Peter Sutovsky - US grants

The proposed research is aimed to contribute the data on the role of microtubules in human oogenesis and fertilization, using Rhesus monkey as a model animal. Four basic questions will be addressed: 1. What is the organization of microtubules and meiotic apparatus during oocyte maturation and fertilization? 2 Is there evidence for a paternal centrosome contribution during normal fertilization and polyspermy? 3. Will parthenogenetically activated Rhesus oocytes organize a bipolar mitotic apparatus at the time of first division? 4. What is the role of microtubules during Rhesus fertilization? The methods used in the study include conventional immunofluorescence, laser scanning confocal microscopy, transmission electron microscopy, time lapse video microscopy, ultrastructural immunocytochemistry and the methods of in vitro maturation and fertilization. The immunocytochemical methods will take profit from the availability of new antibodies against alpha, beta - tubulins, kinetochores, centrosomes and microtubule organizing centers. The expected results should accomplish the knowledge about basic processes occurring during the oogenesis and fertilization, which belong to the most important problems of human reproduction.

DESCRIPTION (provided by applicant): This R21 exploratory proposal addresses a major gap in U.S. reproductive medicine, namely the lack of reliable, objective methodology for accurate diagnostics of human male infertility. Our goal is to validate the sperm-specific thioredoxin SPTRX3 as a novel biomarker of human sperm quality. We will examine how sperm SPTRX3 levels in male infertility patients correlate to a failure to conceive, to spontaneous abortion and to unwanted multiple births in their female partners treated by assisted reproductive therapies (ART). Current methods of human semen analysis and diagnostics of human male infertility rely heavily on subjective light microscopic evaluation. This is inaccurate because of a significant overlap between semen parameters of fertile and infertile men. Objective, automated methods for sperm analysis exist but are expensive and confined to specialized reference laboratories, as opposed to an inexpensive doctor's office fertility test. We have already established that SPTRX3 protein is carried exclusively by defective, morphologically deviant human spermatozoa. Our preliminary data demonstrate that the increased semen content of SPTRX3-carrying spermatozoa coincides with reduced pregnancy rates after ART. In contrast, ART couples with male partners exhibiting low semen SPTRX3 levels produced significantly more multiple births. This two year project has ONE SPECIFIC AIM, i.e. to demonstrate a statistical relationship between semen SPTRX3 levels and the occurrence of fertilization failure, spontaneous pregnancy loss and multiple pregnancies in couples from a general infertility clinic population. Our HYPOTHESIS is that the female partners of men with low SPTRX3 levels are more likely to conceive by ART and less likely to suffer a spontaneous abortion, but they are also more prone to multiple births. Semen SPTRX3 content will be evaluated in raw and gradient/swim-up purified semen samples from infertile couples by automated flow cytometric analysis and epifluorescence-light microscopic analysis of immunolabeled spermatozoa, and by semi-quantitative western blotting/densitometry technique. The influence of high semen SPTRX3 content will be evaluated against basic measures of treatment outcome, including fertilization and first embryo cleavage rates, incidence of spontaneous abortion following a successful assisted fertilization and embryo transfer, and the incidence of multiple pregnancies in couples treated by ART. To gain mechanistic understanding of how SPTRX3 carryover affects human sperm function, we will correlate sperm chromatin structure, likely affected by the presence of SPTRX3-containing nuclear vacuoles, with flow cytometric SPTRX3 data. We will also assess male pronuclear development after heterologous ICSI of sperm from high/low SPTRX3 samples into hamster ova, an assay commonly used to predict human sperm fertilizing ability and developmental potential. Based on feedback from our clinical collaborators, one of whom will participate in this project, we anticipate that the SPTRX3 based test will allow clinicians to make a treatment decision between intrauterine insemination (IUI) versus IVF/ICSI in the general infertility clinic-population. In the IVF/ICSI treated couples, a decision will be facilitated for how many embryos should be transferred to obtain a singleton pregnancy, as opposed to no pregnancy or an unwanted multiple birth. Upon validation by the proposed research, this novel biomarker can be quickly transferred to clinical practice in the form of probe for a reference laboratory test, or a lateral flow cassette (dipstick) for doctor's office use;an over the counter home fertility test could also be developed. PUBLIC HEALTH RELEVANCE: This project will explore and validate a novel biomarker of human male infertility, the recently discovered sperm borne protein SPTRX3. Preliminary data suggest that this biomarker can be used to more accurately diagnose male infertility and also to predict the likelihood of multiple births and spontaneous pregnancy losses after assisted fertilization. The proposed exploratory research is necessary for future diagnostic technology development leading to the introduction of this innovative diagnostic tool in US infertility clinics. Annually, more than 135,000 infertile couples are treated for infertility in US clinics, where the treatment-decision making still relies on outdated, highly subjective light microscopic method of semen evaluation.

Sutovsky, Peter - PD SUMMARY The present project is directly relevant to the goals of RFA PAR-13-204, Dual Purpose with Dual Benefit: Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Species. It will be the first project to systematically link the genetic traits affecting male fertility to sperm phenotypes easily measurable in a human/animal semen sample. Whole genome sequencing has identified a large number of gene polymorphisms with the potential to affect male fertility in humans and livestock. However, there is very little understanding of how these polymorphisms may affect the phenotypes of spermatozoa, their fertilizing ability and their influence on pre-embryo development and early embryo loss. We hypothesize that unique mutations/polymorphisms in genes expressed during spermatogenesis and pre- embryo development (further ?fertility associated genes?) are responsible for male subfertility and for early embryo loss during pregnancy. Our goal is to link these genetic traits to sperm phenotypes measurable in a semen sample and reflected by the males? fertility in vivo and in vitro. We chose artificial insemination (AI) bulls as model system because their genotypes are publically accessible, they have extensive fertility records from AI services, and their fertility in vivo correlates with their in vitro fertility. We have identified 3,601 candidate fertility-related genes with loss-of-function LOF) polymorphisms in the whole genome sequences of 219 bulls representing 18 breeds and performed phenotype analysis on several candidate gene products that revealed significant differences between fertile but under-performing bulls with less than satisfactory AI fertility and top bulls with highest AI fertility. Both genes also show non-synonymous polymorphisms in humans. AIM 1 of this project will employ genotyping and in silico search to identify genetic differences between fertile and subfertile bulls, including polymorphisms associated with high/low conception rates in AI, as well as those prevalent in yearling bulls that failed Breeding Soundness Evaluation (BSE). We hypothesize that fertile bulls fall on the opposite ends of fertility range due to identifiable polymorphisms in relatively few genes controlling spermatogenesis, sperm phenotype and sperm function. AIM 2 will use a combination of proteomic and cell biological approaches, including but not limited to innovative image based flow cytometry (IBFC), to link genetic fertility traits to the sperm phenotypes easily measurable in a semen sample. Based on the identification of sperm antigens expressed only or predominantly by carriers of the fertility-affecting mutations, we will develop multiplex, flow cytometry-based high throughput semen quality assays for routine use in field AI. We hypothesize that a non-synonymous change in a gene controlling spermatogenesis or sperm function will alter the sperm phenotype via change in the quantity, localization and/or function of a sperm or embryo protein encoded by this gene, thus influencing fertility of the carrier male. Altogether, we expect to identify and validate sperm phenotype biomarkers encoded by fertility associated polymorphic genes, and to improve sire management by genetic selection and objective, automated semen evaluation. This project will also yield new methods and potentially new treatments for human male and idiopathic infertility. The field of reproductive biology will be advanced through better understanding of the link between male genotype and sperm phenotype.