MicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

The protein pattern of the follicular fluid (FF) and the ultrastructure of the inner cumulus–oocyte complex (COC) has been analysed in single antral follicles (n = 146) of buffalo B. Bubalis ovaries. The protein population of FF was fractionated by SDS-PAGE; the resulting pattern was Coomassie stained and processed for densitometry. Comparartive analysis of sera and autologous FFs showed a marked difference in the level (measured as the percentage of total proteins) of one 21 kDa polypeptide band, called ‘L’. Concentration of L, which was mainly higher in the serum (2.05 ± 1.5%) than in the surrounding FF(0.98 ± 0.94%), fluctuated widely in fluids from the in the same ovary. On gel filtration of FF and SDS-PAGE of ther factions collected, the L polypeptide was found and eluted together with a 36 kDa polypeptide, called ‘H’, with an exclusion volume lower than that of albumin. The levels of both polypeptides in the eluted fractions were measured by gel densitometry and the same ratio H/L was found (2:1). These data suggest that H and L are subunits of a complex high-molecular-weight protein. The presence of L levels in male sera comparable to those detected in females indicates that this putative protein does not originate in the ovary but is transported from the blood. Moreover, a correlation between the increase in the percentage of Lf (calculated as %L in FF/%L in serum) and atresia was observed. COCs (n = 86) obtained during the collection of the single FF samples were processed for transmission electron microscopy. The ultrastructure of each COC was compared with the SDS-PAGE data of the associated FF. Healthy COCs were found to be related to very low levels of Lf (between 0 and 14% of those measured in serum). COCs with an early atretic ultrastructure undetectable at the dissection microscope, were associated with FFs having Lf levels between 24% and 60%; advanced atresia was associated with Lf values up to 70%. Finally, the acrosome reaction of buffalo precapacitated spermatozoa in vitro was monitored by adding one volume of FF with high (FF +; Lf = 80%) or undetectable (FF - ) values of Lf to the sperm suspension. It was found that 96.2% of spermatozoa treated with FF - showed a normal acrosome reaction, without any change in their motility pattern; spermatozoa treated with FF +, in contrast, exhibited poor motility, and only 66.4% of them underwent the acrosome reaction. The ionophore A23187, in a control experiment, resulted in lower percentages of acrosome reactions (38%). In conclusion, our results suggest that enhanced influx of L from serum into the follicle antrum causes an increase in the polypeptide level in FF. This might be due to a loss of selectivity in the blood-follicle barrier of early atretic follicles. The use of the L polypeptide as a biochemical marker for identifying the earlier stage of follicular atresia is proposed.

The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus–oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p < 0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (1 arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p < 0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.