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Gawecka J.E.,University of Hawaii at Manoa | Ribas-Maynou J.,Autonomous University of Barcelona | Ward W.S.,Institute for Biogenesis Research | Ward W.S.,University of Hawaii at Manoa
Asian Journal of Andrology | Year: 2015

The highly condensed chromatin of mammalian spermatozoa is usually considered to be biologically inert before fertilization. However, we have demonstrated that even in this compacted state, sperm chromatin is subject to degradation at open configurations associated with the nuclear matrix through a process we have termed sperm chromatin fragmentation (SCF). This suggests that a mechanism exists to monitor the health of spermatozoa during transit through the male reproductive tract and to destroy the genome of defective sperm cells. The site of DNA damage in SCF, the matrix attachment sites, are the same that we hypothesize initiate DNA synthesis in the zygote. When sperm that have damaged DNA are injected into the oocyte, the newly created zygote responds by delaying DNA synthesis in the male pronucleus and, if the damage is severe enough, arresting the embryo's development. Here we present a model for paternal DNA regulation by the nuclear matrix that begins during sperm maturation and continues through early embryonic development. © 2015 AJA, SIMM & SJTU. All rights reserved.


Owens J.B.,Institute for Biogenesis Research | Urschitz J.,Institute for Biogenesis Research | Stoytchev I.,Institute for Biogenesis Research | Dang N.C.,Institute for Biogenesis Research | And 6 more authors.
Nucleic Acids Research | Year: 2012

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24 of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy. © The Author(s) 2012.


News Article | January 6, 2016
Site: phys.org

The researchers, including Dr. Monika Ward, post-doctoral fellows Yasuhiro Yamauchi and Jonathan Riel, and PhD student Victor Ruthig, who are all from of the Institute for Biogenesis Research (IBR), have discovered that only three genes from the Y chromosome are needed for male mice to make sperm able to fertilize oocytes and generate offspring after Intracytoplasmic Sperm Injection (ICSI), a fertilization technique developed at the John A. Burns School of Medicine at UH Mānoa. Two years ago the same group reported it successfully obtained offspring from male mice that had only two Y chromosome genes, testis determinant Sry and spermatogonial proliferation factor Eif2s3y. These males did not produce sperm and, to achieve fertilization, researchers had to use the immature precursor cells, spermatids, and a technique called Round Spermatid Injection (ROSI). The Practice Committee of the American Society of Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology considers ROSI an experimental procedure and do not recommend it for treatment of male infertility. ICSI, however, is used commonly worldwide, with thousands of children born annually. At the IBR, researchers considered which of the Y chromosome genes may be responsible for turning spermatids into sperm. In an international collaboration with Paul Burgoyne's group from Francis Crick Institute in London, England, and Michael Mitchell from INSERM, in Marseille, France, they hypothesized that the key gene is Zfy2 (zinc finger protein 2). They added the Zfy2 transgene to males already transgenic Sry and Eif2s3y and lacking the Y chromosome. The resulting males carrying only three Y chromosome genes were producing sperm. These males were not able to reproduce on their own because their sperm number was too low. But when the researchers harvested sperm from the testes and injected them into the oocytes, they become fertilized. And when the embryos were transplanted to surrogate mothers, young were born with the same efficiency as from males with normal intact Y chromosome. Demonstration that three Y chromosome derived genes are enough for a formation of sperm functional in assisted fertilization is an important finding advancing current knowledge about Y chromosome gene function "Considering that ICSI, and not ROSI, is commonly used in human infertility treatment, the findings bear translational significance," said Dr. Ward. "Transformation of round spermatids into sperm is a key developmental process gaining a lot of attention due to newly ascribed roles for the sperm epigenome in fertilization and transgenerational inheritance." Dr. Ward's study points to Zfy2 being a key regulator in this process, including the function of its end product—spermatozoa. Dr. Ward's team described the discovery in a manuscript published by the leading genetics journal PLoS Genetics. An accompanying manuscript from Burgoyne and Mitchell groups will appear in PLoS One. Explore further: Two Y genes can replace the entire Y chromosome for assisted reproduction in mice


