Polycystic ovary syndrome (PCOS) is a complex reproductive and endocrine disorder affecting 5–10% of women of reproductive age, but the pathophysiology of PCOS still remains unknown. Here, the aim of our study was to analyze the effects of rapamycin treatment that may regulate impaired hormonal levels and folliculogenesis in dehydroepiandrosterone (DHEA)-treated PCOS mouse. We hypothesized that rapamycin may ameliorate the negative effects of PCOS in DHEA-induced PCOS mouse model. The target of rapamycin (TOR) gene product is a serine/threonine kinase that has been implicated in the control of cell growth, proliferation and autophagy, and rapamycin is a potent inhibitor of mTORC1 pathway. In this study, for the first time, mTORC1 and activation products are presented at protein and mRNA levels after rapamycin treatment in DHEA-induced PCOS mouse ovary. We showed that rapamycin treatment may regulate follicular development, hormonal levels and provide ovulation in DHEA-induced PCOS mouse. Additionally, we assessed decreased primordial follicle reserve, increased number of primary and secondary follicles, corpus luteum structure forms again after 10 days of rapamycin treatment. This study presented here suggests rapamycin treatment regulates hormonal phenotype and folliculogenesis in the ovary and also mTOR signalling pathway in granulosa cells of DHEA-induced PCOS mouse ovary which may have potential to attenuate understanding the mechanism of dominant follicle selection and anovulatory infertility.

Conjugated linoleic acid (CLA) is a mixture of positional isomers of linoleic acid found in ruminant products and meat. The diet supplementing with CLA is an emerging area, requiring studies to elucidate its effects on animals and human reproduction, as well as its side effects. Therefore, the aim of this study was to evaluate the effects of CLA gastric administration, during the pregestational and gestational period in biometric and reproductive parameters, as well as in ovarian morphophysiology. Animals were distributed in three groups: (1) control (n = 10); (2) fish oil (n = 10); and (3) CLA (n = 10), that daily received, by gavage, phosphate-buffered saline, fish oil and CLA, respectively, carried out over 50 days (before mating, mating and pregnancy). There was an increment in the nasoanal distance and Lee index of the CLA and fish oil-treated groups during the first weeks (P > 0.05). CLA administration did not affect the ovarian follicle mobilization (P > 0.05), the number of follicles (P > 0.05) and the integrated density of lipid content of oocytes included in antral follicles (P > 0.05). There was no effect of CLA administration on the litter weight (P > 0.05; F2 and F3), however, an increment (P < 0.05) in the number of pups per litter (F2) was observed. Overall, this study demonstrated the absence of side effects of the CLA gastric administration on mice reproductive performance and suggests that this treatment would transgenerationally enhance fertility in this species.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

This study evaluated the effect of fibroblast growth factor-2 (FGF-2) on the morphology, primordial follicle activation and growth after in vitro culture of domestic cat ovarian tissue. Ovaries (n = 12) from prepubertal domestic cats were collected and fragmented. One fragment was fixed for histological analysis (fresh control). The remaining fragments were incubated in control medium alone or with 10, 50 or 100 ng/ml FGF-2 for 7 days. After in vitro culture, the following endpoints were analyzed: morphology, activation by counting primordial and developing follicles, and growth (follicle and oocyte diameters). Treatment with 100 ng/ml FGF-2 maintained (P > 0.05) the percentage of normal follicles similar to fresh control. Follicle survival was greater (P < 0.05) after culture in 100 ng/ml FGF-2 than in 50 ng/ml FGF-2. The percentage of primordial follicles decreased (P < 0.05) and the percentage of developing follicles increased (P < 0.05) in all treatments compared with fresh tissue. The proportion of developing follicles increased (P < 0.05) in tissues incubated with 100 ng/ml FGF-2 compared with control medium and other FGF-2 concentrations. Furthermore, culture in 10 or 100 ng/ml FGF-2 resulted in increased (P < 0.05) follicle and oocyte diameters compared with fresh tissues and MEM+. In conclusion, FGF-2 at 100 ng/ml maintains follicle survival and promotes the in vitro activation and growth of cat primordial follicles.

