In the present experiment, bovine cumulus expansion was obtained with 1000 ng/ml pFSH in serum-free TCM-199 media. Although not significant, expansion in TCM with 100 ng/ml pFSH and 10% FCS tended to be greater than in 100 ng/ml pFSH alone. Earlier studies suggested that FSH-dependent cumulus expansion occurred only when serum was present in the culture media  and the effects of FSH and serum were additive over FSH or serum alone . However, older studies may have been done with sub-optimal culture media. The presence of glutamine and glucose or glucosamine in culture media (as precursors for hyaluronic acid synthesis) enhances FSH-induced cumulus expansion . TCM-199, commonly used for bovine IVM, contains both glutamine and glucose. Therefore, in the absence of serum, adequate concentrations of glucose and glutamine may be necessary for FSH to induce cumulus expansion in vitro.
Cumulus expansion was induced with only 1 ng/ml rhFSH in serum-free media, which is in contrast to the high concentration pFSH required for cumulus expansion. These results agree with recent studies reporting low rhFSH concentrations (0.01–1 IU/ml (1–100 ng/ml)) are effective for in vitro maturation in several species [4, 25, 26]. It is not clear why bovine cumulus expands at lower doses of rhFSH compared to pFSH. Recombinant human FSH has been shown to be more effective than urinary human FSH for superovulation of women , and it was suggested that its more basic isoforms and lesser degradation (4% vs. 40%) may be responsible for its increased effectiveness. Both rat and human FSH are reported to bind well to the human FSHr but ovine FSH binds poorly ; whereas the rat FSHr was reported to generate cAMP and testosterone more efficiently with recombinant human FSH than with recombinant rat FSH . However, only one published study has compared the binding efficiency of FSH of various species with the bovine FSHr. In that study, rhFSH bound better to calf testis than rat FSH . It is possible that porcine FSH does not efficiently bind the bovine FSHr, which could explain why high concentrations are required to initiate bovine cumulus expansion. Nonetheless, the majority of bovine superovulation protocols utilize pFSH for ovarian stimulation. While cumulus expansion is not necessary to achieve nuclear maturation, expansion of the cumulus may be an outward sign of favorable cytoplasmic maturation. In vivo, cumulus expansion is necessary to achieve ovulation and fertilization. However, during bovine IVF, when several thousand sperm are added for each egg, there may be sufficient sperm to fertilize oocytes with unexpanded cumulus, resulting in unaffected developmental rates.
Multiple FSHr transcript sizes are detected by Northern analysis in human testis , rat granulosa  and bovine ovary . Multiple transcripts may be a result of alternative splicing and the use of different polyadenylation sites. Primers used to amplify FSHr from bovine cumulus-oocyte complexes were identical to those used by van Tol et al.,  and produced the same two main product sizes, although others were detectable. A common mammalian FSHr splice isoform is missing exon 9 and this isoform was detected here and in other bovine studies [8, 34].
The abundance of both FSHr mRNA isoforms decreased in COCs after 6 h of maturation in IVM medium containing gonadotropins, estradiol and serum. This is consistent with the downregulation of FSHr mRNA and FSH binding in rat granulosa after a pre-ovulatory gonadotropin stimulus , decreased FSH binding after culture of mouse COCs in media containing serum , and decreased FSHr mRNA in bovine granulosa after culture with FSH . Abundance of high FSHr mRNA isoform, in particular, was higher in better quality COCs. The role of the exon 9-deleted FSHr isoform in FSH binding and signaling is unclear; other isoforms missing exon 2, 5 or 6 either do not bind FSH, or do not interfere with activities of the full-length receptor in cotransfection studies [35, 36].
Several LHr transcript sizes have been detected in mammals, which may result from different transcription start sites, alternative splicing and different polyadenylation signals. Although LHr mRNA was recently detected by RT-PCR only in the wall of bovine follicles, and not in granulosa or cumulus ; there were two mismatches in the primers derived from porcine sequence that may have affected binding and amplification efficiency of those primer sets. In the current studies, LHr primers consistently amplified a product of 533 bp from bovine cumulus and corpus luteum. These LHr primers were located in exon 11 and thus would detect only mRNAs encoding isoforms with a complete transmembrane domain, and not other truncated isoforms reported in ruminants [37, 38]. However, these primers would not distinguish the F isoform from the full-length mRNA. The F isoform, which differs from the full-length isoform in the absence of exon 10 (27aa), is the only LHr mRNA expressed in monkey testis, yet binds hCG and generates second messengers similarly to the full-length human LH receptor . In bovine corpora lutea, the full-length isoform LHr mRNA is expressed at approximately a 2–3:1 ratio to the F isoform [37, 38], and expression of all isoform mRNAs appears to be regulated coordinately in bovine corpora lutea throughout the estrous cycle . Because β-actin and other primer sets amplify across intron-exon borders and yet larger product sizes were not detected, and because expression of LHr mRNA tended to increase during the culture period, the detection of LHr mRNA in these experiments was not likely a result of genomic DNA contamination. Thus, the design of primers and probes, as well as which isoforms are expressed by a tissue, may also lead to the discrepancies in the ability to detect the LHr mRNA in various studies.
There was an effect of COC quality grade on abundance of LHr mRNA, as better quality COCs had greater LHr mRNA levels. There was a trend towards increased LHr mRNA abundance from 6 h to 24 h coincident with advancing maturation. The increase in LHr mRNA expression in cultured bovine COCs is corroborated by an increase in LH binding after culture of mouse COCs with FSH . Advancing nuclear maturation of oocytes may allow increased LHr mRNA expression in cumulus, as, although germinal vesicle stage mouse oocytes suppress granulosa LHr mRNA expression, mature oocytes are much less effective . It is likely that the increased abundance of LHr mRNA during maturation reflects increased luteinization of the cumulus cells.
Abundance of Cx43 mRNA was significantly lower in poorer quality COCs compared to better quality COCs. Differential expression of the Cx43 mRNA among varying COC classes indicates that this gene product may be a useful marker of oocyte competence. Reduced Cx43 mRNA could be inherent to the lower quality of these COCs, as when Cx43 mRNA is decreased in antisense experiments; Cx43 protein decreased, cumulus-oocyte dye transfer was lower and oocyte maturation rate was reduced . Cx43 mRNA abundance also decreased in COCs from 6 h to 18–24 h of maturation. This agrees with decreased Cx43 protein observed in the outer cumulus layers of COCs when matured for 12 h .
Marker genes that predict developmental competence could be used in the design of more appropriate maturation and culture media and selection of better oocytes for culture and embryos for transfer. This study indicates that levels of mRNAs encoding FSHr, LHr and Cx43 mRNAs are dependent on time of maturation and oocyte quality. These data may indicate differences in gonadotropin signaling among varying COC qualities that could reflect variation in their developmental competence.