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This study aimed to investigate the effects of eugenol on growth, viability, antrum formation and mRNA expression of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, bovine ovaries were collected from a local slaughterhouse and in the laboratory the follicles were isolated from the ovarian cortex. The follicles were then cultured in TCM-199+ alone or supplemented with different concentrations of eugenol (0.5, 5.0 and 50.0 μM). Follicular diameters and antrum formation were evaluated on days 0, 6, 12 and 18. Viability analysis was performed using calcein and ethidium homodimer. Real-time PCR was used to quantify mRNA levels for SOD, CAT, GPX1 and PRDX6 in cultured follicles. Follicular diameters and mRNA levels in follicles cultured in vitro were compared using analysis of variance and Kruskal–Wallis tests, while follicular survival and antrum formation were compared using the chi-squared test (P < 0.05). The results showed that secondary follicles cultured with eugenol maintained similar morphology and viability to follicles cultured in the control group. A progressive increase in follicular diameter was observed between days 0 and 12 for all treatments, except for follicles cultured with 50 µM eugenol. Eugenol (5.0 and 50.0 μM) increased mRNA levels for GPX1 in cultured follicles, but 0.5 μM eugenol reduced mRNA levels for SOD. The addition of eugenol did not influence mRNA expression for CAT and PRDX6. In conclusion, eugenol supplementation reduces mRNA levels for SOD and increases mRNA levels of GPX1 in bovine secondary follicles cultured in vitro.
This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150–200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal–Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.
The present study evaluated the effect of knockout serum replacement (KSR), fetal bovine serum (FBS) and bovine serum albumin (BSA) on the viability and growth of bovine secondary follicles cultured in vitro for 12 days. To this end, secondary follicles were isolated (185–202 μm) and cultured in vitro in TCM-199+ medium supplemented with KSR (5% and 10%), FBS (5% and 10%) or BSA (3 mg/ml) at 38.5°C with 5% CO2 in air. Follicular diameters were evaluated on days 0, 4, 8 and 12. After 12 days of culture, follicular survival analysis was performing by using calcein-AM and ethidium homodimer. Before and after culture, follicles were fixed in paraformaldehyde for histological evaluation. Follicular diameter at different days of culture were compared using the Kruskal–Wallis test, while the percentages of viable follicles were analyzed by chi-squared test (P < 0.05). Results showed that follicles cultured in the presence of KSR at both concentrations presented higher follicular survival rates than those cultured in control medium alone or supplemented with FBS or BSA. Conversely, the presence of KSR, BSA or FBS did not increase follicular diameter after 12 days of culture. Histology analysis showed that, among the tested treatments, follicles cultured in the presence of KSR had preserved rounded oocytes, juxtaposed granulosa cells and intact basal membrane. In conclusion, supplementation of culture medium with KSR increases the follicular survival of bovine secondary follicles cultured in vitro.
This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus–oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.
This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus–oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.
The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM – control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.
Relatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.
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