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Qinglei Li, Fermin Jimenez-Krassel, James J Ireland and George W Smith

The molecular mechanisms of ovulation and luteinization have not been well established, partially due to lack of a comprehensive understanding of functionally significant genes up-regulated in response to an ovulatory stimulus and the signaling pathways involved. In the present study, transcripts increased in bovine preovulatory follicles following a GnRH-induced LH surge were identified using microarray technology. Increased expression of 368 and 878 genes was detected at 12 (368 genes) and 20 h (878 genes) following GnRH injection. The temporal, cell specific and prostanoid-dependent regulation of selected genes (ADAM10, DBI, CD36, MTSS1, TFG, and RABGAP1) identified from microarray studies and related genes (ADAM17 and AREG) of potential significance were also investigated. Expression of mRNA for DBI and CD36 was simultaneously up-regulated in theca and granulosa cells (GC) following the LH surge, whereas temporal regulation of ADAM10, MTSS1, TFG, and RABGAP1 was distinct in the two cell compartments and increased granulosa TFG and RABGAP1 mRNA were prostanoid dependent. AREG mRNA was increased in theca and GCs at 12 and 24 h following GnRH injection. ADAM17 mRNA was increased in theca, but reduced in GCs 24 h following GnRH injection. The increased ADAM17 and AREG mRNA were prostanoid dependent. ADAM10 and ADAM17 protein were increased specifically in the apex but not the base of preovulatory follicles and the increase in ADAM17 was prostanoid dependent. Results reveal novel information on the regulation of preovulatory gene expression and suggest a potential functional role for ADAM10 and ADAM17 proteins in the region of follicle rupture.

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Tirtha K Datta, Sandeep K Rajput, Gabbine Wee, KyungBon Lee, Joseph K Folger and George W Smith

Upstream stimulating factor 1 (USF1) is a basic helix–loop–helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8–16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation of JY1, GDF9, and FST genes associated with oocyte competence.

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Jennifer L Juengel, Michelle C French, Laurel D Quirke, Alexia Kauff, George W Smith and Peter D Johnstone

We hypothesised that cocaine- and amphetamine-regulated transcript (CARTPT) would be differentially expressed in ewes with differing ovulation rates. Expression of mRNA for CARTPT, as well as LHCGR, FSHR, CYP19A1 and CYP17A1 was determined in antral follicles ≥1 mm in diameter collected during the follicular phase in ewes heterozygous for the Booroola and Inverdale genes (I+B+; average ovulation rate 4) and ++ contemporaries (++; average ovulation rate 1.8). In ++ ewes (n = 6), CARTPT was expressed in small follicles (1 to <3 mm diameter), where 18.8 ± 2.5% follicles expressed CARTPT. CART peptide was also detected in follicular fluid of some follicles of ++ ewes. In I+B+ ewes, 5/6 ewes did not have any follicles that expressed CARTPT, and no CART peptide was detected in any follicle examined. Expression pattern of CYP19A1 differed between I+B+ and ++ ewes with an increased percentage of small and medium follicles (3 to <4.5 mm diameter) but decreased percentage of large follicles (≥4.5 mm diameter) expressing CYP19A1 in the I+B+ ewes. Many of the large follicles from the I+B+ ewes appeared non-functional and expression of LHCGR, FSHR, CYP17A1 and CYP19A1 was less than that observed in ++ ewes. Expression of FSHR and CYP17A1 was not different between groups in small and medium follicles, but LHCGR expression was approximately double in I+B+ ewes compared to that in ++ ewes. Thus, ewes with high ovulation rates had a distinct pattern of expression of CARTPT mRNA and protein compared to ewes with normal ovulation rates, providing evidence for CART being important in the regulation of ovulation rate.

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Qinglei Li, Leanne J Bakke, J Richard Pursley and George W Smith

