Ph Chow

Male genital tract antioxidant enzymes : Their ability to preserve sperm DNA integrity

Male germ cells are unique because they lose a bulk of their cytoplasm as cytoplasmic droplets when they develop, leading to a decrease in endogenous antioxidant and hence a dependence on extracellular antioxidant system to overcome oxidative stress. Spermatozoa are particularly vulnerable to oxidative stress because their plasma membrane is rich in polyunsaturated fatty acids and membrane-bound NADPH oxidase. To protect spermatozoa from oxidative attack, an optimal amount of reactive oxygen species is maintained by balancing the reactive oxygen species generated during sperm maturation in the epididymidis and antioxidants in secretions of the male reproductive tract. The male accessory sex glands secretions have been shown to be the major source of antioxidant enzymes in the ejaculate and have the important function of preserving sperm DNA integrity from oxidative stress experienced in the uterine environment. In our in vivo golden hamster model, ablation of the five major male accessory sex glands, namely the ampullary glands, coagulating glands, dorsolateral prostate, ventral prostate and seminal vesicle, was found to cause higher incidence and greater degree of DNA damage in spermatozoa. These damaged sperm are able to undergo fertilization at the same rate as intact ones; however, the outcome of embryos sired is seriously affected.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up‐regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down‐regulated at this time point. Semiquantitative reverse transcriptase‐polymerase chain reaction and Western blot analyses confirmed the data obtained from the two‐dimensional electrophoresis gels. Furthermore, we were able to up‐regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up‐regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp‐4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down‐regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference‐mediated silencing of Prdx1 expression, indicating that Prdx1 down‐regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock‐down cells revealed that the level of NF‐kappaB inhibitor epsilon (IκBε) was dramatically reduced. Furthermore, we found an increase in NFκB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS‐induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival‐promoting activities. Developmental Dynamics 233:266–281, 2005.

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.

CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR−/−) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.

Total ablation of paternal accessory sex glands curtails developmental potential in preimplantation embryos in the golden hamster

The effects of total removal of paternal accessory sex glands (TX) on preimplantation embryonic development was studied in the golden hamster model. Cell numbers of the two groups of embryos did not differ up to 60 h p.c., but at 66 and 70 h p.c., each TX embryo has 2 and 3 cells less respectively (P<0.05, TX vs SH). At 70 h p.c., 46.6±4.4 of the TX embryos blastomeres were labelled with the terminal deoxynucleotide transferase – mediated dUTP-nickend-labelling technique, compared with 31.5±2.1 in the SH group (P<0.01, TX vs SH). No difference was found in the SDS-PAGE profiles of two-cell embryos from the two groups. An extra band corresponding to 136.5 kDa was consistently found in the four-cell TX embryos. The nascent proteins profiles of four-cell embryos from the two groups were similar. As the embryos progressed from two to four cells, the protein content decreased by 16% in the SH embryos (P<0.05) and 7% in the TX embryos. These observations suggest that total ablation of paternal accessory sex glands could result in developmental aberrations from the two-cell to morula stages and a higher incidence of apoptosis at 70 h p.c.

Ablation of paternal accessory sex glands is detrimental to embryo development during implantation

The accessory sex glands are present in most mammals, but their function(s) have not yet been clearly defined. In the golden hamster, removal of all the glands or the ventral prostate alone have been shown to considerably reduce fertility, while the effect is milder if the ampullary glands only are removed. In this study, embryo development from the 5th to the 7th day after mating are examined. Structural and morphometric criteria such as cell number, cell density, embryo volume, volume fraction of proamniotic cavity further revealed that abnormalities can be demonstrated as early as day 5 in the embryos sired by males with the ventral prostate gland alone or all glands ablated. Twin implantation and deviation from normal implanted axis are also observed. This is likely to be attributed to attenuated cell proliferation, as indicated by proliferating cell antigen labelling and more necrotic cell death. Taken together, exposure of sperm to secretions of the male accessory sex glands in particular, the ventral prostate, is important for differentiation and multiplication of cells after the embryo has implanted.

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.

Absence of paternal accessory sex gland secretions disturbs epigenetic reprogramming and expression of Igf2 and Dlk1 in golden hamster embryos.

