Effects of sperm DNA damage on the levels of RAD51 and p53 proteins in zygotes and 2-cell embryos sired by golden hamsters without the major accessory sex glands.

We previously reported that the male accessory sex gland (ASG) secretion is the main source of antioxidants to safeguard sperm genomic integrity and functional competence. Removal of all ASGs in the golden hamster can reduce male fertility by increasing embryo wastage. This study aims to investigate whether the oxidative DNA-damaged sperm from hamsters without all ASGs (TX) could successfully fertilize oocytes and to qualify the status of DNA repair by the expression of RAD51 and p53 proteins. Here we demonstrated a significantly higher DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine) in sperm from TX males than those from sham-operated males. Comet assays demonstrated that all female pronuclei in both zygotes were intact, but single- and double-strand DNA damage was found in decondensed sperm in TX males only. DNA damage could also be detected in both nuclei of the TX 2-cell embryos. RAD51, a DNA repair enzyme, was found to be evenly distributed in the cytoplasm and nuclei in oocytes/zygotes, while at the 2-cell stage, a strong expression of p53 protein and a larger clear perinuclear area without RAD51 expression were found in TX embryos. In conclusion, we demonstrated for the first time DNA damage in decondensed sperm of zygotes and blastomeres of 2-cell stage embryos sired by TX males, resulting in the activation of DNA repair. Sperm DNA damage could induce the increase in p53 expression and the reduction of RAD51 expression in the TX 2-cell stage embryos.

Growth-differentiation factor-8 (GDF-8) in the uterus: its identification and functional significance in the golden hamster.

Transforming growth factor-beta superfamily regulates many aspects of reproduction in the female. We identified a novel member of this family, growth-differentiation factor 8 (GDF-8) in the 72 h post coital uterine fluid of the golden hamster by proteomic techniques. Uterine GDF-8 mRNA decreased as pregnancy progressed while its active protein peaked at 72 h post coitus (hpc) and thereafter stayed at a lower level. At 72 hpc, the GDF-8 transcript was localized to the endometrial epithelium while its protein accumulated in the stroma. Exogenous GDF-8 slowed down proliferation of primary cultures of uterine smooth muscle cells (SMC) and endometrial epithelial cells (EEC). In addition, GDF-8 attenuated the release of LIF (leukaemia inhibiting factor) by EEC. As for the embryo in culture, GDF-8 promoted proliferation of the trophotoderm (TM) and hatching but discouraged attachment. Our study suggests that GDF-8 could regulate the behavior of preimplantation embryos and fine-tune the physiology of uterine environment during pregnancy.

Absence of paternal accessory sex glands dysregulates preimplantation embryo cell cycle and causes early oviductal-uterine transit in the golden hamster in vivo.

We hypothesize that deletion of paternal accessory sex glands compromised developmental potential of preimplantation embryos because of dysregulation of blastomeric cell cycle regulators. Quantification by immunocytochemistry showed disrupted cyclicity of proliferating cell nuclear antigen, augmented p53 and p21 expression, and premature transit from oviduct to uterus.

[Identification of sperm-binding proteins in the ventral prostate of the golden hamster].

OBJECTIVE: To investigate the binding of secretory proteins in the ventral prostate to the surface of sperm. METHODS: We used different techniques to demonstrate the possibility of ventral prostate secretory proteins binding to sperm in golden hamsters. Polyclonal antibodies against crude secretion of the ventral prostate cultured in rabbits were used to detect the antigens in hamster epididymal, uterine and oviductal spermatozoa by indirect immunofluorescence technique. The uterine and oviductal spermatozoa were collected after mating with the males with or without ventral prostate glands. The ventral prostate secretory proteins were isolated and transblotted to the membrane, which was incubated with the biotinylated epididymal sperm membrane proteins, and then the biotinylated binding proteins were stained. RESULTS: An immunoreaction restricted to the middle piece was observed in the sperm incubated with the ventral prostate secretion and ejaculated sperm recovered from the uteri and oviducts. The rate of the epididymal sperm bound with the ventral prostate secretory proteins was (80 +/- 5) %, and the rats of the sperm binding to the ventral prostate secretory proteins were (30.0 +/- 4.6) % from the uterus and (16.0 +/- 3.6) % from the oviduct after mating with the males with ventral prostate glands, significantly higher than after mating with those without prostate glands (P < 0.01). Five bands were identified by Western blot analysis in vitro of the ventral prostate secretory proteins incubated with biotinylated epididymal sperm membrane proteins. CONCLUSION: The present data indicate that ventral prostate secretory proteins bind to the middle piece of sperm in golden hamsters.