The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are potential regulators of the focalized extracellular matrix degradation required for ovulation. The objectives of the present study were to determine localization and temporal regulation of TIMP-3 and TIMP-4 mRNA and protein in bovine preovulatory follicles. Ovaries containing preovulatory follicles were collected at 0, 12 and 20 h after GnRH injection for real-time PCR quantification of TIMP-3 and TIMP-4 mRNAs and immunohistochemical localization studies. Additional samples collected at 0, 6, 12, 18 and 24 h post GnRH injection were subjected to Western analysis to determine temporal changes in TIMP-3 and TIMP-4 proteins in the apex and base of preovulatory follicles. Results indicate the gonadotropin surge regulates TIMP-3 and TIMP-4 expression. TIMP-3 and TIMP-4 mRNAs increased within 12 h after GnRH injection. TIMP-3 protein was localized to granulosal and thecal layers of preovulatory follicles and adjacent ovarian stroma, whereas TIMP-4 immunoreactivity was localized to granulosal and thecal cells and ovarian blood vessels. Amounts of TIMP-3 and TIMP-4 proteins in the follicular apex peaked within 12 h post GnRH injection and subsequently declined by 24 h. However, amounts of TIMP-3 and TIMP-4 proteins in the base were not elevated after GnRH administration. Results demonstrate that mRNA and protein for both TIMP-3 and TIMP-4 are increased in bovine preovulatory follicles following the gonadotropin surge. Coordinate expression of TIMPs and MMPs may help regulate the extracellular matrix remodeling characteristic of the ovulatory process.

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Qinglei Li, Fermin Jimenez-Krassel, Yasuhiro Kobayashi, James J Ireland and George W Smith

A growing body of evidence supports an obligatory role for intrafollicular prostanoids in the mechanism of ovulation. However, the prostanoid-dependent mediators of the follicular extracellular matrix degradation required for ovulation are unknown. The objectives of this study were to determine the cellular compartment(s) in which the gonadotropin surge-induced regulation of select extracellular matrix degrading enzymes and their cognate inhibitors occurs in bovine preovulatory follicles, and to test whether such regulation is blocked by intrafollicular administration of the prostanoid synthesis and ovulation inhibitor, indomethacin (INDO). Follicular fluid prostaglandin E2 concentrations were elevated in diluent-treated follicles before ovulation (24 h after GnRH injection), but the increase was blocked in INDO-treated follicles. Real-time PCR analysis revealed the specific follicular cell types where gonadotropin surge-induced increases in mRNA abundance for members of the matrix metalloproteinase/tissue inhibitor of metalloproteinase and plasminogen activator families occurred. INDO treatment increased thecal cell mRNA for tissue inhibitor of metalloproteinase-4 and its protein abundance in the apex of preovulatory follicles before ovulation, but suppressed granulosal cell mRNA and activity for tissue plasminogen activator in follicular fluid and the follicle apex. Plasmin activity was also suppressed in the follicular fluid of INDO-treated follicles. Effects of INDO injection on select matrix metalloproteinases were not observed. The results suggest that gonadotropin surge-induced regulation of tissue inhibitor of metalloproteinase-4 and tissue plasminogen activator may be prostanoid dependent, and support a potential role for increased tissue plasminogen activator expression and decreased tissue inhibitor of metalloproteinase-4 expression in the mechanism of ovulation.

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Osman V Patel, Anilkumar Bettegowda, James J Ireland, Paul M Coussens, Patrick Lonergan and George W Smith

Poor oocyte competence contributes to infertility in humans and livestock species. The molecular characteristics of such oocytes are generally unknown. Objectives of the present studies were to identify differences in RNA transcript abundance in oocytes and early embryos associated with reduced oocyte competence and development to the blastocyst stage. Microarray experiments were conducted using RNA isolated from germinal vesicle stage oocytes collected from adult versus prepubertal animals (model of poor oocyte competence). A total of 193 genes displaying greater mRNA abundance in adult oocytes and 223 genes displaying greater mRNA abundance in prepubertal oocytes were detected. Subsequent gene ontology analysis of microarray data revealed significant overrepresentation of transcripts encoding for genes in hormone secretion classification within adult oocytes and such genes were selected for further analysis. Real-time PCR experiments revealed greater abundance of mRNA for βA and βB subunits of inhibin/activin and follistatin, but not the α subunit in germinal vesicle stage oocytes collected from adult versus prepubertal animals. Cumulus cell follistatin and βB subunit mRNA abundance were similar in samples collected from prepubertal versus adult animals. A positive association between time of first cleavage (oocyte competence) and follistatin mRNA abundance was noted. Follistatin, βB, and α subunit mRNAs were temporally regulated during early bovine embryogenesis and peaked at the 16-cell stage. Collectively, results demonstrate a positive association of follistatin mRNA abundance with oocyte competence in two distinct models and dynamic regulation of follistatin, βB, and α subunit mRNAs in early embryos after initiation of transcription from the embryonic genome.