Monika A. Ward - US grants

health science research support; sperm; artificial fertilization; biomedical resource; biomedical facility; assistive reproductive technique; clinical research; laboratory mouse;

The major goal of this application is to develop a simple and efficient method for preservation of valuable mouse genomes by using ICSI with preserved ejaculated spermatozoa. The hypothesis of this application is that ejaculated spermatozoa can be repeatedly retrieved from a male, preserved, and then used for ICSI yielding live offspring with high efficiency. The collection of spermatozoa from mice is limited to techniques that involve euthanasia and result in a death of the male, precluding him from further breeding. A method for repeatedly obtaining ejaculates allows for keeping the male alive and able to breed. In the Specific Aim 1 we will test if ejaculated preserved spermatozoa injected into the oocytes by ICSI allow obtaining live offspring with high efficiency. We will repeatedly obtain spermatozoa from several C57BL/6 males and preserve them by conventional eryopreservation and by simple freezing without cryoprotection. We will use ICSI to inject these spermatozoa into the oocytes from C57BL/6 females to produce embryos. We will evaluate the effects of these methods on sperm DNA integrity by analysis of patemal chromosomes in the zygotes and perform embryo transfer and produce live offspring. Finally, we will apply the proposed technology to one mutant mouse strain (azh - abnormal spermatozoon head-shape) with fertility problems to demonstrate its usefulness in preservation of a medically important mouse strain. In the Specific Aim 2 we will evaluate the mechanism of chromosome degradation in ejaculated preserved mouse spermatozoa. We will explore the biological significance of observed phenomenon that spermatozoa exposed to uterine content are more susceptible to DNA damage. We will establish if the unknown factor present in the uterus after mating affects live spermatozoa or rather interferes with the conditions of unprotected sperm freezing. We will also analyze independently the effects of two compounds of the uterine content: uterine fluid and seminal vesicle fluid. The significance of this proposal is that it will vigorously test the novel approach of using ICSI with preserved ejaculated spermatozoa to maintain valuable mouse genomes. This method will allow increasing the overall efficiency of reconstituting mouse strains and will accelerate sharing of given genetic material (i.e. a novel mutation) within the scientific community. Our expectations are that the results of this application will allow us to recommend ICSI with preserved ejaculated spermatozoa as a simple and efficient method for preservation of valuable mouse genomes.

DESCRIPTION (provided by applicant): The major goal of this application is to evaluate the phenomenon of sperm DNA degradation observed as the result of various sperm treatments. The hypothesis is that there exists a mechanism in mammalian fertilization that prevents the transmission of potentially damaged DNA to the embryo by disruption of the paternal chromosomes that this mechanism is based on the activity of endogenous nucleases, and that it can be active within sperm cells. In our preliminary studies we have shown that when spermatozoa are treated with various chemicals (detergents, DTT, exogenous DNA) or are subjected to mechanical insults (freeze drying and freezing without cryoprotection), and then injected into the oocytes, the paternal chromosomes in the zygotes are broken. We have also shown that this DNA degradation could be partially or completely prevented when ion chelators, EGTA or/and EDTA, were present in the sperm handling medium, which suggests the involvement of endogenous nuclease/s in this process. The idea that spermatozoa may have nuclease-dependent mechanism for DNA degradation differs from current thinking on the function of mammalian male gametes. In this application we will initiate the exploration on the origin and function of sperm DNA damage by expanding our preliminary data and answering the first major questions that this data brought. In Specific Aim 1, we will determine the causes of sperm DNA damage, and whether they are inherent to the cell or, are the result of experimental manipulation. We will explore what are the conditions under which the paternal chromosome breaks, examine early post-fertilization events after ICSI with treated spermatozoa, and test if paternal chromosome breakage can be observed in the absence of ICSI, to exclude the possibility that observed chromosome breakage is an artifact of ICSI combined with manipulation. Specific Aim 2 will answer the most crucial question of this application: Does DNA degradation occur in the spermatozoa or in the oocyte? We will test whether paternal DNA breakage can be induced in the oocytes after fertilization. We will analyze sperm DNA integrity before and after DNA synthesis. We will also evaluate if maternal (oocyte) DNA is degraded in the result of insults to which sperm, oocyte, or both are exposed. Finally, we will establish whether spermatozoa are able to induce degradation of DNA other than their own, a direct test to confirm the possibility that DNA degradation mechanism exist within sperm heads. In Specific Aim 3, we will test if paternal DNA degradation depends on the activity of endogenous Ca2+ and Mg2+dependent nucleases or topoisomerase II. We will establish if stresses to which spermatozoa are exposed can be neutralized by the presence of on chelators and other nuclease inhibitors, what is the range of nuclease activity, and what are the conditions under which the maximum protection is provided. We will also establish if endogenous Ca2+ and Mg2+dependent nucleases are present in the oocytes, if they can be inhibited there, and what will be the effects of this inhibition on paternal DNA integrity. The significance of this proposal is that it will test a novel idea that spermatozoa are active cells able to respond to their environment. This response is suspected to be a part of the mechanism that allows preventing the transmission of potentially damaged DNA to the embryo during fertilization. The study will have impact on basic science by advancing our understanding of biological system of mammalian reproduction. It will also have significance for clinical research and health and welfare of infertile couples providing clues for human assisted reproduction clinics as to what treatments evoke DNA-degrading mechanisms in spermatozoa and therefore should be avoided.

