Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes

Abstract

Contents Growth differentiation factor 9 (GDF‐9) and bone morphogenetic protein 15 (BMP‐15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199‐Hepes medium as (a) Control group; (b) GDF‐9 antibody (Ab); (c) BMP‐15 Ab; (d) recombinant human (rh) GDF‐9; (e) rh BMP‐15 or (f) rh BMP‐15 and GDF‐9. Data were evaluated by ANOVA. The Abs against GDF‐9 or BMP‐15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF‐9 Ab (64.4 ± 2.1%) or BMP‐15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF‐9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP‐15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF‐9 (3.7 ± 0.4%) or rhBMP‐15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF‐9 (14.7 ± 3.1) and BMP‐15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs.

GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process

“…In the present study, we found a clear difference in the expression of the GDF-9 and BMP-15 genes in CCs according to the phases of the cycle, however, we did not find such differences in meiotic maturation rates (see Table 2). Meiotic development in oocytes from follicles at different estrous phases was low and within the range reported for in vitro-matured canine oocytes [12,14,27,47]. Although the percentages of MII oocytes were not different among the reproductive phases, the most remarkable result of the meiotic development analyses was the relationship of the cumulus expansion with the percentage of oocytes at the MII stage, but only during the estrus phase.…”

“…At the same time, there was a significantly lower expression of both genes in expanded CCs from IVM COCs retrieved at estrus in comparison to non-cultured COCs, suggesting that other factors possibly play a role in the meiotic stimulus at the final stage of development. However, these proteins seem to be important in acquiring meiotic competence, because the addition of recombinant GDF-9 and BMP-15 in oocyte maturation media was shown to enhance the competence of canine oocytes matured in vitro [14]. This effect is possible achieved in the early development stages.…”

“…Cumulus-oocyte complexes (~400) at each phase of the estrous cycle were in vitro-maturated for 72 h in accordance with our previous studies [14]. In brief, COCs were incubated in 100 µL drops of Tissue Culture Medium 199 (TCM199-25 mM Hepes; Earle's salt, Invitrogen, Carlsbad, CA, USA), with 10% fetal calf serum (FCS) (Merck KGaA), 0.25 mM pyruvate, 10 IU/mL of hCG, 300 IU/ mL of penicillin, 20 µg/mL of streptomycin and, 2 µg/mL of ß-estradiol (E 2 ) (E8875-1G.…”

“…GDF-9 and BMP-15 were identified in canine granulosa/theca cells [11] and in canine oocytes during in vitro maturation (IVM) [12], and their changes during follicular development throughout the dog estrous cycle were also reported [13]. Recent studies using recombinant GDF-9 and BMP-15 in vitro demonstrated that both growth factors in combination also play a significant role in meiotic development in dogs [14].…”

A Comparative Analysis of Oocyte Development in Mammals

“…GDF9 and BMP15 may cooperate in the process of oocyte meiosis resumption in dogs. Supplementing in vitro cultures with both recombinant human proteins (supplied as homodimers of the mature region) increases the rates of oocytes reaching metaphase II, while this improvement is not observed with either GDF9 or BMP15 added individually [128]. However, the pathways by which GDF9 and BMP15 may interact synergistically to improve oocyte meiosis competence is currently unknown.…”