Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.

We evaluated the relationship between plasma levels of anti-Müllerian hormone (AMH) and the number of antral follicles at the restart of the follicular wave in crossbred Holstein cows reared under extensive grazing systems over 2500 m above sea level. The study included 140 cows from 15 farms that were in average at the 75.3 ± 2.10 d post partum. Animals were synchronized according to the following regime: day 0 = intravaginal progesterone releasing device (IPD) + estradiol benzoate (EB); day 7: withdrawal of IPD + prostaglandin; and day 8: EB, for restart of the follicular wave on day 11. On this day 11, antral follicle counts (AFCs) were made by transrectal ultrasound, and a plasma sample was taken for the determination of AMH. The mean AMH plasma level was 0.06 ± 0.03 ng/ml and the mean AFC was 17.26 ± 0.38 follicles. A strong positive linear correlation was found between these two variables (r = 0.783, r = 0.613, P < 0.0001). Cows were categorized according to AMH concentration as high (>0.09 ng/ml), intermediate (0.09–0.05 ng/ml) or low (<0.05 ng/ml). Cows with high AMH presented a higher AFC (25.0 ± 2.21 follicles) than those with low AMH (14.08 ± 2.68 follicles; P < 0.001. Our results suggest that the cut-off value of AMH = 0.09 ng/ml may be useful for selecting donors in multiple ovulation embryo transfer programs involving cows with these characteristics. Our data further suggest that AMH plasma concentration correlates with AFC and can be used as an endocrine biomarker of the number of antral follicles present at a given moment of the estrous cycle in crossbred Holstein cows raised at altitudes above 2500 m.

This study aimed to investigate the effects of IL1β and TNFα on growth and maturation of oocytes from small follicles (1–3 mm) during in vitro culture. To this end, cumulus–oocyte complexes (COCs) with diameters of ~110 µm were cultured in TCM-199 medium alone or supplemented with IL1β (10 ng/ml), TNFα (10 ng/ml) or both for 48 h. The oocytes were measured at the beginning and at the end of the culture period. COCs were cultured for 20 h in pre-maturation medium and then half of the COCs of each group was destined for in vitro maturation and the remaining COCs were used to evaluate meiotic progression, mitochondrial distribution and the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. The results showed that COCs cultured with TNFα alone or together with IL1β had higher diameters than those cultured in control medium alone or supplemented with IL1β. Control oocytes isolated from large antral follicles (>5 mm) had heterogeneous distribution of mitochondria. Oocytes isolated from small antral follicles, that had been grown in vitro in TCM-199 alone or supplemented with TNFα had similar heterogeneous mitochondrial distribution before in vitro maturation (IVM). After IVM, mitochondria were heterogeneously distribution when cultured in TCM-199. However, when cultured with TNFα and/or IL1β, mitochondria were homogeneously distributed. Presence of TNFα and/or IL1β in TCM-199 culture medium did not influence the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. In conclusion, TNFα and a mixture of TNFα and IL1β both stimulated the growth of bovine oocytes during their in vitro culture, but do not influence gene expression in grown oocytes.

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.