Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.
Insufficient cytoplasmic maturation of MII oocytes may be one reason of the low rate of embryo production in vitro. To solve this problem, a two-step procedure of IVM is tested. In this case, there is a prematuration period (PMP) in which meiosis is arrested, followed by a resumption period (RP) during which the oocytes are liberated from the inhibition of meiosis. Preliminary studies have shown that a defined medium (DM) inhibits nuclear oocyte maturation (NM) (Unpublished data). Thus, our purpose was to verify if meiosis arrest is followed by an inhibition of cytoplasmic maturation (CM). Herein we investigated the effects of DM on NM and CM using the two-step procedure for IVM. COCs were selected from bovine ovaries and cultured in non-defined medium (NDM), composed of TCM-199 suplemented with 10% fetal calf serum and 200 ηg mL-1 FSH or in a defined medium (DM), MEMα supplemented with 0.1% Polyvinyl Alcohol. Both media are from Invitrogen-Gibco/BRL, Grand Island, NY, USA. In the PMP (experiment 1), COCs (n = 232) were distributed in G1 (NDM) or G2 (DM) and cultured for 24 h as usual. After this, NM and CM were analyzed. As inhibition of meiosis was observed in G2, a second step of culture (experiment 2) was performed to evaluate the ability of the COCs to reverse meiosis arrest. Thus, COCs (n =202) were distributed in G1 (NDM) or G3 (DM +NDM). COCs from G1 were cultivated in NDM for 24 h, and that ones from G3 in DM for 24 h followed by cultivation in NDM for an additional 24 h (RP). After this, NM and CM were analyzed. The CM evaluation (distribution of organelles) was performed in fresh oocytes (0 h) and in G1, G2, and G3 after usual preparation for transmission electron microscopy (TEM) analysis. The NM data were analyzed by Chi-square test. After the PMP period, G1 COCs presented 83.17% (84/101) of complete nuclear maturation whereas G2 presented 74.05% (97/131) of NM inhibition (oocytes in GV or GVBD) (P < 0.05). This meiosis arrest observed in DM was reversed after 24 h of cultivation in NDM, 79.17% (90/110) of G3 COCs were at MII. The analysis of CM has shown fresh COCs with ultrastructural characteristics of immaturity, as presence of mitochondria in the periphery of the ooplasm, and cortical granule clusters in the ooplasm; G1 COCs with mature cytoplasmic characteristics as mitochondria spread throughout the ooplasm and cortical granules located close to the oolema and G2 COCs with both immature and mature characteristics as presence of ooplasm granule clusters and presence of mitochondria spread throughout the ooplasm. G3 have shown mature cytoplasmic characteristics in the end of RP. In conclusion, a two-step procedure of IVM using a DM was able to maintain meiosis arrest while the cytoplasm undergoes maturation (G2). This could be a strategy to improve the rate of viable embryos obtained at in vitro embryo production. FAP-DF, Finatec, CNPq, Capes.
Granulosa cells (GC) and theca cells (TC) luteinize in culture because of the presence of serum in the medium and, as a consequence, the production of progesterone (P4) increases and that of 17β-estradiol (E2) decreases. The follicular phase of the bovine estrous cycle is characterized by the presence of E2, as well as aromatase activity, that is turned off in the atresia or in the luteal phase of the bovine estrous cycle. We propose to characterize 2 co-culture systems utilizing slices of follicular walls (explants) containing both GC and TC and to cultivate them in nondefined medium (NDM; TCM199+serum) or defined medium (DM; α MEM+polyvinyl alcohol). To prepare the explants, 80 follicles measuring 4 to 6 mm in diameter were selected. Concentrations of P4 and E2 were evaluated by RIA after 24, 48, and 72 h of culture and values were corrected by tissue weight (10 mg). Morphological analysis of the explants was stained with hematoxylin and eosin. The total mRNA from GC-TC explants was purified and the expression of the following genes was estimated by RT-PCR: bovine 3 beta-hydroxysteroid dehydrogenase (HSD3B1), cholesterol side-chain cleavage cytochrome P450 (CYP11A1), 17|3-hydroxylase (CYP17), aromatase cytochrome P450 (CYP19), steroidogenic acute regulatory protein (StAR), follicle-stimulating hormone receptor (FSHr), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) as internal control. The results were analyzed by two-way ANOVA followedby Bonferroni least-significant tests. Microscopic analyses showed that cells cultured in NDM lost their polyhedral shape and acquired a fibroblast-like form. Defined medium maintained GC morphology with low cytoplasm:nucleus ratio and GC-GC contact, similar to in vivo cells. In NDM, the concentration of P4 increased as opposed to that of E2, which decreased after 48 h of culture. In DM, the concentrations of P4 and E2 were maintained in the first 48 h of culture. The level of P4 in NDM was higher than in DM during all periods tested. Our data show that CYP11A1 expression remains absent in NDM for follicles explants that initially did not express CYP11A1, whereas in DM the expression of this gene appears after 24, 48, or 72 h of culture, evidence that indicates rescue from atresia. It is important to notice that gene expression of steroidogenic enzymes in DM were higher than in NDM. Very low levels of CYP11A1 and CYP19 were observed in the NDM group. On the other hand, in the DM group there was a progressive increase of all the steroidogenic enzymes and FSHr, which reached a peak at 48 h of culture. In conclusion, our results suggest that DM showed a stimulating effect on steroidogenic activity of GC-TC co-culture similar to that observed in vivo for functional growing follicle, whereas in NDM the explants seemed to mimic luteinizing follicles. Grants from FAP-DF, Finatec, CNPq, and Capes.
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