Exogenous GSH Supplementation to Raw Semen Alters Sperm Kinematic Parameters in Infertile Patients.

Glutathione is an important antioxidant found in all mammalian cells. Sperm motility is positively correlated with seminal reduced glutathione (GSH) levels, and infertile men are known to have lower GSH levels. Studies on GSH supplementation in improving sperm functions in infertility patients are limited. Here, we re-investigate the effect of exogenous GSH supplementation on human sperm motility and kinematic parameters. Residual semen samples from 71 infertility patients who came for routine semen analysis for infertility assessment were studied. Liquefied raw semen was supplemented with GSH (0-10 mM) for 1 h. The untreated sample was the blank control. Only a 5 mM concentration was tested in all 71 samples. After two washes, the sperm was incubated and then analyzed for sperm motility and kinematic parameters by computer-assisted semen analysis (CASA), followed by adenosine triphosphate (ATP), reactive oxygen species (ROS) levels, free thiols, and DNA damage analyses. At 2 hrs post-treatment, GSH supplementation significantly altered many of the kinematics, compared to the control. Straight line velocity (VSL) (p = 0.0459), curvilinear velocity (VCL) (p < 0.0001), average path velocity (VAP) (p < 0.0001), and lateral head amplitude (ALH) (p < 0.0001) were decreased, whereas straightness (STR) (p = 0.0003), linearity (LIN) (p = 0.0008), and beat cross frequency (BCF) (p = 0.0291) were increased in 5 mM group. Wobble (WOB) (p = 0.4917), motility (MOT) (p = 0.9574), and progressive motility (PROG) (p = 0.5657) were unchanged. ATP level was significantly increased in the 5 mM group (p < 0.05). It is concluded that exogenous GSH supplementation does alter sperm kinematics in humans. These altered kinematic parameters together with increased energy (ATP) may have a positive role in influencing the success rates of ART procedures.

A novel embryo quality scoring system to compare groups of embryos at different developmental stages.

PURPOSE: To construct a new embryonic quality scoring system to compare groups of embryos at different developmental stages. METHODS: Based on a hypothesis that the implantation potential of any embryo in an ovum pickup (OPU) cycle remains the same at any stage of development, be it day 2, 3, or 5, a new embryo quality scoring (EQS) system was designed. It was based on the analysis of the clinical results of 1610 single embryo transfers. We validated this scoring system in the comparison of embryonic quality between groups by evaluating the mean scores calculated at day 2, day 3, and day 5 for 957 embryos (150 cycles) from 3 different groups. We then compared EQSs of patients with pregnancy favorable factors (group A) such as young age and high AMH levels, with the patients with contra features (group B). RESULTS: We confirmed that each mean EQS assessed at different stages of embryonic development within the same group was similar. The mean EQSs on day 3 and day 5 in group A were significantly higher than the mean EQSs on days 2, 3, and 5 in group B. CONCLUSION: The novel EQS system proposed by us enables embryonic quality comparison between groups of embryos at different developmental stages.

Role of peptidylarginine deiminase 4 (PAD4) in pig parthenogenetic preimplantation embryonic development.

Arginine modification to citrulline (citrullination) is catalyzed by peptidylarginine deiminases (PADs) and one of the isomers PAD4 is shown to be involved in the gene regulation. In our previous paper we studied the localization and expression of PAD4 and the target of PAD4 in mammalian gametes and preimplantation embryos. In this study the role of PAD4 was examined in the pig diploid parthenogenetic preimplantation embryonic development. Knockdown of PAD4 by RNAi resulted in delayed development. Inhibition of PAD4 by a potent PAD4 inhibitor Cl-amidine from the time of activation for 24 h resulted in developmental arrest at the first cleavage. Inhibition at the later stages of development resulted in delayed or arrested development. A shorter exposure to Cl-amidine for 6 h at any stage of growth resulted in slow development. Thus, this study suggests that PAD4 activity is essential for the normal development of the embryos.

Localization and expression of peptidylarginine deiminase 4 (PAD4) in mammalian oocytes and preimplantation embryos.

Post-translational modifications generally involve the addition or removal of various functional groups to or from the protein residues. However, citrullination, which is catalyzed by the peptidylarginine deiminases (PADs), involves conversion of one kind of amino acid residue into another. One of five isoforms, PAD4 is a nuclear enzyme known to play a role in development, differentiation and apoptosis through gene regulation. To investigate the possible role of PAD4 in mammalian preimplantation embryonic development, we first studied localization and expression of PAD4 and citrullinated proteins in pig and mouse oocytes, and parthenogenetic or in vitro fertilized (IVF) embryos. Immunofluorescence study revealed that PAD4 primarily localizes in the cytoplasm in pig oocytes and parthenogenetic embryos. However, the nuclear translocation of PAD4 was observed in late germinal vesicle (GV) stage oocytes prior to GV breakdown and was localized around the metaphase (M)I and II spindle. Nucleus localized PAD4 was noticed partially again in blastocysts. In mouse IVF embryos, nuclear translocation started from the 2-cell stage and gradually increased up to blastocyst. Western blot studies confirmed that PAD4 was expressed in oocytes, and parthenogenetic embryos of pig. Citrullinated proteins were detected in granular form on the chromatin in GV, MI and MII oocytes and nuclei in all the stages of the embryos studied. It was found that the target of citrullination was histone protein (H3), not B23. Therefore the presence of PAD4 and citrullinated histone H3 in oocytes and embryos suggested a possible role for PAD4 in preimplantation embryonic development.

Broad, ectopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice.

