G.M. Silva

Steady-state level of messenger RNA and immunolocalization of aquaporins 3, 7, and 9 during in vitro growth of ovine preantral follicles

Aquaporins (AQPs) are a well-conserved family of small (approximately 30 kDa) membrane channel proteins that facilitate rapid movement of fluids and have a unique tissue-specific pattern of expression. These proteins have been found in the female reproductive systems of humans, rats, and mice. However, the expression and cellular localization of AQPs have not extensively been studied in the female reproductive system of sheep. Therefore, this study aimed to evaluate, by real-time polymerase chain reaction and immunohistochemistry respectively, the levels of messenger RNA and the immunolocalization of AQP3, AQP7, and AQP9 in large isolated ovine secondary follicles over a period of IVC. Our analysis revealed that AQP3 and AQP9 were present predominately in follicles that exhibited antrum formation, suggesting a crucial role of these AQPs in the formation of the antrum. Interestingly, AQP7 was only expressed in follicles that had not formed an antrum by Day 12 of culture. In conclusion, the presence of protein channels (AQP3 and AQP9) seems to be essential for the formation of the antrum in isolated ovine secondary follicles cultured in vitro and thus plays an important role during folliculogenesis in this species.

Gene Expression and Immunolocalization of Fibroblast Growth Factor 2 in the Ovary and Its Effect on the In Vitro Culture of Caprine Preantral Ovarian Follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.

Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production

OBJECTIVE Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). METHODS Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. RESULTS GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). CONCLUSIONS Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production.

Effect of morphological integrity, period, and type of culture system on the in vitro development of isolated caprine preantral follicles

The aims of this study were the following: (1) to define an optimal period for the IVC of isolated caprine preantral follicles, (2) to verify the relationship between follicular morphology (intact, extruded, and degenerate follicles) and estradiol production, and (3) to evaluate the effects of the bidimensional (2D) and three-dimensional (3D) culture systems on the in vitro development of caprine preantral follicles. Three experiments were performed. In experiments 1 and 2, the isolated secondary follicles were cultured for 18, 24, and 30 days or 30, 36, and 42 days, respectively. In experiment 3, the optimal culture period from experiment 2 was used for 2D and 3D culture systems. After culture, the oocytes were submitted to IVM. The morphological integrity, antral cavity formation rates, follicular diameter, presence of healthy, grown oocytes (≥110 μm), rates of resumption of meiosis, and estradiol concentrations were evaluated. In experiment 1, the percentage of oocytes that resumed meiosis was higher in oocytes cultured for 30 days (48.84%) than in oocytes cultured for 18 and 24 days (15% and 20.93%, respectively). In experiment 2, the percentage of oocytes that resumed meiosis was significantly higher in oocytes cultured for 30 and 36 days (47.5% and 50%, respectively) than in oocytes cultured for 42 days (20%). The estradiol concentrations on Day 12 of culture were similar for normal and extruded follicles and higher than those observed in degenerate follicles at the end of the culture period. In conclusion, the 36-day culture period resulted in the highest rates of meiosis resumption. In addition, because the loss of follicular integrity affects the patterns of estradiol production, follicular integrity is a good predictor of follicular quality.

Bovine dominant follicular fluid promotes the in vitro development of goat preantral follicles

The aim of this study was to evaluate the effect of follicular fluid collected from bovine dominant follicles (bFF) on the in vitro development of goat preantral follicles and determine the best time to add this supplement to the culture medium. The preantral follicles were isolated and randomly distributed into four treatments in absence (control) or presence of 10% of bFF added on Days 0 (FF0-18), 6 (FF6-18) or 12 (FF12-18) of culture onwards. After 18 days, follicular development was assessed based on follicular survival, antral cavity formation, increased follicular diameter as well as fully grown oocyte (>110 μm) viability and meiosis resumption. The oocytes from the cultured follicles were in vitro-matured and processed for fluorescence or ultrastructural analysis. The results showed that on Day 18 the treatment FF0-18 had a significantly higher (P<0.05) survival than control and FF12-18, but not FF6-18. The addition of bFF at the beginning of culture (FF0-18 and FF6-18) promoted a high percentage of follicular growth, meiosis resumption and early antrum formation. Moreover, this study described for the first time the ultrastructural analysis of caprine oocytes grown in vitro. This evaluation revealed that in the presence of bFF on (FF0-18) the in vitro-grown oocytes presented normal organelle distribution and well-defined, intact plasma and nuclear membranes. In conclusion, bFF originating from dominant follicles maintain the survival and promote the in vitro growth of goat preantral follicles when added at the beginning of culture.

Bone Morphogenetic Protein-6 (BMP-6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre-antral Follicles Cultured In Vitro†

BMP-6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP-6) and recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP-6 (Experiment 2). Secondary follicles were cultured in αMEM(+) alone (control medium) or supplemented with BMP-6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP-6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP-6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non-cultured control and after in vitro culture (MEM and 1 ng/ml BMP-6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP-6 at 1 ng/ml treatment. In conclusion, the low BMP-6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.

High-frequency oscillations (HFOs) in electroencephalography: Review and case study

This article presents a review focusing technological aspects of EEG recordings considering the frequency band 40-500 Hz. This includes the “Gamma” (40-80 Hz) and “Supergamma” (80-100 Hz) rhythms, which has been extensively analysed for animal models since the 80s. On the other hand, the band 100-500 Hz, abbreviated by “High-Frequency Oscillations”-HFO, has been studied since 1995, focusing epilepsy in both human and rat models. Current challenges are highlighted from the literature, leading to guidelines for those starting in the field, so that at the end we present two examples of HF0 recorded (1-100 Hz) in normal individual and in a patient from our local university hospital.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50ng mL-1 AMH and/or 100ng mL-1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0-9) and second (Days 10-18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH+FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P<0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P>0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.