and human sperm.