Mammalian metaphase II (mII) exit and embryogenesis are induced at fertilisation by a signal thought to come from the sperm protein, phospholipase C-zeta (PLCZ1). Meiotic progression can also be triggered without sperm, as in parthenogenesis, although the classic mouse in vivo parthenogenetic model, LT/Sv, fails in meiosis I owing to an unknown molecular etiology. Here, we dissect PLCZ1 specificity and function in vivo and address its ability to interfere with maternal meiotic exit. Wild-type mouse Plcz1 expression was restricted to post-pubertal testes and the brains of both sexes, with region-specifying elements mapping to a 4.1 kb Plcz1 promoter fragment. When broad ectopic PLCZ1 expression was forced in independent transgenic lines, they initially appeared healthy. Their oocytes underwent unperturbed meiotic maturation to mII but subsequently exhibited autonomous intracellular free calcium oscillations, second polar body extrusion, pronucleus formation and parthenogenetic development. Transfer of transgenic cumulus cell nuclei into wild-type oocytes induced activation and development, demonstrating a direct effect of PLCZ1 analogous to fertilisation. Whereas Plcz1 transgenic males remained largely asymptomatic, females developed abdominal swellings caused by benign ovarian teratomas that were under-represented for paternally- and placentally-expressed transcripts. Plcz1 was not overexpressed in the ovaries of LT/Sv or in human germline ovarian tumours. The narrow spectrum of PLCZ1 activity indicates that it is modulated by tissue-restricted accessory factors. This work characterises a novel model in which parthenogenesis and tumourigenesis follow full meiotic maturation and are linked to fertilisation by PLCZ1.

Epigenetic discrimination by mouse metaphase II oocytes mediates asymmetric chromatin remodeling independently of meiotic exit.

In mammalian fertilization, paternal chromatin is exhaustively remodeled, yet the maternal contribution to this process is unknown. To address this, we prevented the induction of meiotic exit by spermatozoa and examined sperm chromatin remodeling in metaphase II (mII) oocytes. Methylation of paternal H3-K4 and H3-K9 remained low, unlike maternal H3, although paternal H3-K4 methylation increased in zygotes. Thus, mII cytoplasm can sustain epigenetic asymmetry in a cell-cycle dependent manner. Paternal genomic DNA underwent oocyte-mediated cytosine demethylation and acquired maternally-derived K12-acetylated H4 (AcH4-K12) independently of microtubule assembly and maternal chromatin. AcH4-K12 persisted without typical maturation-associated deacetylation, irrespective of paternal pan-genomic cytosine methylation. Contrastingly, somatic cell nuclei underwent rapid H4 deacetylation; sperm and somatic chromatin exhibited asymmetric AcH4-K12 dynamics simultaneously within the same mII oocyte. Inhibition of somatic histone deacetylation revealed endogenous histone acetyl transferase activity. Oocytes thus specify the histone acetylation status of given nuclei by differentially targeting histone deacetylase and acetyl transferase activities. Asymmetric H4 acetylation during and immediately after fertilization was dispensable for development when both parental chromatin sets were hyperacetylated. These studies delineate non-zygotic chromatin remodeling and suggest a powerful model with which to study de novo genomic reprogramming.

A restricted role for sperm-borne microRNAs in mammalian fertilization.

Prototypical microRNAs (miRNAs) are 21 approximately 25-base-pair RNAs that regulate differentiation, carcinogenesis, and pluripotency by eliminating mRNAs or blocking their translation, in a process that is collectively termed RNA interference (RNAi). In zebrafish, RNAi mediated by miRNAs regulates early development, and in mice embryos that lack the miRNA precursor processor Dicer are nonviable. However, the roles of miRNAs in mammalian fertilization are unknown. In this report, we show using microarrays that miRNAs are present in mouse sperm structures that enter the oocyte at fertilization. The sperm contained a broad profile of miRNAs and a subset of potential mRNA targets, which were expressed in fertilizable metaphase II (mII) oocytes. Oocytes contained transcripts for the RNA-induced silencing complex (RISC) catalytic subunit, EIF2C3 (formerly AGO3). However, the levels of sperm-borne miRNA (measured by quantitative PCR) were low relative to those of unfertilized mII oocytes, and fertilization did not alter the mII oocyte miRNA repertoire that included the most abundant sperm-borne miRNAs. Coinjection of mII oocytes with sperm heads plus anti-miRNAs to suppress miRNA function did not perturb pronuclear activation or preimplantation development. In contrast, nuclear transfer by microinjection altered the miRNA profile of enucleated oocytes. These data suggest that sperm-borne prototypical miRNAs play a limited role, if any, in mammalian fertilization or early preimplantation development.

Injection of mammalian metaphase II oocytes with short interfering RNAs to dissect meiotic and early mitotic events.

The manipulation of mammalian metaphase II (mII) oocytes has illuminated the mechanisms of fertilization and early embryogenesis and is central to nuclear transfer. Although RNA interference (RNAi) would greatly facilitate this type of manipulation, its application to mature, developmentally competent mII oocytes has not been evaluated. We report efficient RNAi by the injection of short interfering RNAs (siRNAs) into mII oocytes. The levels of the target mRNA and corresponding protein were rapidly and efficiently reduced. The siRNAs were effective when injected in the subnanomolar to nanomolar range and induced concurrently RNAi of multiple targets, revealing the kinetic parameters of RNAi in mII oocytes. Coinjection of sperm with siRNA functionally abolished the transcripts in the resultant blastocysts and in cloned embryos into which siRNA was coinjected during somatic cell nuclear transfer. The RNAi method was used to dissect the early mitotic roles of meiotic regulators, which suggests that CDC20 is essential for the first mitotic division, while EMI1 and EMI2 are not essential for this process. Our results show that siRNA injection of oocytes confers temporal control of RNAi in the analysis and manipulation of key processes in mammalian meiosis and early embryogenesis.