Xiaofang Ding

Inhibition of human sperm function and mouse fertilization in vitro by an antibody against cation channel of sperm 1: the contraceptive potential of its transmembrane domains and pore region

OBJECTIVE To explore the contraceptive potential of the CatSper1 transmembrane domains and pore region in vitro. DESIGN In vitro study with human sperm and mouse fertilization. SETTING Andrology laboratory of an academic research center. PATIENT(S) AND ANIMAL(S) Normozoospermia and viripotent BALB/c mice. INTERVENTION(S) The specific binding of an anti-CatSper1 IgG antibody (H-300) to CatSper1 was confirmed by Western blot and immunofluorescence. Sperm from humans and mice were incubated with H-300. MAIN OUTCOME MEASURE(S) The effects of H-300 on human sperm progressive motility, abnormal acrosome, hyperactivated motility, and mouse in vitro fertilization rates were analyzed. RESULT(S) A significant decline in sperm progressive motility was observed after 1, 2, and 4 hours of incubation with H-300; the change was mainly ascribed to the decline of fast progressive motility. Significant inhibition of the hyperactivated motility was observed after 5 hours of incubation with H-300. The incubation of mouse sperm with H-300 before insemination reduced the in vitro fertilization rate to 28% of control levels (72% inhibition). CONCLUSION(S) CatSper1 may be a potential target for immunocontraception, and the antibody may be a tool to study the function of ion channels in sperm in which relatively fewer methods can be applied.

Characterization of a piRNA binding protein Miwi in mouse oocytes

Argonaute proteins and Piwi proteins bind with microRNA (mRNA) and Piwi-interacting RNA (piRNA), respectively, to form functional complexes. Piwi proteins are mostly restricted to germ cells and stem cells, and the Piwi-piRNA pathway is required for normal spermatogenesis. Although piRNAs were also recently identified in mammalian oocytes, expression of Piwi proteins in the ovary has not been well characterized. Previous studies did not detect mRNA of Miwi, a murine homologue of Piwi proteins, in total RNA of mouse ovary tissue. We demonstrated herein the presence of Miwi in murine oocytes. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence based on quantum dots immune labeling technique were used to investigate the expression profile of Miwi in oocytes of adult and neonatal females at 0, 1, 2, 3, and 4 weeks postpartum. Although RT-PCR was negative in total RNA of the adult ovary, both RT-PCR and Western blot detected Miwi in oocytes of adult mice, and ovaries of neonatal females. Miwi transcript and protein peaked at 1 and 2 weeks postpartum, respectively. Miwi mRNA was detectable in newborn mouse ovaries, implying its transcription was initiated at least in the primordial follicle. Its protein was strong in late primary and secondary follicles, but appeared to decrease as maturation proceeded. The exclusion of anti-Miwi immunofluorescence from some cytoplasmic granules was observed. Given that diverse biologic and molecular functions have been revealed for the Piwi-piRNA pathway in germline cells of many species, Miwi might be an important functional protein in murine folliculogenesis.

Genome-wide promoter methylation profile of human testis and epididymis: identified from cell-free seminal DNA

BackgroundDNA methylation analysis is useful for investigation of male fertility in mammals, whereas the reliance on tissues limits the research on human. We have previously found the presence of high concentration of cell-free seminal DNA (cfsDNA) in human semen. We proposed that some testis and epididymis-specific methylated promoters could be detected in human cfsDNA, and thus hold promise as noninvasive epigenetic biomarkers for male infertility, of which most cases are caused by defects in testicular sperm production or epididymal sperm maturation.ResultsThe ejaculate of successfully vasectomized men does not contain any secretion from testis and epididymis. Here we compared genome-wide promoter methylation profiles in cfsDNA between health donors and post-vasectomy men. Promoters of 367 testis and epididymis-specific hypomethylated genes and 134 hypermethylated genes were identified. Subsequent validation by Methyl-DNA immunoprecipitation and MethyLight analysis confirmed the result of promoter microarray. Gene Ontology analysis revealed many genes involved in male reproduction.ConclusionWe detected the testis and epididymis-specific methylated promoters in human cfsDNA, which may be used for noninvasive epigenetic biomarkers for the study and diagnosis of male infertility.

Alterations of testis-specific promoter methylation in cell-free seminal deoxyribonucleic acid of idiopathic nonobstructive azoospermic men with different testicular phenotypes

OBJECTIVE To explore the feasibility of quantification of testicular DNA methylation from cell-free seminal DNA (cfsDNA), and analyze promoter methylation alterations in men with idiopathic nonobstructive azoospermia (NOA). DESIGN Comparison between testicular DNA and paired cfsDNA, and among NOA patients with different testicular phenotypes. SETTING Academic research institute and andrology practice. PATIENT(S) Eighty-eight idiopathic NOA patients with different testicular phenotypes and 24 normozoospermic men. INTERVENTION(S) Testicular biopsies and semen analysis. MAIN OUTCOME MEASURE(S) Five testis-specific methylated promoters were selected. Promoter methylation was quantified using MethyLight in testicular DNA and paired cfsDNA, and the mRNA level was determined by real-time quantitative polymerase chain reaction. RESULT(S) Correlations of methylation of the selected five promoters between testicular DNA and paired cfsDNA were observed; and promoter methylation was negatively related to the messenger RNA level in testis. The cfsDNA methylation of these promoters showed different dynamic changes among the subtypes of NOA and normozoospermia. Among them, CCNA1 and DMRT1 promoter methylation in the hypospermatogenesis group was higher than in other groups and showed diagnostic potential for the patient with hypospermatogenesis. CONCLUSION(S) Cell-free seminal DNA could be a novel, noninvasive biomarker for the detection of testicular epigenetic aberrations. Epigenetic information in cfsDNA related to spermatogenesis may serve to predict successful testicular sperm retrieval in NOA patients.

Two B-cell epitope vaccines based on uPA effectively inhibit fertility in male mice

uPA, a trypsin-like serine protease, was found to take active part in male reproduction. Our previous work had demonstrated the antifertility effects of its full length protein immunization, but with immune tolerance and other latent side effects. Here we discovered two effective B-cell epitopes of uPA for male contraception in growth factor-like domain and kringle domain respectively. Together with carrier protein, immunization of these two epitope peptides could induce high titers of specific antibodies in male mice. Significant reduction of fertility was observed in these two groups in mating trial without evident systemic illness or abnormal mating behavior. Epididymal sperms of immunized males exhibited impaired progressive motility and ability to fertilize eggs in vitro. The immunization of another predicted epitope in serine protease domain and the control groups showed no similar positive results. Importantly, T cells were not activated after the challenge of these B-cell epitopes itself, which suggests that these vaccines do not induce cell-mediated autoimmunity. Taken together, our study discovered two uPA B-cell epitopes as novel targets for male immunocontraception with minimum side effects. Considering their high identity with human uPA protein, these two epitope vaccines hold great promise to be developed for man use in the future.