Reproductive Strategies in Animal Species Emphasizing Developmental Biology

Basic Sciences

Investigators

Abstract

The reproductive system of model species such as the domestic cat and its non-domestic relatives offers an unusual opportunity for understanding the changes and adaptations of genes that mediate species isolation and survival. The Laboratory of Genomic Diversity (LGD) has undertaken a comparative physiology approach to describe the aspects of feline reproduction that discriminate between species and allow for behavioral co-adaptation. In addition, empirical methods to develop cryopreservation have been assessed to optimize assisted reproductive technologies in these species, including artificial insemination, in vitro fertilization (IVF) and embryo transfer between these closely related species.<BR><BR>Models of how to use new technologies to assess reproductive fitness are emerging to help insure gene diversity and propagate endangered species. Non-invasive hormone metabolite monitoring assays, artificial insemination techniques and genome resource banking have been developed to aid in studies examining the adaptive differences among the Felidae. Significant discoveries include the finding that standard cooling techniques for cat sperm result in extensive cell membrane damage, allowing the creation of slower, more effective cooling procedures. Sperm from males producing many malformed cells are less likely to survive cooling-freezing-thawing stress. Egg freezing studies reveal that cat eggs are highly sensitive to cool temperatures. Investigations also continue on the transmission of Feline Immunodeficiency Virus (FIV) (related to HIV) in cat semen. <BR><BR>A related challenge is the high incidence of abnormally shaped sperm found in the semen of some domestic cats and many endangered cat species. This condition, known as teratospermia and common in men, limits fertilization capacity. Although sperm from teratospermic cats were found to have the same amount of DNA as normal sperm, the former have decreased amounts of protamine, a class of nuclear proteins that play an important role in DNA stabilization. Semen has also been collected and cryopreserved from several wildtype and disease cat models for testing through IVF studies<BR><BR>Cats have the short generation times and large litter sizes required for development of transgenic and knock-out research models. Successful adaptation of these techniques to the cat would greatly increase the ability of researchers to develop feline models of human genetic diseases. In collaboration with James Kehler (University of Pennsylvania, School of Veterinary Medicine), we are determining the conditions necessary to propagate and to genetically manipulate feline embryos and to establish feline embryonic stem (ES) and embryonic germ (EG) cells. A retroviral vectors has been used successfully to transduce feline embryos and a putative feline EG cell line. This proof of principle demonstration paves the way for performing gain and loss of function experiments during embryonic development of the cat. Initial studies will determine whether expression of the transcription factor <I>Oct4</i> is required for the maintenance of pluripotency in the pre-implantation cat embryo and for formation of feline ES cells, and will determine if <I>Oct4</i> is required for the survival of cat primordial germ cells and the formation of EG cells. While in mice the transcription factor <I>Oct4</i> plays a role in maintenance of pluripotency in ES cells and in the survival of primordial germ cells, the role of <I>Oct4</i> during development of other mammals has not been established. In addition, <i>Oct4</i> has also been implicated as an oncogene required to maintain the pluripotentiality of some human testicular germ cell tumors (TGCTs). A novel in vitro assay is being developed using human TGCT cell lines to screen for <i>Oct4</i> inhibitory compounds. We are developing the cat as a comparative model system for studying the role of <I>Oct4</i> and other genes during mammalian development. <p></p>Several key resources have been established during the past year to facilitate these developmental studies using cat models. Specifically, laparoscopic oocyte collection, IVF and embryo transfer experiments have been successfully performed to generate cat embryos for exeriments at the NIHAC in Dickerson, MD. Studies are ongoing through September of 2007, to establish and characterize potential feline ES and EG cell lines. In addition, we are beginning to collaborate with Dr. Ronald McKay at the NINDS to isolate epiblast stem cell lines from post-implantation cat embryos. In parallel to the establishment of potential pluripotent cell lines from cat embryos, feline embryonic fibroblast lines have been established and characterized. These somatic cell lines will be used in the coming year for gene targeting through homologous recombination and subsequent nuclear transfer experiments to develop new knock-out models of cats. Towards this goal, an electroporator device has been acquired and a protocol has been developed to introduce foreign DNA constructs into cat cell lines. The recent publication of the annotated 2X Feline Genome Project that was spear-headed by our group will facilitate the production of genetic constructs to target specific loci within the cat genome