Pituitary porcine FSH, and recombinant bovine and human FSH differentially affect growth and relative abundances of mRNA transcripts of preantral and early developing antral follicles in goats.

Three different sources of FSH (porcine pituitary, pFSH; recombinant bovine, rbFSH; and recombinant human, rhFSH) were compared during in vitro culture of preantral and early antral follicles of goats for 18 days. Treatments were: base medium supplemented with no FSH (control), 10, 50, or 100 mIU/mL pFSH (pFSH10, pFSH50, and pFSH100, respectively), 100 ng/mL rbFSH (rbFSH), and 50 mIU/mL rhFSH (rhFSH). There were evaluations of follicle morphology, antrum formation, growth rate, estradiol production, oocyte viability and chromatin configuration, and follicle wall relative abundance of mRNA transcript for MMP-9, TIMP-2, CYP17, CYP19A1, FSHR, Insulin-R, and BAX/BCL-2 ratio. Follicle degeneration rates were similar among all treatment groups at the end of culturing. When there were treatments with pFSH, however, there was a lesser (P < 0.05) percentage of intact follicles and estradiol production, and greater (P < 0.05) extrusion rates. Furthermore, with only pFSH10 (antral follicles) and pFSH100 (preantral and antral follicles) treatments, there was a lesser (P < 0.05) follicle growth. For preantral follicles, when there was addition of pFSH10, pFSH100, and rhFSH there was lesser (P < 0.05) oocyte meiotic resumption compared to control and rbFSH treatments. For antral follicles, when there were treatments with rhFSH and pFSH10 there was greater (P = 0.08 - P < 0.05) oocyte maturation. In conclusion, the source of FSH differentially affected gene expression, as indicated by mRNA abundances, and follicular dynamics of preantral and antral follicles in vitro. Addition of FSH during the in vitro culture improved the developmental outcomes only for antral follicles.

Mitotic index and morphological characteristics of ovarian small follicles from goats submitted to nutritionally unbalanced regimens.

The objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding-refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Progesterone and Follicle Stimulating Hormone interact and promote goat preantral follicles survival and development in vitro / Progesterona e Hormônio Folículo-Estimulante interagem e promovem a sobrevivência e o desenvolvimento in vitro de folículos pré-antrais caprinos

We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a significant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a significant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies confirmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles.

Este trabalho verificou os efeitos da progesterona e do hormônio folículo-estimulante (FSH) na sobrevivência e no crescimento de folículos pré-antrais caprinos. Fragmentos de tecido ovariano foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (MEM) sozinho ou contendo progesterona (1, 2.5, 5, 10 ou 20ng/mL), FSH (50ng/mL) ou a combinação entre esses dois hormônios. O tecido fresco (controle não-cultivado) e o cultivado foram processados para análise histológica e ultra-estrutural. Após 7 dias a adição de FSH a todas as concentrações de progesterone manteve o percentual de folículos normais similar ao controle fresco. No dia 7 de cultivo, um alto percentual de folículos em desenvolvimento foi observado somente no tratamento com 2,5ng/ml de progesterona associada ao FSH ou com 10ng/ml de progesterona sozinha, em relação ao controle fresco. Do dia 1 para o dia 7 de cultivo, um aumento significativo no percentual de folículos em desenvolvimento foi observado no MEM sozinho e adicionado de 2,5ng/ml de progesterona + FSH. Além disso, após 7 dias, em todos os tratamentos, houve um aumento significativo no diâmetro folicular em relação ao controle, exceto nos tratamentos com MEM sozinho, 5ng/ml de progesterona + FSH ou 10ng/ml de progesterona sozinha. A análise ultra-estrutural confirmou a integridade follicular após 7 dias de cultivo no tratamento com 2,5ng/ml de progesterona + FSH. Em conclusão, este estudo demonstrou que a interação entre progesterona e FSH mantém a integridade ultra-estrutural, estimula a ativação de folículos primordiais e o posterior crescimento de folículos pré-antrais caprinos cultivados in vitro.

Vitrification of bovine ovarian tissue by the solid-surface vitrification method.

Exposure time and addition of sucrose to the vitrification medium as well as the solid-surface vitrification (SSV) on the morphology of bovine preantral follicles were evaluated. Ovarian tissue was exposed for 1, 5, or 10 min to 4.0 M ethylene glycol with or without the addition of 0.5 M sucrose. Subsequently, the tissue was washed out from cryoprotectants or vitrified by the SSV method. Independently of the presence of sucrose, exposure to vitrification solution for 10 min did reduce the percentages of normal follicles when compared with control. However, the highest rates of normal follicles were attained when tissue was previously exposed to the vitrification solution, with sucrose added or not, for 10 min. Although the SSV is a promising procedure to be applied in ovarian tissue, an optimal vitrification solution for bovine ovarian tissue needs to be developed.

Steady-state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro.

The aims of this study were to investigate steady-state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT-PCR was used to analyze caprine steady-state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1-3 mm) and large (3-6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM(+)) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT-PCR demonstrated an increase in steady-state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady-state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM(+) after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth.

Dimethyl sulfoxide perfusion in caprine ovarian tissue and its relationship with follicular viability after cryopreservation.

Ovarian cortical fragments (3 x 3 x 1 mm) were exposed to dimethyl sulfoxide (DMSO) in different concentrations for further analysis of cryoprotectant perfusion by applying high-performance liquid chromatography (HPLC) and conventional cryopreservation. This simple perfusion test can predict the efficiency of the cryopreservation procedure.

Quantification of dimethyl sulfoxide perfusion in sheep ovarian tissue: a predictive parameter for follicular survival to cryopreservation.

The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ∼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.

Effect of cryopreservation on viability, activation and growth of in situ and isolated ovine early-stage follicles.

Isolated or cortical tissue-enclosed (in situ) sheep early-stage follicles were exposed to 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or unexposed, or frozen/thawed in the presence of these cryoprotectants and then cultured for 5 days in enriched minimal essential medium (MEM) or not cultured. Cultured and uncultured follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. Follicular diameter was measured and the percentages of primordial and developing follicles calculated. Exposure of isolated or in situ follicles to DMSO or EG led to a marked decrease in the percentage of viable follicles. The percentage of viable isolated and in situ follicles further decreased when they were in vitro-cultured for 5 days, EG-exposed follicles generally showing a more damaging effect than DMSO-exposed follicles. Cultured follicles, both isolated and in situ, which were exposed to EG and DMSO, as well as in situ follicles, which had been frozen/thawed in the presence of one of these cryoprotectants, showed similar growth rates as cultured, untreated follicles, while in these groups significantly lower percentages of primordial follicles and higher percentages of more advanced follicular stages were observed. Among the treated groups, the highest percentage (71-75%) of developing follicles was observed after culturing cryoprotectant-exposed isolated follicles. In contrast, when cryopreserved, isolated follicles were cultured, they did not increase in diameter and did not develop into more advanced stages. In conclusion, exposure to or cryopreservation in the presence of EG and DMSO, as well as their further in vitro culture, negatively affected the viability of ovine isolated and in situ early-stage follicles. In vitro growth of early-stage follicles and activation of primordial follicles were better maintained when follicles had been frozen/thawed and cultured in situ.