Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

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The objective of this study was to determine the effects of TNF-α and IL-1β on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO in air, for 18 days, in TCM-199 alone (cultured control) or supplemented with 10 ng/ml IL-1β, 10 ng/ml TNF-α or both TNF-α and IL-1β. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1β and a mixture of IL-1β and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1β or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1β, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.

The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.

SummaryThis 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.

This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml - Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

Esse estudo objetivou comparar dois protocolos hormonais para transferência de embriões em tempo fixo (TETF) em vacas de leite baseados no momento da aplicação de PGF2α. Um total de 2.172 vacas mestiças Bos indicus foram utilizadas. De acordo com o protocolo de sincronização das receptoras de embriões, as vacas foram divididas em dois tratamentos: o Convencional e o MMSINCRO (tratamento teste). O protocolo Convencional consistiu na inserção de um dispositivo contendo progesterona e administração de benzoato de estradiol no Dia 0. No Dia 8, a progesterona foi removida e aplicado PGF2α, eCG e cipionato de estradiol. No Dia 10, os animais receberam GnRH. A TETF foi realizada no Dia 17. O MMSINCRO consistiu na inserção da progesterona e administração de benzoato de estradiol no Dia 0. No Dia 7, animais receberam PGF2α. No Dia 9, a progesterona foi removida e os animais receberam eCG e cipionato de estradiol. No dia 11, foi aplicado GnRH. A TETF foi realizada no Dia 18. A taxa de concepção das receptoras do tratamento MMSINCRO (52%) foi significativamente superior a do protocolo Convencional (33 %). A avaliação qualitativa do corpo lúteo mostrou uma melhor qualidade no tratamento MMSINCRO. Dessa forma, foi possível concluir que para TETF em vacas leiteira, aconselha-se a utilização do protocolo MMSINCRO nas receptoras, o qual foi capaz de aumentar a taxa de concepção. Esse achado proporciona uma melhora na eficiência reprodutiva de bovinos leiteiros, e consequentemente, em uma maior produção de leite e derivados nas fazendas de vacas leiteiras.