College of Agriculture & Natural Resources
Permanent URI for this communityhttp://hdl.handle.net/1903/1598
The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
Browse
3 results
Search Results
Item Derivation of pluripotent stem cells from blastocysts and somatic cells in the domestic cat (Felis Catus)(2018) Zhou, Ran; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pluripotent stem cells in the domestic cat model represent a huge potential for disease modeling, drug screening and regenerative medical treatments for feline species as well as for humans. However, current knowledge on deriving and maintaining stem cells has been obtained primarily from studies in mouse, rat, and human. Difficulties in attaining similar results in cats indicate the necessity to better understand pluripotency in this species. The hypothesis was that inadequate cytokine supplementation results in pluripotency loss along with declining transcription factors expression. The main goal of this project was to assess the effects of selected growth factors and inhibitors, in maintaining pluripotency in embryonic cells, and to attain pluripotency from fibroblasts by controlling expression of exogenous transcription factors. In the first study, conventional cytokine cocktails, leukemia inhibitory factor coupling with glycogen synthase inhibitor 3, and mitogen-activated protein kinase inhibitor (LIF and 2i) could partially maintain pluripotency regulatory circuitry in the cat. In this condition, embryonic cells reached a state that was not fully defined (neither naive nor primed). Overall, cell characterizations revealed a trend of pluripotency loss over time. In the second study, pluripotency was attained by forced expression of inducible exogenous transcription factors (NANOG, POU5F1, CMYC, and SOX2) and cultured in medium supplemented with the same cytokine combination identified in the first study. Notably, unlike previous reports in the cat, colonies with partial pluripotent features could be maintained after the transgenes were silenced. In addition to the protein and transcript markers for pluripotency, lineage marker dynamics were examined in pluripotent cells and embryoid bodies. The outcome suggested the cells generated with LIF and 2i had developed beyond the undifferentiated stage of ICM in expanded blastocyst. Collective results not only challenged the efficacy of the cytokines combinations LIF and 2i in maintaining feline pluripotency, but also suggest direction of research towards the species-specific signaling requirement in embryonic progression and stem cell derivation.Item THE IMPACT OF CULTURE MEDIA ON THE IN VITRO PRODUCTION OF CAT BLASTOCYSTS AND EXPLANT QUALITY(2012) Nestle, Emily; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continued improvements in embryo culture media composition allow for the growth of high quality blastocysts, which can be used to derive embryonic stem cells (ESCs). ESCs are capable of becoming any cell type in the body making them a valuable research tool for therapeutic and regenerative research, while furthering our understanding of embryonic development and cell differentiation. The domestic cat is an important model species for both human medicine and wild felids. Cat embryo culture produces blastocysts at a rate far below that of the mouse and initial attempts at deriving cat ESCs have resulted in embryonic stem-like cells, which cannot be maintained indefinitely. In this study we assessed and compared the quality of cat blastocysts produced in vitro using two commercial human blastocyst growth media, and the maintenance of pluripotency markers OCT-4 and NANOG in inner cell mass explants from in vitro produced blastocysts over 14 days.Item Priming with Oral Progestin Before Ovulation Induction Facilitates Ovarian Function in the Cat (Felis catus)(2007-11-21) Bauer, Rosemary Aileen; Ottinger, Mary Ann; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Artificial insemination (AI) has been developed in multiple felid species as a tool for retaining gene diversity in threatened or endangered populations. Yet, pregnancy success remains low (< 5%) following AI in most felids, particularly in species that spontaneously ovulate. This failure has been attributed to variable ovarian status at the time of insemination and adverse residual effects caused by exogenous gonadotropins used to induce ovulation. Using the domestic cat as a research model, a new AI regimen that incorporated short-term ovarian suppression with oral progestin (altrenogest; ALT) before ovulation induction was investigated. The hypothesis was that oral progestin priming would prevent spontaneous ovulation, improve ovarian responsiveness to exogenous gonadotropins and mitigate adverse effects caused by persistent gonadotropin actions. Specific objectives were to: (1) increase fundamental understanding of the mechanisms controlling ovarian function; and (2) characterize how oral progestin priming prior to exogenous gonadotropin treatment influences ovarian responsiveness, fertilization, early embryonic development and luteal function in the cat. Fecal hormone monitoring was used to establish an ALT dosage that provides rapid, reversible ovarian suppression with no residual effects on estrous cyclicity. With this information, the influence of progestin priming on ovarian responsiveness to exogenous gonadotropin dosage was investigated. Priming increased ovarian sensitivity to gonadotropins, supporting the use of lower dosages for ovulation induction. Next, in vivo fertilization success and in vitro early embryonic development was characterized following laparoscopic, intrauterine AI in cats treated with ALT. Progestin-primed females demonstrated a good ovarian response to ovulation induction and more consistent embryonic development, compared to cats treated with gonadotropins alone. Furthermore, endocrine data revealed that normal luteal progesterone levels were maintained only in queens primed with the oral progestin. Finally, histology and quantitative RT-PCR were used to characterize the differential effects on luteal function observed. Aberrant CL progesterone production was not associated with changes in ovarian morphology, or the expression of six specific genes associated with luteal function and progesterone biosynthesis. Overall, these studies increased knowledge of domestic cat reproductive physiology and improved understanding of ovarian suppression for enhanced AI efficiency in felids.