Accessory sex gland (ASG) secretion is known to exert an effect on sperm that is heritable in hamster embryos. We hypothesized that ASG secretion changes the sperm epigenome, which in turn is propagated in sired embryos. To test our hypothesis, we produced male hamsters that were devoid of either all ASG (TX) or only the ventral lobe of the prostate gland (VPX). A sham-operated control group (SH) was also established. These males were mated with normal females; uterine sperm, fertilized oocytes, and pre-implantation embryos were harvested from the females after mating. Epididymal sperm were collected at the end of experiments. Immunofluorescent staining was performed on these harvested specimens using antibodies against 5-methylcytosine, Dnmt1, Dnmt3a, Dnmt3b, protamine 1, protamine 2, and aectyl-H4K5. Expression of Igf2 and Dlk1 were analyzed by real-time RT PCR and in situ hybridization. We demonstrated that the DNA methylation pattern changed dynamically in SH, TX, and VPX fertilized oocytes. In VPX and TX embryos, DNA demethylation was slower and remethylation was delayed when compared with SH embryos. In addition, Dnmt3b expression was also abnormal. When sperm from VPX and TX males were exposed to whole ASG secretion in vivo, the resulting embryos all methylated normally. Immunofluorescent staining revealed that there was no difference in protamine packaging of uterine sperm from VPX and TX males. The staining also showed a lower level of acetyl-H4K5 expression in the male pronuclei of TX produced embryos. Furthermore, the VPX and TX embryos also expressed higher levels Igf2, and Dlk1. We concluded that interactions between ASG and sperm affected: (1) histone acetylation in male pronuclei; (2) DNA methylation in fertilized oocytes; and (3) Igf2 and Dlk1 expression embryos.

The growth arrest specific gene (gas6) protein is expressed in abnormal embryos sired by male golden hamsters with accessory sex glands removed.

Expression of growth arrest specific gene (gas6) and its receptors in embryonic and uterine tissues in normal pregnancy and pregnancy that produces abnormal embryos sired by hamsters with partial or total deletion of male accessory sex glands was studied by in situ hybridization, immunohistochemistry, reverse-transcription polymerization reaction and enzyme-linked immunoabsorbant assay. At oestrus, very strong gas6 mRNA and Gas6 expression were seen only in the uterine epithelium and endometrial glands. Upon implantation, both of them could be demonstrated in the decidualizing stroma. From day 4 to day 7 p.c, gas6 mRNA was present in the embryo, but Gas6 immunoreactivity was only found in those showing features of degeneration. The gas6:β-actin mRNA ratio was low in oestrus and at day 4 of pregnancy but rose as the embryo grew. As for the receptors, Rse was detected in embryonic cells during days 5–7 p.c., and decidual cell from days 4 to 7 p.c., but Mer could be found in decidual cells and trophoblasts. It was concluded that gas6 had a role in endometrial transformation during decidualization and trophoblastic invasion. In the embryo, gas6 was transcribed, but the protein was only produced in response to need, such as when normal progression of development was threatened.

Tissue specific expression and sequence analysis of a stress responsive gene Bre in adult golden hamster (Mesocricetus auratus)

Bre (brain and reproductive organ-expressed) is a new and putative stress-modulating gene of yet unknown function. BRE has previously been shown to interact with type 1 tumor necrosis factor receptor (TNFR1) and modulate the action of TNF. Apart from the brain and reproductive organs, Bre and BRE are highly expressed in steroid producing tissues such as the adrenal gland. Here we report for the first time the cloning of the Bre gene from golden hamster, a model organism extremely valuable for reproduction and steroid research, and examination of its tissue specific expression. Sequence analysis demonstrated that the peptide sequence of BRE in hamster shares ~99% homology with those of human, monkey and mouse. The hamster Bre gene transcribed a ~1.8-kb mRNA which translated a 44-kDa protein. Bre was strongly expressed in neurons and luminal epithelia of urogenital, digestive and respiratory organs. Bre was also detected in lymphoid tissues and endocrine glands. Immunohistochemistry demonstrated a similar protein expression pattern. Exceptions to this included the adrenal gland, where a high level of Bre was accompanied by weak immunoreactivity; as well as the oocytes and islets of Langerhans, where BRE protein but not the mRNA was localized. These data indicated that Bre gene products were expressed in a wide variety of tissues other than the brain and reproductive organs, as was originally described. Based on our findings, we propose that Bre is a housekeeping gene in tissues that are constantly subjected to environmental hazards such as luminal epithelia. Our results further support the proposed role for BRE in endocrine and immune functions.