Induction of growth arrest and polycomb gene expression by reversine allows C2C12 cells to be reprogrammed to various differentiated cell types.

Reversine is a small, cell permeable synthetic chemical that has the ability to reprogram C2C12 myogenic cells to become various differentiated cell types. However, we still do not know how reversine works or the genes and proteins involved. Hence, we have used comparative proteomic techniques to address this issue. We have identified several proteins that were associated with cell cycle progression which were downregulated by reversine. Simultaneously, there were proteins associated with the induction of growth arrest that were upregulated. Consequently, we investigated the effects of reversine on C2C12 cell growth and established that it inhibited cell growth. Reversine had little affects on cell survival. We also investigated whether expressions of the polycomb genes, polycomb repressive complex 1 (PHC1) and Ezh2, were affected by reversine. Polycomb group genes are normally involved in chromatin based gene silencing. We found that PHC1 and Ezh2 expressions were enhanced by reversine and that it correlated with the inhibition of muscle specific transcriptional factor genes, myogenin, MyoD, and Myf5. Therefore, we believe that reversine is able to reprogram C2C12 cells to various differentiated cell types by inducing cell growth arrest, and promoting PHC1 and Ezh2 expressions.

Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart.

In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.

Comparative proteomic analysis reveals a function of the novel death receptor-associated protein BRE in the regulation of prohibitin and p53 expression and proliferation.

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.

Ultrastructural characterization of whole hydrosalpinx from infertile Chinese women.

Hydrosalpinx (HSP) has been shown to be detrimental to the outcome of assisted reproduction, but little is known of its pathology. This prospective study examined and detailed ultrastructural characterization of HSP of infertile women presenting for assisted reproductive treatments. Both light and electron microscopies were used to characterize HSP. Hematoxylin and eosin staining of HSP showed areas without epithelial cell lining or with abnormalities such as flattening of the epithelial layer and exfoliation of epithelial cells with occasional normal columnar epithelial lining. HSP muscle fibers were atrophic and occasionally replaced by fibrous tissues, or separated by areas of severe edema. Inflammatory cells could be found in hydrosalpinx fluid (HF) in the lumen in areas with flattened to no epithelial cells, without epithelial lining, as well as in dilated blood vessels and/or lymph vessels. Scanning electron microscopy of the epithelial surface revealed epithelial denudation-severe loss of both cilia and microvilli and stomata exuding globular bodies on eroded ampulla surfaces. Severe chronic inflammation and damage to the epithelial lining and musculature of Fallopian tubes and the presence of inflammatory cells provides an explanation for HF formation, and thus for the detrimental effects of HF on reproductive processes and IVF outcome.

Increased cystic fibrosis transmembrane conductance regulator (CFTR) expression in the human hydrosalpinx.

BACKGROUND: Hydrosalpinx (HSP), characterized by abnormal fluid accumulation in the Fallopian tube, is one of the main causes of infertility in women; however, the mechanism underlying the formation of hydrosalpinx fluid (HF) remains elusive. The present study investigated the possible involvement of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, in the pathogenesis of hydrosalpinx. METHODS: Masson's trichrome staining was used to characterize epithelial transformation in human HSP; RT-PCR, immunohistochemistry and immunofluorescence staining were used for CFTR expression and localization. RESULTS: Masson's trichrome staining showed areas of epithelial transformation, focally attenuated and pseudostratified. Immunostaining showed enhanced CFTR immunoreactivity in the focally attenuated and pseudostratified areas of HSP epithelium. RT-PCR revealed that CFTR expression in HSP was significantly greater than that in normal Fallopian tubes. CONCLUSIONS: These results indicate that HSP epithelium undergoes epithelial transformation with elevated CFTR expression, which may lead to increased transepithelial electrolyte and fluid secretion resulting in HF formation. The present findings may lead to the development of new treatment strategies for infertile patients with HSP.

Male genital tract antioxidant enzymes: their source, function in the female, and ability to preserve sperm DNA integrity in the golden hamster.