[unreadable] DESCRIPTION (provided by applicant): It has been argued that most Y chromosome coded genes are likely to have roles in sperm production or function. However, it is not clear if these genes provide essential spermatogenic function or just potentiate the spermatogenic process. The goal of this application is to determine the minimum Y chromosome gene requirement that is compatible with successful reproduction by assisted reproductive technologies (ART). The hypothesis of this application is that a Y gene complement of as few as two or three genes is enough to enable the production of male `gametes' (round spermatids or sperm) that are capable of participating in fertilization if delivered into the oocytes via injection. In preliminary data we provide evidence that in the mouse the presence of only two Y-coded genes, Sry and Eif2s3y, allows formation of testes, ongoing spermatogonial proliferation, and completion of meiosis to generate round spermatids. We suggest that further addition of one copy of Zfy is sufficient to allow the production of some sperm, albeit with morphologically abnormal heads. We also show that assisted reproduction by ICSI and IVF enable the generation of live offspring from subfertile and infertile males with Y chromosome deficiencies. In this application we will focus on analyzing various mouse models with limited Y gene complements: (1) males with an almost intact Y short arm but complete absence of Y long arm genes; (2) males with no Y long arm genes and the known Y short arm genes limited to Sry, Eif2s3y, a single copy of Zfy, and a reduced number of copies of Rbmy; and (3) males with only Eif2s3y and Sry. In Specific Aim 1 we will generate these males with limited Y gene complements and define more precisely at what stage of spermiogenesis cells arrest or become abnormal. We will perform histological analysis of the testes, confirm the presence of specific spermatogenic cell types by immunostaining, and examine acrosome development as a marker of spermiogenic stage. In Specific Aim 2 we will use sperm and/or round spermatids from these models for ICSI and/or ROSI. We will observe early post-fertilization events after injection, obtain embryos, and produce live offspring. We will genotype progeny to test which sperm genotypes were successful in supporting fertilization and embryo development. The significance of this application is that it will advance the understanding of the role of Y chromosome encoded genes in spermatogenesis and sperm function; it will also provide valuable information for those using ART to treat human infertility associated with Y gene deficiencies. PUBLIC HEALTH RELEVANCE: In this application we seek to determine the minimum Y gene complement that is compatible with the generation of sperm competent in fertilization via ART (ICSI and ROSI). The study will add to the understanding of the functions of Y chromosome coded genes by defining which Y genes provide essential spermatogenic function rather than just potentiating the spermatogenic process. The application also has significance for those utilizing ART to treat male infertility. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Y chromosome was once thought to be responsible only for turning on sex determination towards the male pathway via the testis determinant gene Sry. However, subsequent studies revealed a number of other genes located on this chromosome that are likely to encode information important for formation and function of the male gametes. The Y chromosome was once thought to be responsible only for turning on sex determination towards the male pathway via the testis determinant gene Sry. However, subsequent studies revealed a number of other genes located on this chromosome that are likely to encode information important for formation and function of the male gametes. The goal of this project is to define the roles of certain Y encoded genes. More specifically, we seek to (1) unveil the function and mechanism of action of genes encoded on the Y chromosome long arm, (2) test whether testis determinant gene Sry plays a role in sperm function;(3) define the minimum Y gene complement that is sufficient to generate gametes capable of achieving successful fertilization. This will be done through the analysis of unique mouse models lacking various parts of the Y chromosome, and use of assisted reproduction technologies (ART): intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF), in combination with molecular biology approaches. Deletions within the male specific region of the Y chromosome are a common genetic cause of spermatogenic failure, making men carrying affected Y chromosome a target group for infertility treatment via ART. Given the known and potential problems associated with the use of ART in humans, it is essential to continue efforts on identifying the underlying causes of infertility. Our work will advance understanding of the Y chromosome encoded genes role in spermatogenesis and sperm function and ultimately may help to combat infertility, a major health problem in U.S.A.