Two years ago, the University of Hawaii (UH) team led by Monika A. Ward, Professor at the Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawai'i, demonstrated that only two genes of the Y chromosome, the testis determinant factor Sry and the spermatogonial proliferation factor Eif2s3y, were needed for male mice to sire offspring with assisted fertilization. Now, the same team, with a collaborating researcher from France, Michael Mitchell (INSERM, Marseille), took a step further and produced males completely devoid of the entire Y chromosome. In this new study scheduled for online publication in the journal Science on Jan. 29, 2016, Ward and her UH colleagues describe how they generated the "No Y" males, and define the ability of these males to produce gametes and sire offspring. The UH researchers first replaced the Y chromosome gene Sry with its homologue and direct target encoded on chromosome 11, Sox9. In normal situation, Sry activates Sox9, and this initiates a cascade of molecular events that ultimately allow an XY fetus to develop into a male. The researchers used transgenic technology to activate Sox9 in the absence of Sry. Next, they replaced the second essential Y chromosome gene, Eif2s3y, with its X chromosome encoded homologue, Eif2s3x. Eif2s3y and Eif2s3x belong to the same gene family and are very similar in sequence. The researchers speculated that these two genes may play similar roles, and it is a global dosage of both that matters. They transgenically overexpressed Eif2s3x, increasing dose of the X gene beyond that provided normally by X and Y. Under these conditions, Eif2s3x took over the function of Eif2s3y in initiating spermatogenesis. Finally, Ward's team replaced Sry and Eif2s3y simultaneously, and created XOSox9,Eif2s3x males that had no Y chromosome DNA. Mice lacking all Y chromosome genes developed testes populated with male germ cells. Round spermatids were harvested and a technique called round spermatid injection (ROSI) was used to successfully fertilize oocytes. When the developed embryos where transferred to female mouse surrogate mothers, live offspring were born. The offspring derived from the "No Y" males were healthy and lived for normal life span. The daughters and grandsons of the "No Y" males were fertile and capable of reproducing on its own without further technological intervention. Ward's team produced three consecutive generations of "No Y" males using ROSI showing that males lacking Y chromosome genes can be repeatedly propagated with technical assistance. "Most of the mouse Y chromosome genes are necessary for development of mature sperm and normal fertilization, both in mice and in humans," Ward said. "However, when it comes to assisted reproduction, we have now shown that in the mouse the Y chromosome contribution is not necessary." The study provides new important insights into Y chromosome gene function and evolution. It supports the existence of functional redundancy between the Y chromosome genes and their homologues encoded on other chromosomes. "This is good news," Ward said, "because it suggests that there are back-up strategies within genomes, which are normally silent but are capable of taking over under certain circumstances. We revealed two of these strategies by genome manipulation. Whether such alternative pathways would ever be activated without human help, for example in response to environmental changes, is unknown. But it is certainly possible and has already happened for two rodent species which lost their Y chromosomes. " The development of assisted reproduction technologies (ART) allows bypassing various steps of normal fertilization by using immotile, non-viable, or immature gametes. The newest study as well as Ward's preceding report (Science 2014 Jan 3; 343 (6166: 69-72) support that in the mouse ROSI is a successful and efficient form of ART. In humans, ROSI is considered experimental due to concerns regarding the safety of injecting immature germ cells and other technical difficulties. The researchers hope that the success in mouse studies may spark the re-evaluation of human ROSI for its suitability to become an option for overcoming male infertility in the future. Explore further: Fortunately for men, size doesn't matter (much) More information: Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction, DOI: 10.1126/science.aad1795


Gelber K.,Institute for Biogenesis Research | Tamura A.N.,Institute for Biogenesis Research | Alarcon V.B.,Institute for Biogenesis Research | Marikawa Y.,Institute for Biogenesis Research
Journal of Assisted Reproduction and Genetics | Year: 2011

Purpose To assess the impact of embryonic stem cell culture medium (ESCM) on the pre- and postimplantation development of the mouse embryo, as a mammalian model, in comparison with the conventional culture medium, a potassium simplex optimized medium (KSOM). Methods Development in ESCM versus KSOM was compared in terms of embryo morphology, cleavage, cavitation, hatching, cell number, expression of TE and ICM transcription factors (Cdx2 and Oct4, respectively), implantation, and development in utero. Results An enriched medium like ESCM can be beneficial for in vitro embryo development when cultured from the 8- cell stage, as evidenced by promotion of blastocyst development with respect to cavity expansion, hatching, and cell division. Such benefits were not observed when embryos were cultured from the 2-cell stage. Conclusions ESCM may augment in vitro embryo development from the 8-cell stage. Using different culture media at different stages may be beneficial to achieve more effective human in vitro fertilization. © Springer Science+Business Media, LLC 2011.


PubMed | Institute for Biogenesis Research
Type: Journal Article | Journal: PloS one | Year: 2016

Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1 was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1 in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1 expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1 was expressed in adult male and female germ cells. In the testis, Hif1 was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1 was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1 transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1 protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1 is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1 in early progenitor cells.

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