Histone demethylase KDM4A overexpression improved the efficiency of corrected human tripronuclear zygote development.

Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, eight-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000 ng/µl lysine-specific demethylase 4A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.

In vitro study on vertical transmission of the HIV-1 gag gene by human sperm.

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.

Melatonin protects against oxybenzone-induced deterioration of mouse oocytes during maturation.

Oxybenzone (OBZ), an ultraviolet light filter that is widely used in sunscreens and cosmetics, is an emerging contaminant found in humans and the environment. Recent studies have shown that OBZ has been detected in women's plasma, urine, and breast milk. However, the effects of OBZ exposure on oocyte meiosis have not been addressed. In this study, we investigated the detrimental effects of OBZ on oocyte maturation and the protective roles of melatonin (MT) in OBZ-exposed mouse models. Our in vitro and in vivo results showed that OBZ suppressed oocyte maturation, while MT attenuated the meiotic defects induced by OBZ. In addition, OBZ facilitated H3K4 demethylation by increasing the expression of the Kdm5 family of genes, elevating ROS levels, decreasing GSH, impairing mitochondrial quality, and disrupting spindle configuration in oocytes. However, MT treatment resulted in significant protection against OBZ-induced damage during oocyte maturation and improved oocyte quality. The mechanisms underlying the beneficial roles of MT involved reduction of oxidative stress, inhibition of apoptosis, restoration of abnormal spindle assembly and up-regulation of H3K4me3. Collectively, our results suggest that MT protects against defects induced by OBZ during mouse oocyte maturation in vitro and in vivo.

Effects of hepatitis B virus S protein on human sperm function.

BACKGROUND: Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function. METHODS: Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed. RESULTS: HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs-HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 microg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential. CONCLUSIONS: HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.

The integrated HIV-1 provirus in patient sperm chromosome and its transfer into the early embryo by fertilization.

Complete understanding of the route of HIV-1 transmission is an important prerequisite for curbing the HIV/AIDS pandemic. So far, the known routes of HIV-1 transmission include sexual contact, needle sharing, puncture, transfusion and mother-to-child transmission. Whether HIV can be vertically transmitted from human sperm to embryo by fertilization is largely undetermined. Direct research on embryo derived from infected human sperm and healthy human ova have been difficult because of ethical issues and problems in the collection of ova. However, the use of inter-specific in vitro fertilization (IVF) between human sperm and hamster ova can avoid both of these problems. Combined with molecular, cytogenetical and immunological techniques such as the preparation of human sperm chromosomes, fluorescent in situ hybridization (FISH), and immunofluorescence assay (IFA), this study mainly explored whether any integrated HIV provirus were present in the chromosomes of infected patients' sperm, and whether that provirus could be transferred into early embryos by fertilization and maintain its function of replication and expression. Evidence showed that HIV-1 nucleic acid was present in the spermatozoa of HIV/AIDS patients, that HIV-1 provirus is present on the patient sperm chromosome, that the integrated provirus could be transferred into early embryo chromosomally integrated by fertilization, and that it could replicate alongside the embryonic genome and subsequently express its protein in the embryo. These findings indicate the possibility of vertical transmission of HIV-1 from the sperm genome to the embryonic genome by fertilization. This study also offers a platform for the research into this new mode of transmission for other viruses, especially sexually transmitted viruses.

In vivo study on vertical transmission of the HIV-1 gag gene via mouse oocytes.

Acquired immunodeficiency syndrome (AIDS) is a major public health problem worldwide. This study was performed to explore the feasibility of vertical transmission of human immunodeficiency virus-1 (HIV-1) gag gene via oocyte. The recombinant plasmid (pIRES2-EGFP-gag) was injected into mouse ovaries to transfect germ cells. Induction of superovulation and then animal mating were performed to collect oocytes and two-cell embryos. Positive FISH signals for HIV-1 gag DNA were detected in the nuclei of oocytes and embryos, and in chromosomes of mature oocytes, indicated integration of the gene into the oocyte genome and gene replication in the embryo. HIV-1 gag cDNA positive bands detected by RT-PCR in oocytes and embryos indicated successful gene transcription, while positive immunofluorescence signals for HIV-1 gag protein indicated successful translation in both oocytes and embryos. The HIV-1 gag gene was transmitted vertically to the next generation via oocytes and it retained its function in replication, transcription and translation following at least one mitotic division in embryos.