Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles.

This study aimed to investigate the effects of different concentrations of N-acetylcysteine (NAC) on the growth, antrum formation, viability, and ultrastructure of bovine secondary follicles cultured in vitro for 18 days. To this end, the follicles were cultured in TCM-199+ medium alone or supplemented with 1.0, 5.0, or 25.0 mM NAC. Follicular growth, antrum formation, viability (calcein-AM and ethidium homodimer-1) and ultrastructure were evaluated at the end of culture period. The results showed that 1.0 mM NAC increased the percentage of growing follicles and the fluorescence intensity for calcein-AM when compared to other treatments (p < 0.05). On the other hand, follicles cultured with 25.0 mM NAC had higher fluorescence intensity for ethidium homodimer-1, which is a sign of degeneration. Ultrastructural analysis showed that oocytes from follicles cultured in control medium alone or with 1 mM NAC had intact zonae pellucidae in close association with oolemmae, but the ooplasm showed mitochondria with a reduced number of cristae. On the other hand, oocytes from follicles cultured with 5 or 25 mM NAC had extremely vacuolated cytoplasm and no recognizable organelles. In conclusion, 1 mM NAC increases cytoplasmic calcein staining and the growth rate in bovine secondary follicles cultured in vitro, but the presence of 5 or 25 mM NAC causes damage in cellular membranes and organelles, as well as reducing the percentages of growing follicles.

Effects of Aloe vera extract on growth, viability, ultrastructure and expression of mRNA for antioxidant enzymes in bovine secondary follicles cultured in vitro.

This study aimed to investigate the effects of Aloe vera extract on follicular growth, viability, ultrastructure, and mRNA levels for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, secondary follicles were mechanically isolated from the ovarian cortex and cultured at 38.5 °C, with 5% CO2 in air, for 18 days in TCM-199+ alone or supplemented with 2.5%, 5.0%, 10.0% and 20.0% Aloe vera extract. Follicular growth, morphology and antrum formation were evaluated every 6 days, while ultrastructure was evaluated at the end of culture. Analysis of viability was performed by calcein-AM and ethidium homodimer-1, while mRNA levels for SOD, CAT, GPX1 and PRDX6 were evaluated by real-time PCR at the end of culture. The results show that follicles cultured with 2.5% Aloe vera had increased the rate of antrum formation, while 2.5% and 5.0% Aloe vera improved follicular viability rate. Follicles cultured with 2.5% and 10.0% Aloe vera increased the levels of mRNA for SOD and GPX1 respectively, but the levels of CAT were reduced in follicles cultured with 2.5%, 5.0%, 10.0% and 20.0%. Additionally, follicles cultured with 2.5% of Aloe vera had their ultrastructure well preserved, while those cultured with 5.0%, 10.0% and 20.0% exhibited increased oocyte vacuolization and damaged organelles. In conclusion, 2.5% Aloe vera increases antrum formation, viability and expression of mRNA for SOD in cultured secondary follicles, but higher concentrations of Aloe vera have negative effects on follicular ultrastructure.

Protective Effect of Cimicifuga racemosa (L.) Nutt Extract on Oocyte and Follicle Toxicity Induced by Doxorubicin during In Vitro Culture of Mice Ovaries.