Many studies have shown that oestrogen affects late follicular development, but whether oestrogen is involved in other aspects of folliculogenesis remains unclear. In this study, two antagonists of oestrogen, tamoxifen and G15, were used to determine the effects of oestrogen on folliculogenesis. Mouse preantral follicles and cumulus–oocyte complexes (COCs) were cultured in vitro. The results showed that follicle growth stimulated using pregnant mare serum gonadotrophin (PMSG) was inhibited using tamoxifen, whether in vivo or in vitro. The average diameters, the maximum diameters of follicles and the numbers of follicles with a diameter of more than 300 μm decreased significantly following a 4-day culture with tamoxifen. G15, the antagonist of oestrogen via the membrane receptor, did not change follicular growth stimulated by PMSG in vitro. Results of in vitro maturation of COCs showed that germinal vesicle breakdown (GVBD) occurred spontaneously (95.1%) after 2 h in culture, and the GVBD ratio changed little with the addition of either oestrogen or 10 μM G15. However, first polar body (PBI) extrusion was driven by oestrogen markedly and supplementation with 10 μM G15 inhibited PBI extrusion (82.4% vs 55.0%) significantly. These results demonstrated that oestrogen promotes follicle growth through the nuclear receptor during follicle growth and then triggers the transition of metaphase to anaphase through the membrane receptor during meiotic resumption. So oestrogen plays a progressive role in the two phases of follicle growth and oocyte meiotic resumption.

Preantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

We compare the efficiency of mechanical or enzymatic methods, and their combination, for the isolation of ovarian preantral follicles (PFs) from collared peccaries. The ovaries from six females were subjected to the different methods investigated here. For the enzymatic method, ovary fragments were exposed to collagenase type IV in TCM-HEPES medium; the mechanical procedure was based on ovarian cortex dissociation by using a scalpel blade. The residual solution obtained after the mechanical isolation was subjected to the enzymatic procedure. The number of isolated PFs was quantified and classified as primordial, primary, or secondary; their viability was assessed using trypan blue dye assay. To confirm the results, PFs derived from the most efficient method were evaluated for integrity using scanning electron microscopy (SEM) and subjected to a 24 h in vitro culture for subsequent evaluation of viability by using fluorescent probes. A higher number of PFs (P < 0.05) was obtained from the enzymatic method (961.7 ± 132.9) in comparison with the mechanical method (434.3 ± 88.9), but no difference was observed between the two methods and their combination (743.2 ± 92.8). The trypan blue assay showed that the enzymatic method (98.7 ± 0.6%) provided the highest percentage of viable follicles (P < 0.05). Furthermore, SEM confirmed the ultrastructural integrity of the surface architecture of peccary PFs isolated by the enzymatic procedure; epifluorescence microscopy was used to confirm their viability (86.0%). In conclusion, we suggest that the enzymatic method investigated here is useful for the isolation of viable ovarian PFs from collared peccaries.

This study investigated the in vitro culture of bovine follicles included in ovarian tissue for 2 or 6 days (D2 or D6), with the addition of different concentrations of follicle-stimulating hormone (FSH) (0, 10, 50, 100 or 200 ng/ml). Data were compared for follicular development, morphological integrity and diameter of follicles and oocytes. Ovaries (n = 10) from Nelore cows (n = 5) were divided into fragments (n = 11 per ovary) and were immediately fixed in Bouin’s solution (D0) or were individually cultured for 2 or 6 days in one of the described concentrations of FSH and then processed for histology. Compared with the rates of follicular development at D2 for minimal essential medium (MEM) (75.0%) and 50 ng/ml of FSH (71.1%), the best rates of follicular development at D2 were obtained with 10 (84.7%), 100 (87.5%) and 200 ng/ml of FSH (85.0%; P<0.05). After 6 days of cultivation, there were no differences among treatments regarding follicular growth. The morphological integrity of preantral follicles was better maintained by 100 ng/ml FSH for 2 and 6 days of cultivation (51.2 and 40.4%, respectively; P<0.05) than that for MEM (D2: 30.9%, D6: 20.8%), 10 (D2: 39.2%, D6: 22.8%), 50 (D2: 30.4%, D6: 28.8%) and 200 ng/ml FSH (D2: 45.2%, D6: 36.8%). FSH at 100 ng/ml provided the highest mean diameter averages: 34.5±10.8 µm at D2 and 33.2±12.5 µm at D6 (P<0.05). We concluded that the medium supplemented with 100 ng/ml FSH during in vitro culture provided appropriate conditions for the development and morphological integrity of preantral follicles in cattle.

