Doctoral Dissertations

Date of Award

12-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Animal Science

Major Professor

James D. Godkin

Committee Members

Judith M. Grizzle, Jeffrey A. MacCabe, F. Neal Shrick, Donald S. Torry

Abstract

Vitamin A (retinol) and its natural metabolites, isoforms of retinoic acid, are collectively known as retinoids and are important signaling molecules in vertebrate development and (differentiation. A growing body of evidence suggests retinoids play an important role in reproduction. Systemic and intercellular transport of retinol is mediated by retinol binding protein (RBP). The cellular binding proteins, cellular retinol binding proteins (CRBPs) and cellular retinoic acid binding proteins (CRABPs), participate in retinol storage and metabolism. The actions of retinoids are mediated through nuclear retinoic acid receptors which belong to the family of nuclear ligand inducible transcription factors. The goals of this study were to localize the retinoid binding proteins in the oviduct and ovary and examine the effect of retinoids on oocyte maturation/competence and early embryonic development. Retinol binding protein was immunolocalized to the luminal epithelia of oviductal mucosa of both the ampulla and isthmus. Retinol binding protein expression was considerably higher on day 1 than days 5 or 10 of the estrous cycle. Synthesis, secretion and gene expression of RBP was modulated by ovarian steroids. Within the ovary, RBP was localized IV primarily in the thecal cells of non atretic follicles with some diffuse staining in the granulosa cells and stromal cells. Retinol binding protein staining was also present in the cytoplasm of oocytes from some antral but not pre-antral follicles. Cellular retinol binding protein was localized in thecal cells of non atretic follicles with diffuse staining in the stromal layer. Cellular retinol binding protein was observed in large cells of the corpus luteum while RBP was identified in both large and small luteal cells. Only CRABP exhibited intense staining in nuclei of oocytes from primordial follicles, but was not observed in oocytes or follicular cells of all other size follicles. Cellular retinoic acid binding protein was absent in all cells of the corpus luteum but was present in the stromal layer encapsulating it. In cattle, ovaries from normal cycling animals were analyzed for the presence of RBP and CRBP. Retinol binding protein mRNA was present in thecal but not granulosa cells of antral follicles. However, both thecal and granulosa cells synthesized RBP in vitro. No relative differences were observed in RBP mRNA concentrations or synthesis in luteal tissue between days 2, 6, 10 or 15. Retinol binding protein and CRBP were immunolocalized exclusively to large luteal cells. Experiments were conducted to identify effects of retinoid treatments on superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with retinoids or vehicle on the first and last day of FSH treatment and embryos surgically recovered and cultured in vitro until occurrence of blastocyst formation and hatching. Treatment of ewes with retinol during superovulation resulted in a dramatic increase in both blastocyst formation and embryonic hatching in comparison with retinoic acid, 9 cis retinoic acid or vehicle. Retinol treatment also significantly improved the number of embryos that progressed through the 8-cell in vitro block. The final experiment was conducted to evaluate the effects of the retinoids, retinol (100 µM and 10 µM ROH) and retinoic acid (10 µM and 1 µM RA) on the maturation, fertilization and subsequent development of cattle oocytes/embryos in vitro. Addition of 100 µM ROH or 10 µM RA to either maturation and fertilization or embryonic culture media resulted in no blastocyst formation or reduced blastocyst formation. The presence of retinoids in the lower concentrations during oocyte maturation either had no effect or diminished blastocyst formation. Embryos cultured in the presence of 10 µM ROH or 1 µM RA, regardless of maturation and fertilization treatment, exhibited increased blastocyst formation in comparison with culture medium alone. Collectively, these experiments identify retinoid VI binding proteins in the ovary and oviduct and provide evidence that retinoids may influence oocyte development/competence and early embryonic development. These results suggest that retinol has the potential to positively impact reproductive efficiency and assisted reproduction protocols in domestic animals.

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