William S.B. Yeung

Sperm-borne microRNA-34c is required for the first cleavage division in mouse

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin–treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3′ UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti–Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3′UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida.

Fertilization in humans is initiated by binding of spermatozoa to a selectin ligand on the egg’s extracellular matrix. Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewisx sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewisx sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewisx sequence represents the major carbohydrate ligand for human sperm-egg binding.

Effect of perinatal and postnatal bisphenol A exposure to the regulatory circuits at the hypothalamus–pituitary–gonadal axis of CD-1 mice

Bisphenol A (BPA) is used in the manufacture of many products and is ubiquitous in the environment. Adverse effects of BPA on animal reproductive health have been reported, however most of the studies relied on the approaches in the assessment of conventional histology and anatomical features. The mechanistic actions of BPA are not clear. In the present study, a murine model was used to study potential effects of BPA exposure during perinatal and postnatal periods on endocrine functions of hypothalamic-pituitary-gonadal (HPG)-axis. At the hypothalamic-pituitary level, BPA exposure resulted in the up-regulation of the expression levels of KiSS-1, GnRH and FSH mRNA in both male and female pups. At the gonadal levels, BPA caused inhibition in the expressions of testicular steroidogenic enzymes and the synthesis of testosterone in the male pups. Conversely exposure to BPA resulted in a greater aromatase expression level and the synthesis of estrogen in the female pups. BPA is a weak estrogen agonist and its effects reported on animal studies are difficult to reconcile with mechanistic action of estrogen. In this study we hypothesized that the effects of BPA on reproductive dysfunction may be due to its actions on gonadal steroidogenesis and so the anomalous releases of endogenous steroid hormones. This non-ER-mediated effect is more potent in affecting the feedback regulatory circuits in the HPG-axis.

A randomized double blind comparison of real and placebo acupuncture in IVF treatment.

BACKGROUND Acupuncture has been used during IVF treatment as it may improve outcome, however, there are concerns about the true efficacy of this approach. This randomized double blind study aimed to compare real acupuncture with placebo acupuncture in patients undergoing IVF treatment. METHODS On the day of embryo transfer (ET), 370 patients were randomly allocated to either real or placebo acupuncture according to a computer-generated randomization list in sealed opaque envelopes. They received 25 min of real or placebo acupuncture before and after ET. The endometrial and subendometrial vascularity, serum cortisol concentration and the anxiety level were evaluated before and after real and placebo acupuncture. RESULTS The overall pregnancy rate was significantly higher in the placebo acupuncture group than that in the real acupuncture group (55.1 versus 43.8%, respectively, P = 0.038; Common odds ratio 1.578 95% confidence interval 1.047-2.378). No significant differences were found in rates of ongoing pregnancy and live birth between the two groups. Reduction of endometrial and subendometrial vascularity, serum cortisol concentration and the anxiety level were observed following both real and placebo acupuncture, although there were no significant differences in the changes in all these indices between the two groups. CONCLUSIONS Placebo acupuncture was associated with a significantly higher overall pregnancy rate when compared with real acupuncture. Placebo acupuncture may not be inert. Trial registered with HKClinicalTrials.com: number HKCTR-236.

Effects of chinese green tea on weight, and hormonal and biochemical profiles in obese patients with polycystic ovary syndrome : A randomized placebo-controlled trial

Objectives: To study the effects of green tea on body weight, and biochemical and hormonal profiles in obese Chinese women with polycystic ovary syndrome (PCOS). Methods: Thirty-four obese Chinese women with PCOS were randomized into either treatment with green tea capsules or placebo for 3 months. The anthropomentric measurements, and biochemical and hormonal profiles before and after treatment in each group were compared. Results: The body weight of the green tea group decreased by a nonsignificant 2.4% after treatment; whereas the body weight, body mass index (BMI), and body fat content of the control group were significantly higher after 3 months. There were no differences in any of the hormone levels measured in either group. The biochemical profiles of the two groups were also similar except that there was a small but significant rise in the triglyceride level in the green tea group. Fewer patients in the green tea group remained amenorrhoeic, but this was not significantly different from the control group. Conclusions: Green tea supplementation did not significantly reduce body weight in obese women with PCOS, nor did it alter the glucose or lipid metabolism.

