Ph.D. - Developmental and Reproductive Biology

There have been decades of work on the Y chromosome and how it relates to maleness and male reproduction. In light of the increasing decline of human male fertility and dependence on assisted reproduction more knowledge about the causes behind male infertility is needed to find more solutions for affected men. This dissertation details histological investigations aiming to elucidate function of Y chromosome genes in male development and fertility using a mouse model. The testis and the seminiferous tubules within were explored and spermatogenesis was quantitatively assessed. Males lacking Y chromosome and transgenic for either the key Y-derived transgenes (Sry and Eif2s3y) or transgenes of their non Y-derived homologues (Sox9 and Eif2s3x) were shown to produce haploid germ cells that could be used for assisted reproduction technologies (ART) and yield live offspring. Another Y chromosome gene, Zfy2, was identified as a gene allowing for transformation of round spermatids into sperm. While in males with only two Y chromosome genes, Sry and Eif2s3y, spermatogenesis progressed to round spermatid stage, addition of Zfy2 allowed for complete spermatogenesis and formation of sperm capable of generating offspring with ART. Investigations into the testicular abnormalities in mice with limited Y chromosome gene contribution showed relationship between the number of Y genes present and the severity and distribution of cellular abnormalities in the seminiferous epithelium and defects of the testis interstitium. Human spermatogenesis was also histologically investigated utilizing testis biopsies from normal and infertile men, some with Y chromosome azoospermic factor (AZF) deletions, validating mice with Y chromosome deficiencies as a model for human male Y-linked infertility. Gross anatomy investigations into human male urogenital anatomy were also undertaken using a novel dissection method in order to grow the repository of male urogenital teaching tools at the medical school. Both examined specimens had common male genital pathologies (direct inguinal hernia, varicocele) and so also showcase physical abnormalities that can affect male reproductive health.

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The role of sperm DNA damage in the origin of infertility associated with Y chromosome long arm deletions in the mouse model

([Honolulu] : [University of Hawaii at Manoa], [December 2013], 2013-12) Riel, Jonathan M.

In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with severe non-pairing Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injections (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. Our initial study tested if NPYq deficiency is associated with sperm DNA damage-a known cause of poor ICSI results. We observed that epididymal sperm from mice with severe NPYq deficiency are impaired in oocyte activation ability, and have an increased incidence of oocyte arrest and paternal chromosome breaks. Comet assays revealed increased DNA damage in both epididymal and testicular sperm, and transmission electron microscopy showed sperm having impaired membrane integrity and abnormal chromatin condensation. We therefore concluded that the increased DNA damage associated with NPYq deficiency might be a consequence of disturbed chromatin remodeling taking place during spermiogenesis. There are four distinct multi-copy genes found in the long arm of the Y chromosome. One of them, Sly, is known to control the expression of sex chromosome genes after meiosis; Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has also been shown to be the underlying cause of sperm head anomalies and infertility associated with NPYq gene loss. We therefore hypothesized that Sly is our target gene. To test this, we examined mice with transgenically (RNAi) silenced Sly. Our analysis of Sly-deficient mice demonstrated similar 'sperm DNA damage' phenotype. This confirmed that lack of Sly is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions. This project provides the first evidence of DNA damage in sperm from mice with NPYq deficiencies and that the multi-copy NPYq-encoded Sly gene plays a key role in processes regulating chromatin remodeling and thus maintaining DNA integrity in sperm.

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The potential role of hypoxia inducible factor 1 alpha in multipotent germ cells and neural stem/progenitor cells

([Honolulu] : [University of Hawaii at Manoa], [December 2014], 2014-12) Takahashi, Natsumi

Hypoxia inducible factor 1 alpha (HIF1α) is a key transcription factor which transcriptionally activates an array of genes involved in adaptive responses to physiological hypoxia. HIF1α has been implicated in many disease conditions as well as normal development of many organs including the heart, the brain and the bones. In recent years, a number of studies have revealed that HIF1α also maintains undifferentiated state of many types of stem cells such as embryonic stem cells, hematopoietic stem cells and mesenchymal stem cells. In this study, we sought to investigate whether HIF1α is localized in intestinal stem cells, hair follicle stem cells and germline cells, in which HIF1α expression has not yet been reported. Although we did not detect any HIF1α signal in intestinal and hair follicle stem cell compartments, we observed a clear expression of HIF1α in both male and female germ cells. Furthermore, our results showed the presence of HIF1α throughout the fetal and neonatal development of both male and female germ cells, raising a possibility that HIF1α may play a role as a transactivator of glycolytic genes in highly proliferative cells such as primordial germ cells and spermatogonial cell as those cell types heavily depend on glycolysis for energy production. Lastly, we investigated whether HIF1α regulates telomerase activity in the cultured neural stem/progenitor cell (NSPCs) population, as our previous study revealed that HIF1α transactivates mTert and subsequently regulates telomerase activity in mouse embryonic stem cells. Our shRNA-mediated Hif1α knockdown assay resulted in the decreased levels of telomerase activity, strongly suggesting that HIF1α transactivates mTert in NSPCs. Regulation of telomerase activity may be important to ensure the long term survival of NSPCs and continuous neurogenesis throughout the life of an organism. Together, the results of this study illustrate the involvement of HIF1α in normal development and maintenance of types of stem/progenitor cells in which the localization and function of HIF1α was never extensively discussed, pointing out that further investigation may lead to better understanding of the roles of HIF1α in multipotent cells.

Ph.D. - Developmental and Reproductive Biology

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