Mammalian reproduction conventionally requires oocytes provided by a female and sperm provided by a male to achieve fertilization. We go beyond this convention and propose the hypothesis that an adult individual of any sex can be induced to produce gametes of the opposite sex, and that these gametes are functional in assisted fertilization. The premise for this hypothesis comes from our recently published studies demonstrating that genetically engineered male mice with limited or no Y chromosome genes can successfully reproduce by assisted fertilization (ART), and from significant advancements in field of cell reprogramming and differentiation. Our laboratory has shown that in the mouse only two Y chromosome genes, testis determinant Sry and spermatogenesis driver Eif2s3y, are sufficient for a male to produce haploid male gametes functional in ART. We have subsequently demonstrated that the function of these two genes could be replaced by that of their homologues encoded on other chromosomes, and that a mouse with a single X chromosome (XO) lacking all Y chromosome genes can produce male gametes and sire healthy offspring after assisted fertilization. The laboratory of our collaborator, Mitinori Saitou, has shown that both male and female gametes can be obtained from induced pluripotent stem cells (iPSC) differentiated into primordial germ cell like-cells (PGCLC). In this proposal, we marry our findings and expertise and ask 3 questions: Aim 1. Can an adult female mouse produce male gametes functional in assisted fertilization? Aim 2. Can an adult male mouse produce female gametes functional in assisted fertilization? Aim 3. Can an adult mouse of either sex sire uniparental offspring? To address these questions we will develop somatic cell lines from an adult mouse of a given sex, reprogram to iPSC, identify the clones that have lost one sex chromosome and became XO, and differentiate into PGCLC. To produce male gametes we will transgenically add a spermatogenesis driver and transplant PGCLC to testes from neonatal males. To produce female gametes we will reconstitute ovaries in vitro and transplant them under ovarian bursa of recipient females. We will test the function of such derived male and female gametes using assisted reproduction. To produce uniparental offspring, male and female gametes derived from the same individual will be used for fertilization. The findings from this project will impact on our understanding of sex specific differences, especially pertaining to effects of sex chromosomes and X and Y genes on germline development. If successful, we will also provide the field with a proof-of-principle that offspring of both sexes can be obtained from a single individual, which will impact on species preservation.

Project Summary/Abstract The mammalian Y chromosome is a symbol of maleness as it encodes the Sry gene driving male sex determination, a battery of other genes thought to be involved in various aspects of male reproduction, and other genes playing roles of broadly expressed regulators of transcription, translation and protein stability. In spite of these clearly important functions, the knowledge linking the roles of specific Y chromosome genes to specific reproductive and other physiological processes remains limited. This is due to the unusual genomic structure of this chromosome, which for decades rendered it resistant to any targeting attempts. With the emergence of TALEN and CRISPR/Cas9 technologies addressing Y chromosome gene function directly became possible. The oddity of the Y chromosome structure made it also defiant to sequencing. The mouse Y chromosome sequence was finally defined in 2014, 12 years after the remainder of the mouse genome was characterized. When the mouse Y sequence was revealed two new single copy genes were identified on the Y short arm: Prssly (Y-linked serine-like protease) and Teyorf1 (testis expressed, chromosome Y open reading frame 1). These genes were classified as ?acquired genes? and were proposed to be the candidates for male fertility genes. Neither of these genes has been targeted and their function remains unknown. In Preliminary Data we show that Prssly and Teyorf1 expression is restricted to testicular postmeiotic germ cells. We also analyzed PRSSLY and TEYORF1 predicted protein structure and function and found that both predicted proteins resemble proteins that are known to play roles in spermatogenesis. PRSSLY belongs to the peptidase S1 family of proteins that include serine proteases PRSS21 and PRSS55 essential for sperm motility and sperm ability to fertilize oocytes. TEYORF1 belongs to the Claudin family of proteins members of which were reported important for assembly and maintenance of blood-testis barrier, a tissue barrier critical for normal spermatogenesis. Acquisition to the Y chromosome, testis specific expression, and resemblance to proteins of known reproductive functions prompted us to propose a hypothesis that Prssly and Teyorf1 play roles in male reproduction. We will address this hypothesis pursuing two specific aims: Aim 1: Generate Prssly and Teyorf1 knock-out (KO) and Prssly and Teyorf1 reporter tag knock-in (KI) mice using CRISPR/Cas9 system and zygote electroporation. Aim 2: Characterize phenotype of these mice focusing on assessment of fertility, sperm parameters, sperm function in vitro, and spermatogenesis progression. The combined results will allow to define whether Prssly and Teyorf1 play roles in spermatogenesis and fertility. The development of KI mice will open the door to future mechanistic investigations of these factors. The project will impact on understanding of Y chromosome gene function and genetic basis of male fertility. It will also contribute to discussions regarding the evolution of Y chromosomes and the importance of acquired Y genes.