Effects of Native Human Zona Pellucida Glycoproteins 3 and 4 on Acrosome Reaction and Zona Pellucida Binding of Human Spermatozoa

Abstract Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca2+ channels, and extracellular Ca2+ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca2+ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding..

Gene expression profiling of human peri-implantation endometria between natural and stimulated cycles

OBJECTIVE To investigate the effect of high serum E(2) levels in gonadotropin-stimulated cycles (hCG+7) on the gene expression patterns of human endometrium compared with natural cycles on the seventh day of LH surge (LH+7) and elucidate the underlying molecular changes that may be related to endometrial receptivity. DESIGN Observational study. SETTING University Hospital. PATIENTS(S) Infertile patients with normal menstrual cycles undergoing IVF treatment. INTERVENTION(S) Gonadotropin stimulation and endometrial biopsy. MAIN OUTCOME MEASURE(S) Gene expression by microarray and quantitative polymerase chain reaction (qPCR). RESULT(S) Endometrial samples from natural (n = 5) and stimulated (n = 8) cycles were collected. Patients in the stimulated cycles were classified as moderate (n = 4) or excessive (n = 4) responders if their serum E(2) levels on the day of administration of hCG were <or=20,000 pmol/L or >20,000 pmol/L, respectively. The RNA transcripts were profiled by Affymetrix HG-U133A microarray. Clustering and principal component analysis demonstrated a significant difference (>or=2-fold) in the expression patterns of 411 genes among the three groups. Putative estrogen response elements or progesterone response elements were identified in the promoter regions of 49 differentially expressed genes of diverse biologic functions. The qPCR confirmed the microarray result in 47 endometrial samples. CONCLUSION(S) High serum E(2) and/or progesterone modulate the gene expression profiles of human endometrium and may affect endometrial receptivity.

A Comparative Study of Gene Expression in Murine Embryos Developed In Vivo, Cultured In Vitro, and Cocultured with Human Oviductal Cells Using Messenger Ribonucleic Acid Differential Display

Abstract The objectives of this study were to compare the mRNA expression patterns in early mouse embryos in different culture conditions by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Embryos developed in vivo, cultured in vitro, and cocultured with human oviductal epithelial cells were studied at the 2-cell, 4-cell, 8-cell/morula, and blastocyst stages. Messenger RNA profiles were displayed by DDRT-PCR using downstream T11VV (V = A, C, or G) and upstream decamer primers. Total cDNA banding patterns were highly conserved in the three groups studied. Some fragments are unique in different culture conditions. Thirteen out of the 40 selected differentially expressed clones were characterized. The DNA sequence analyses of these clones displayed high sequence homology with cDNA sequences in the mouse expressed sequence tag database. Using semiquantitative RT-PCR, we confirmed differential expression of these DD amplicons in the three groups of embryos. The temporal expression of some of the selected DD amplicons during preimplantation development were studied in the three groups of embryos. In conclusion, DDRT-PCR is an effective tool for contrasting gene expression patterns and characterizing mRNA transcripts in mouse embryo.

The embryotrophic activity of oviductal cell-derived complement C3b and iC3b, a novel function of complement protein in reproduction.

The oviduct-derived embryotrophic factor, ETF-3, enhances the development of trophectoderm and the hatching process of treated embryos. Monoclonal anti-ETF-3 antibody that abolishes the embryotrophic activity of ETF-3 recognized a 115-kDa protein from the conditioned medium of immortalized human oviductal cells. Mass spectrometry analysis showed that the protein was complement C3. Western blot analysis using an antibody against C3 confirmed the cross-reactivities between anti-C3 antibody with ETF-3 and anti-ETF-3 antibody with C3 and its derivatives, C3b and iC3b. Both derivatives, but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as evidenced by the presence of C3 immunoreactivity and mRNA in the human oviduct and cultured oviductal cells. Cyclical changes in the expression of C3 immunoreactivity and mRNA were also found in the mouse oviduct with the highest expression at the estrus stage. Molecules involving in the conversion of C3b to iC3b and binding of iC3b were present in the human oviduct (factor I) and mouse preimplantation embryo (Crry and CR3), respectively. In conclusion, the present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development.

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.