Mahmoud Aarabi

Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice

Mammalian zygotic development is initiated by sperm‐mediated intracellular calcium oscillations, followed by activation of metaphase II‐arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte‐activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm‐induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain‐binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte‐derived WWI domain protein substrate of PAWP for successful fertilization. Sperm‐delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in non‐mammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.—Aarabi, M., Balakier, H., Bashar, S., Moskovtsev, S. I., Sutovsky, P., Librach, C. L., and Oko, R., Sperm‐derived WW domain‐binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice. FASEB J. 28, 4434–4440 (2014). www.fasebj.org

Thrombophilic mutations in Iranian patients with infertility and recurrent spontaneous abortion.

Factor V Leiden (FVL) G1691A, methylenetetrahydrofolate reductase (MTHFR) C677T, and factor II (FII) G20210A mutations are three important causes of thrombophilia, the condition that might be related to infertility and recurrent spontaneous abortion (RSA). In this study we evaluated the presence of these three mutations in 36 female patients with unexplained infertility, 65 female patients with unexplained RSA, and 62 healthy fertile women as control group. DNA was extracted from peripheral blood samples and PCR-RFLP was performed for the molecular diagnosis of each mutation. In addition, activated protein C resistance (APC-R) was also evaluated. The frequencies of FVL, MTHFR, and FII mutations (heterozygous and homozygous) in the control group were 0.0%, 38.7%, and 3.2%, respectively. The frequency of FVL mutation in patients with infertility (30.6%) or RSA (20.0%) was significantly higher than that of the control group. A significantly higher MTHFR mutation rate was also observed in patients with RSA (63.1%) as compared to controls. However, the mutation rate of MTHFR in patients with infertility (50.0%) was not statistically different from that in controls. No significant difference was observed in the frequencies of FII mutations between the patients and controls. Decreased levels of APC-R were observed in 25.0% of infertile patients and 18.9% of patients with RSA. In conclusion, our results show a skew towards higher mutation frequencies of FVL and MTHFR in patients that may necessitate detection of such mutations in these Iranian patients.

Sperm-borne protein, PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release.

We previously reported postacrosomal sheath WW domain binding protein (PAWP) as a candidate sperm borne, oocyte‐activating factor. PAWP enters the oocyte during fertilization and induces oocyte activation events including meiotic resumption, pronuclear formation, and egg cleavage. However, in order to provide proof that PAWP is a primary initiator of zygotic development it is imperative to show that PAWP initiates intracellular calcium signaling, which is considered essential for oocyte activation. Utilizing Xenopus laevis as our model, we injected recombinant PAWP or Xenopus sperm into metaphase II‐arrested oocytes and observed a significant rise in intracellular calcium levels over controls. Concurring intensities and durations of PAWP and sperm‐induced calcium waves, detected by infrared two‐photon laser‐scanning fluorescence microscopy, were prevented by coinjection of a competitive PPGY‐containing peptide derived from PAWP but not by the point‐mutated form of this peptide. This study also correlates PAWP and sperm‐induced calcium release with meiotic resumption in Xenopus. The similar mode of oocyte activation, and the ability of the competitive peptide in blocking both sperm‐ and PAWP‐induced calcium release, provide evidence for the first time that sperm‐anchored PAWP is a primary initiator of zygotic development. Mol. Reprod. Dev. 77: 249–256, 2010.

The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor.

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.

Polymorphisms of plasminogen activator inhibitor-1, angiotensin converting enzyme and coagulation factor XIII genes in patients with recurrent spontaneous abortion

We investigated polymorphisms of plasminogen activator inhibitor-1 (PAI-1), angiotensin converting enzyme (ACE ) and coagulation factor XIII (FXIII) genes and their association with recurrent spontaneous abortion (RSA) in Iranian patients and normal healthy controls. Ten (18.5%) patients were homozygote (4G/4G) for PAI-1 polymorphism, in contrast with two (2%) controls (p = 0.001). Patients with homozygote 4G mutation were significantly more prone to RSA in contrast to others (odds ratio: 11.0, 95% CI: 2.3–52.4). Nineteen (30.2%) patients and 25 (26.6%) controls were homozygote (DD) for ACE polymorphism. We observed only two patients and one control with homozygosity (34leu) for FXIII polymorphism. 4G/4G polymorphism for PAI-1 gene could be a thrombophilic mutation leading to abortion in Iranian population.

High dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR.

Negative biomarker based male fertility evaluation: Sperm phenotypes associated with molecular-level anomalies

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry (FC). A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies (ART) and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA (Lens culinaris agglutinin; reveals altered sperm surface) and PNA (Arachis hypogaea/peanut agglutinin; reveals acrosomal malformation or damage). The Postacrosomal Sheath WWI Domain Binding Protein (PAWP), implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.

Re： Is PAWP the ＇real＇ sperm factor？

This letter is in response to an article written by Michail Nomikos, Karl Swann and F. Anthony Lai in the Research Highlights section of the Asian Journal of Andrology (AJA). The article is entitled, “Is PAWP the ‘real’ sperm factor?” and was written in response to our article entitled, “Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in human and mouse,” published recently in FASEB J.1 According to the Science Editor of AJA, “Research Highlight” pieces are brief articles that are meant to report on publications from the primary literature. Along those lines, we were delighted to read an insightful comment on our FASEB article prepared for AJA by Dr. George L Gerton. In stark contrast, we found that the article by Nomikos et al. was a promotion of phospholipase C zeta (PLCζ) as the “real” sperm factor, a molecule Dr. Lais group has been working on for over a decade. In this article we found several omissions, inaccuracies and mistakes that highlight shortcomings in the study of PLCζ which point to major criteria of a “real” sperm factor that have not yet been addressed for PLCζ.

Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like (WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Testicular MTHFR deficiency may explain sperm DNA hypomethylation associated with high dose folic acid supplementation

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake-DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr+/- mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.