Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation

Abstract

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serumbased IVM system (85.1% IVM, 25.8% blastocyst rate; PO0.05) but were significantly different (P!0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.

FSH and LH receptors are differentially expressed in cumulus cells surrounding developmentally competent and incompetent mouse fully grown antral oocytes

“…Indeed, previous studies showed that, although Fshr and Lhr transcripts are either undetectable (in the mouse; Fu et al 2007) or barely detectable (in the cow (Calder et al 2003;Simões et al 2012), sheep (Tisdall et al 1995;McNatty et al 2000;Logan et al 2002;Cotterill et al 2012) and pig (Shimada et al 2003)) in CCs of COCs isolated from unprimed females, the Lhr gene is upregulated when COCs are cultured in the presence of FSH. Overall, these results indicate that FSH likely has an inducing activity on the Lhr gene in vitro and in vivo.…”

The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro

“…Germinal vesicle stage oocytes ( n = 133) surrounded by three to four layers of tightly compact cumulus cells were aspirated from 3- to 5-mm antral follicles, and MII oocytes ( n = 71) were derived after IVM in the serum-free alpha MEM that contained pre-ovulatory surge levels of ovine LH (100 ng/ml) and ovine FSH (100 ng/ml), bovine transferrin (5 µg/ml), sodium pyruvate (0.47 mM), sodium selenite (5 ng/ml), l -glutamax (3 mM), 0.1% (w/v) BSA, insulin (10 ng/ml) and long R3 IGF-1 (10 ng/ml) (Cotterill et al , 2012). Cumulus enclosed oocytes were matured for 24 h at 39°C in 6% CO 2 , 5% O 2 and 89% N 2 in a humidified atmosphere (Cotterill et al , 2012). Oocytes used for molecular analysis were denuded and snap-frozen in liquid nitrogen in 10 µl of Dulbecco's PBS and kept at −80°C until use (Table I).…”

Time course of the meiotic arrest in sheep cumulus–oocyte complexes treated with roscovitine

“…This result reinforces the evidence that LH and FSH are not essential to resumption and progression of nuclear maturation in vitro (Sanbuissho & Threlfall, 1990). Despite this result, medium supplementation with these substances not only facilitates and accelerates oocyte meiosis but also favours cumulus cell expansion and improves cytoplasmic maturation (Mattioli et al , 1991; Cotterill et al , 2012). As already observed in bovine (Sirard et al , 1988) and goat (Han et al , 2006), our findings also demonstrated that nuclear maturation was not dependent upon cumulus cell expansion in sheep COCs.…”