Kai-Fai Lee

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.

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.

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.

Excessive ovarian stimulation up-regulates the Wnt-signaling molecule DKK1 in human endometrium and may affect implantation: an in vitro co-culture study

BACKGROUND High serum estradiol (E2) levels following ovarian stimulation lead to reduced implantation and pregnancy rates, yet the underlying mechanisms remain unknown. We investigated if aberrant expression of genes in the Wnt-signaling pathway may be involved. METHODS Microarray and real-time PCR analysis were performed to analyze gene expression profiles of endometrial samples taken at day hCG + 7 in stimulated cycles, and days LH + 7 and LH + 10 in natural cycles. Expression of several Wnt-signaling transcripts, including Dickkopf homolog 1 (DKK1), DKK2 and secreted frizzled-related protein 4 (sFRP4), was analyzed throughout the menstrual cycle. JAr spheroid/Ishikawa endometrial cell co-culture experiments were established to study effects of DKK1 on spheroid attachment in vitro. RESULTS We identified 351 differentially expressed genes. Endometrial samples taken at hCG + 7 had similar expression profiles to those at LH + 10. DKK1 transcripts were up-regulated and DKK2 and sFRP4 were down-regulated in the stimulated compared with LH + 7 group (all P < 0.05). DKK1 transcripts were low in proliferative phase (PS) and increased in late-secretory phase (LS, P < 0.05), although DKK2 peaked in mid-secretory phase (P < 0.05). sFRP4 transcripts were high in PS. Treatment of spheroid with recombinant human DKK-1 protein dose-dependently suppressed (P < 0.05 versus control) spheroids attachment onto endometrial cells (associated with decreased beta-catenin protein): this suppression was nullified by anti-DKK1 antibody. CONCLUSION Gene expression patterns in stimulated cycles resembled those of LS in natural cycles, when the implantation window is about to close, suggesting high serum E2 and/or progesterone concentrations may advance endometrial development, altering the implantation window and possibly decreasing pregnancy rate. Aberrant expression of DKK1 might impair embryo attachment and implantation in vivo.

Kidney claudin-19: localization in distal tubules and collecting ducts and dysregulation in polycystic renal disease.

Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin‐19, in kidneys. Mouse claudin‐19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary‐related claudins are claudin‐1 and ‐7, which share ∼75% DNA sequence homology with claudin‐19. Claudin‐19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT‐PCR. Claudin‐19 and zonula occludens‐1 (ZO‐1) are localized at junctional regions of Madin–Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO‐1 is found in the claudin‐19‐associated protein complexes in MDCK cells by co‐immunoprecipitation. Using aquaporin‐1 and aquaporin‐2 antibodies as markers for different renal segment, strong expression of claudin‐19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin‐19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin‐19 immunoreactivity is found co‐localized with the ZO‐1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin‐19 and ZO‐1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin‐19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin‐19 are noticed, suggesting a possible correlation between claudin‐19 and renal disorders. Taken together, claudin‐19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.

Zona-Binding Inhibitory Factor-1 from Human Follicular Fluid Is an Isoform of Glycodelin

Abstract Zona-binding inhibitory factor-1 (ZIF-1), a glycoprotein in human follicular fluid, reduces the binding of spermatozoa to the zona pellucida. ZIF-1 has a number of properties similar to those of glycodelin-A from human follicular fluid. The objective of this study was to compare the biochemical characteristics of these two glycoproteins. N-terminal sequencing and protease-digested peptide mapping showed that ZIF-1 and glycodelin-A have the same protein core. However, these glycoproteins differ in their oligosaccharide chains, as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin-binding ability, and isoelectric focusing. ZIF-1 inhibited spermatozoa-zona pellucida binding slightly more than did glycodelin-A and significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. Indirect immunofluorescence staining revealed specific binding of glycodelin-A and ZIF-1 to the acrosome region of human spermatozoa, where ZIF-1 produced a stronger signal than did glycodelin-A at the same protein concentration. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin that potently inhibits human sperm-egg interaction. Future study on the function role of ZIF-1 would provide a better understanding of the regulation of fertilization in humans.

Gamete/embryo – oviduct interactions: implications on in vitro culture

Fertilization and development of mouse embryos occur in the oviduct. Accumulating data suggested that embryo – maternal communication exists in the preimplantation period, with the female reproductive tract providing the optimal microenvironment conducive to the development of embryos. Signals produced from the developing embryos not only affect their own transport in the oviduct, but the physiology and gene expression patterns of the oviduct. As a step towards understanding the action of embryos on oviductal physiology, both genomics and proteomics approaches are being used to unveil the underlying mechanism of embryo – maternal interaction at the preimplantation stage. Results from recent studies allow us to better understand the roles and the use of oviductal secretory proteins or factors that affect embryo development in vivo and in vitro. It has been shown that in vitro culture alters gene expression of the cultured embryos and may predispose the embryo to certain disease. Therefore, the interaction between gamete/embryo and oviduct in vitro and in vivo, and the long-term effects of embryo culture on foetal development warrant further investigation.

Roles of glycodelin in modulating sperm function

Glycodelin is a glycoprotein with three well-defined isoforms. They are named as glycodelin-S, glycodelin-A and glycodelin-F. The three isoforms have similar protein core but different carbohydrate moieties. Glycodelin-S is abundant in the human seminal plasma. It suppresses sperm capacitation and in doing so, it maintains the spermatozoa in an uncapacitated state before they enter into the uterine cavity. Glycodelin-A is abundant in the amniotic fluid. It is also secreted from endometrial glands into uterine fluid and is produced by the fallopian tube. Glycodelin-A is the first endogenous glycoprotein that was found to inhibit the binding of spermatozoa to the zona pellucida. The immunosuppressive properties of glycodelin-A suggest that the molecule may protect the spermatozoa from immune attack in the maternal reproductive tract. Glycodelin-F was first found in the follicular fluid, hence its name. It also inhibits spermatozoa-zona pellucida binding. In addition, glycodelin-F suppresses progesterone-induced acrosome reaction, and may serve to prevent premature acrosome reaction. Preliminary findings suggest possible presence of yet another glycodelin isoform in the extracellular matrix of cumulus oophorus. Unlike glycodelin-A and -F, it stimulates spermatozoa-zona pellucida binding. In summary, different isoforms of glycodelin have different biological roles on sperm function, and they act in succession to contribute to the success of fertilization.