We aimed to analyze the oogenesis of adult females of the cichlid fish Laetacara araguaiae. The specimens’ gonads were removed and processed for light and transmission electron microscopy. Oogenesis in L. araguaiae showed the following characteristics: a germinal epithelium with three types of oogonia (A-undifferentiated, A-differentiated and B-oogonia), oocytes at meiotic prophase stage and ovarian follicle formation. Oocytes showing primary growth with pre-vitellogenic and cortical alveolus were observed. Similar to data for other cichlids, oocytes in secondary growth or vitellogenesis were characterized by the initial deposition of yolk microgranules. The event that characterizes the maturation stage is nucleolus migration, also called the germinal vesicle, to the oocyte periphery in the direction of the micropyle. The follicular complex undergoes several changes throughout the oocyte stages. To the best of our knowledge this study is the first to describe L. araguaiae oogenesis. Moreover, this study is the first step to better understand the reproductive biology of this species, which shows great potential for use as an ornamental fish.

We aimed in this study to assess whether serum-decreased bovine cumulus–oocyte complex (COC) steroidogenesis during in vitro maturation (IVM) is caused by deficient androgen milieu. For this approach, bovine COCs were cultured in serum-supplemented IVM medium in the presence or absence of 1 μM androstenedione. After 24 h of culture, medium was collected and analyzed for its content of estradiol-17β (E2) and progesterone (P4). Medium E2 content markedly increased after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to the androgen group; 0.32 ± 0.05 ng/ml to the non-androgen group). No significant difference in the P4 content was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to the androgen group; 21.73 ± 1.67 ng/ml to the non-androgen group). Our data provide compelling evidence that bovine COCs steroidogenesis remains functional during culture in serum-supplemented medium and suggest that serum-induced decreased COCs estradiol secretion is caused by deficiency of an aromatizable androgen source.

Ovarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

Dynamic changes in chromatin structure and gene expression occur during follicular and oocyte growth. Epigenetic mechanisms regulate these changes through biochemical reactions that modify the nucleosome structure, and consequently affect transcription. Chromatin remodellers that alter DNA–histone interactions can influence transcriptional activity by facilitating or repressing DNA access. The SWItch/Sucrose NonFermentable (SWI/SNF) complex represents an important chromatin remodelling family, which comprises many protein subunits including the BRG1 (brahma-related gene 1). Our aim in this study was to analyse BRG1 expression patterns in different stages of follicular development. Ovaries (n = 10) were collected from prepubertal gilts and then rinsed in phosphate-buffered saline (PBS). Ovarian fragments of 8 × 8 × 8 mm were cut and placed into a 4% paraformaldehyde solution. For immunofluorescence analysis, samples were incubated with primary antibodies: polyclonal rabbit anti-BRG1 (1/200) or control rabbit IgG at the same concentration, overnight at 4°C. Primary antibodies were detected using Alexa Fluor 594-anti-rabbit 1/1000 diluted secondary antibody. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Positive fluorescence signal for BRG1 was detected in all analysed samples. In primary follicles, the protein was detected only in the oocyte nucleus. However, in growing follicles, BRG1 was identified in granulosa and theca cells in a well defined pattern, according to the proximity of the cells from the oocyte. These results suggest an important role for BRG1 in the regulation of follicular growth, probably modulating granulosa and theca cell proliferation, as well as oocyte growth and maturation.

Three different stages of pig antral follicles have been studied in a granulosa-cell transcriptome analysis on nylon microarrays (1152 clones). The data have been generated from seven RNA follicle pools and several technical replicates were made. The objective of this paper was to state the feasibility of a transcriptomic protocol for the study of folliculogenesis in the pig. A statistical analysis was chosen, relying on the linear mixed model (LMM) paradigm. Low variability within technical replicates was hence checked with a LMM. Relevant genes that might be involved in the studied process were then selected. For the most significant genes, statistical methods such as principal component analysis and unsupervised hierarchical clustering were applied to assess their relevance, and a random forest analysis proved their predictive value. The selection of genes was consistent with previous studies and also allowed the identification of new genes whose role in pig folliculogenesis will be further investigated.

Recent interest in the initial phases of ovarian follicular formation and development has lead to a number of publications in this area, most of which address the autocrine and paracrine factors involved in primordial follicle activation to primary follicle. Primordial follicle assembly (first step in follicle formation) determines the lifetime supply of primordial follicles and remains a poorly understood phenomenon. Despite a number of recent articles that are concentrating on immuno-histochemistry, basic steps in the process are not clear. Hence, we feel it is time to take a step back and see what is available in the literature and identify the gaps in which future research about primordial follicle assembly in humans needs to be directed.