Recently, we reported that male accessory sex gland (ASG) secretions protect sperm genomic integrity by demonstrating that DNA damage was more extensive in sperm not exposed to the secretions. The present study was conducted to find out if ASGs secrete the main antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx or GSH-Px), and catalase (CAT) and if the most abundant one, SOD, can protect those sperm that were not exposed to ASG secretions against NADPH-induced oxidative stress. Four experimental groups of male golden hamsters were used: intact animals with proven fertility, animals with all major ASGs removed (TX), animals that were bilaterally vasectomized, and sham-operated controls. SOD, CAT, and GPx activities were measured in secretions from all 5 ASGs and sperm-free uterine flushing from virgin females and those mated with the experimental males. The alkaline comet assay was used to analyze DNA integrity of the TX group sperm after incubation in a medium containing 50 U/mL of SOD along with 0 to 20 mmol/L NADPH. The main antioxidant enzyme in ASGs was SOD from coagulating glands (P <.05) and GPx together with CAT from ampullary glands (P <.05). Uterine flushing of ejaculates that contained ASG secretions had more SOD and CAT activities than those with epididymal secretions alone (P <.05 and P <.001, respectively), whereas activity of GPx was the same (P >.05). Addition of SOD in vitro dose dependently decreased the incidence of single-strand DNA damage in sperm not exposed to ASG secretions incubated in the presence of 0 to 20 mmol/L NADPH (P <.001). These results indicated that, in terms of abundance, SOD was the main antioxidant enzyme secreted by male ASGs, whereas CAT was the second one. The GPx activity came from both epididymis and ASGs. We conclude that ASG secretions play a significant role in protecting sperm against oxidative stress.

Effect of oral Tadenan treatment on rabbit bladder structure and function after partial outlet obstruction.

PURPOSE: There is increasing evidence that the progressive dysfunction induced by partial outlet obstruction is mediated by ischemia-reperfusion, and bladder decompensation results from ischemia-reperfusion induced damage to the cellular and subcellular organelle membranes of nerve and smooth muscle, mitochondria and sarcoplasmic reticulum. Tadenan, an extract of Pygeum africanum, is a therapeutic prescribed in Europe to relieve symptoms of obstructive bladder dysfunction secondary to benign prostatic hyperplasia. There is excellent experimental evidence that Tadenan treatment of obstructed rabbits reduces and reverses the progression of bladder decompensation. We determined whether Tadenan therapy can reverse the morphological damage associated with obstructive dysfunction. MATERIAL AND METHODS: A total of 36 male New Zealand White rabbits were separated into 6 groups of 6 each. Rabbits in groups 1 and 2 underwent sham operation. For 3 weeks beginning 2 weeks after sham operation group 1 was treated with vehicle and group 2 was treated with 30 mg./kg. Tadenan daily. Rabbits in groups 3 to 6 underwent partial outlet obstruction surgery. Two weeks after obstruction each rabbit was treated for 3 weeks with vehicle in group 3, and with 1, 10 and 30 mg./kg. Tadenan in groups 4, 5 and 6, respectively. After the completion of treatment cystometry was performed on each rabbit and isolated bladder strips were evaluated for contractile responses to field stimulation, adenosine triphosphate, carbachol and KCl. Separate strips were fixed for electron microscopy to determine the location and severity of cellular and subcellular membrane damage. RESULTS: Partial outlet obstruction resulted in reduced compliance, decreased responses of bladder strips to all forms of stimulation tested, and significant and extensive damage to cellular and subcellular organelle membranes consistent with an ischemia-reperfusion etiology. Daily 1 and 10 mg./kg. Tadenan treatments had little effect on the obstruction induced increase in bladder weight or the deleterious changes in bladder function and structure. However, treating obstructed rabbits with 30 mg./kg. Tadenan daily resulted in reduced bladder hypertrophy, improved compliance, improved contractile responses to nearly normal levels of isolated bladder strips to all stimuli tested and reversal of obstruction induced structural damage to cellular and subcellular organelle membranes. CONCLUSION: Tadenan treatment of obstructed rabbits resulted in a dose dependent improvement in bladder ultrastructure in parallel with improved bladder compliance and contractile responses of isolated strips to stimulation, providing support for the hypothesis that damage to cellular and subcellular organelle membranes mediates the contractile dysfunction induced by partial outlet obstruction.