This study evaluated the potential of Cimicifuga racemosa (L.) Nutt extract (CIMI) to reduce the deleterious effects of doxorubicin (DOXO) in oocytes, follicles and stromal cells in mice ovaries cultured in vitro. In experiment 1, mice ovaries were cultured in DMEM+ alone or supplemented with 5, 50 or 500 ng/mL CIMI, while in experiment 2, mice ovaries were cultured in DMEM+ alone or supplemented with 5 ng/mL CIMI (better concentration), 0.3 µg/mL DOXO or both. Thereafter, the ovaries were processed for histological (morphology, growth, activation, extracellular matrix configuration and stromal cell density), immunohistochemical (caspase-3) analyses. Follicle viability was evaluated by fluorescence microscopy (ethidium homodimer-1 and calcein) while real-time PCR was performed to analyses the levels of (mRNA for SOD, CAT and nuclear factor erythroid 2-related factor 2 (NRF2) analyses. The results showed that DOXO reduces the percentage of normal follicles and the density of stromal cells in cultured ovaries, but these harmful effects were blocked by CIMI. The DOXO reduced the percentage of primordial follicles, while the presence of CIMI alone did not influence percentage of primordial follicles. A higher staining for caspase-3 was seen in ovaries cultured in control medium alone or with DOXO when compared with those cultured with CIMI alone or both CIMI and DOXO. In addition, follicles from ovaries cultured with both CIMI and DOXO were stained by calcein, while those follicles cultured with only DOXO were stained with ethidium homodimer-1. Furthermore, ovaries cultured with CIMI or both CIMI and DOXO had higher levels of mRNA for SOD and CAT, respectively, than those cultured with only DOXO. In conclusion, the extract of CIMI protects the ovaries against deleterious effects of DOXO on follicular survival and ovarian stromal cells.

Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and the expression of maturation-related transcripts during prematuration of oocytes from small and medium-sized bovine antral follicles.

This study evaluated the effects of epidermal growth factor (EGF) and progesterone (P4) on growth, the resumption of meiosis and expression of eukaryotic translation initiation factor 4E(eIF4E), poly(A)-specific ribonuclease (PARN), oocyte-specific histone H1 (H1FOO), oocyte maturation factor Mos (cMOS), growth differentiation factor-9 (GDF9) and cyclin B1 (CCNB1) mRNA in oocytes from small and medium-sized antral follicles after prematuration and maturation invitro. Oocytes from small (<2.0mm) and medium (3.0-6.0mm) antral follicles were cultured in medium containing EGF (10ng mL-1), P4 (100 µM) or both. After culture, growth rate, resumption of meiosis and eIF4E, PARN, H1FOO, cMOS, GDF9 and CCNB1 mRNA levels were evaluated. P4 increased cMOS, H1FOO and CCNB1 mRNA levels after the culture of oocytes from small antral follicles, and EGF increased CCNB1 mRNA levels in these oocytes. In the medium-sized antral follicles, P4 alone or in combination with EGF increased oocyte diameter after prematuration invitro. In these oocytes, the presence of either EGF or P4 in the culture medium increased cMOS mRNA levels. In conclusion, P4 increases cMOS, H1FOO and CCNB1 mRNA levels after the culture of oocytes from small antral follicles. P4 and the combination of EGF and P4 promote the growth of oocytes from medium-sized antral follicles, and both EGF and P4 increase cMOS mRNA levels.

Interleukin-1ß and TNF-α systems in ovarian follicles and their roles during follicular development, oocyte maturation and ovulation.

Tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are cytokines that are involved in the development, proliferation and apoptosis of ovarian follicular cells in domestic mammals. The expression of these cytokines in various follicular compartments, depending on the stage of follicle development, demonstrates their involvement in the control of primordial follicle growth up to the preovulatory stage. The mechanism of action of these factors depends on the presence of their receptors that transduce their biological actions. This review shows the expression sites of TNF-α, IL-1ß and their receptors in ovarian follicles, and discusses the mechanism of action of these cytokines during follicle development, oocyte maturation and ovulation in domestic animals.

In vitro culture of secondary follicles and prematuration of cumulus-oocyte complexes from antral follicles increase the levels of maturation-related transcripts in bovine oocytes.

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue.

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α-MEM+ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non-cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.

Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5.

The bone morphogenetic protein system and the regulation of ovarian follicle development in mammals.

The bone morphogenetic protein (BMP) family consists of several growth factor proteins that belong to the transforming growth factor-ß (TGF-ß) superfamily. BMPs bind to type I and type II serine-threonine kinase receptors, and transduce signals through the Smad signalling pathway. BMPs have been identified in mammalian ovaries, and functional studies have shown that they are involved in the regulation of oogenesis and folliculogenesis. This review summarizes the role of the BMP system during formation, growth and maturation of ovarian follicles in mammals.

Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression.

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.

Expression levels of mRNA for insulin-like growth factors 1 and 2, IGF receptors and IGF binding proteins in in vivo and in vitro grown bovine follicles.

This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.