Granulosa Cumulus-oocyte Complexes Research Articles

Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes.

Based on the morphology and expansion of the cumulus cells, several different classes of porcine cumulus-oocyte complexes (COCs) can be distinguished, during their maturation in vitro. The goal of the present study was to find out the rate of each morphologic category in case of COCs and granulosa-cumulus-oocyte complexes (GCOCs), the characteristics of their nuclear progression, cytoplasmic maturation, and the frequency of monospermy after IVF. It was found that the frequency of cumulus expansion is higher in case of GCOCs than that of COCs. Nuclear progression of COCs was more accelerated than that of GCOCs. Oocytes attached to the bottom of culture dish with dark, compact cumulus underwent nuclear and acquired their ability to be activated earlier than that of oocytes showing normal cumulus expansion. The rate of monospermic fertilization after IVF of normal COCs showing normal cumulus expansion was higher than that of COCs attached to the dish. These results suggest that diverse behavior of cumulus cells during in vitro culture affects nuclear and cytoplasmic maturation of porcine oocytes, which also affects IVF results. It can be concluded that granulosa cells promote normal cumulus expansion thus decrease heterogeneity in nuclear and cytoplasmic maturation amongst oocytes.

Meiotic state of bovine oocytes is regulated by interactions between cAMP, cumulus, and granulosa.

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.

Developmental Competence of In Vitro Matured Porcine Oocytes after Electrical Activation.

Developmental competence of in vitro matured porcine oocytes obtained by TCM199- and NCSU 23-based IVM systems after electrical activation were compared. When the nuclear phase of granulosa-cumulus-oocyte complexes (GCOCs) used in the TCM199-based IVM system and cumulus-oocyte complexes (COCs)used in the NCSU 23-based IVM system were observed, most of GCOCs were fully grown dictyate arrest oocytes (GV I, 68.6%), on the other hand, only 33.8% of COCs were at GV I stage. There were no differences in the rates of maturation (93.2%, 82/88 vs 91.3%, 63/69), normal cleavage (70.7%, 58/82 vs 65.1%, 41/63) and development to blastocysts (37.8%, 31/82 vs 34.9%, 22/63) between the in vitro matured oocytes obtained by the two IVM systems. However, the parthenogenetic blastocysts derived from the NCSU23-based IVM system had higher cell numbers than the blastocysts derived from the TCM199-based IVM system (35.6 ± 4.5 vs 22.6 ± 2.0; P<0.01). Transfer of parthenogenetic oocytes derived from the NCSU23-based IVM system resulted in a high developmental rate to somite stage fetuses (26.9%, 45/167). These data demonstrate that the activated porcine oocytes derived from the two IVM systems had equal ability of in vitro development, though there was a difference in the cell number of the parthenogenetic blastocysts. It was also shown that in vitro matured oocytes obtained by the NCSU23-based IVM system had excellent ability of parthenogenetic development to fetuses, indicating that they are usable in porcine developmental engineering.

Effects of epidermal growth factor, insulin‐like growth factor‐I, and dialyzed porcine follicular fluid on porcine oocyte maturation in vitro

Undefined follicular factors that may influence nuclear maturation and/or cytoplasmic maturation are required during in vitro maturation of pig oocytes. Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dialysed porcine follicular fluid (dpFF) were evaluated for their effects on porcine oocyte nuclear maturation in vitro. In Experiment I, eight different maturation media were made in a split-plot factorial design with dpFF (0% vs. 10% v/v dialyzed pFF) as the whole plot component, and EGF (0.0 vs. 50 ng/ml) and/or IGF-I (0.0 vs. 100 ng/ml) as the factorial subplot component. Experiment II was a complete factorial design with dpFF and EGF. Pig follicular granulosa-cumulus-oocyte complexes (GCOC) were obtained from slaughterhouse ovaries, washed, and cultured at 38.5 degrees C in a humidified incubator with 5% CO2 in air for 42 h. Following culture, GCOC were mechanically stripped of granulosa-cumulus cells and evaluated for nuclear maturation by light microscopy. In Experiment I, the percentage of Metaphase II oocytes for control, IGF-I, EGF, and IGF-I+EGF treatments without pFF were 50.7%, 52.6%, 80.9%, and 84.3% (control and IGF-I groups significantly less, P < .001). The same treatments in the presence of pFF were similar and high (84.2, 84.9, 82.1, and 86.8%, respectively). Experiment II gave similar results. These results demonstrate that EGF, in the absence of pFF, promotes a similar level of oocyte nuclear maturation as does pFF alone or pFF with EGF and/or IGF-I. IGF-I does not appear to influence nuclear maturation of GCOC.