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Cattle are deemed less susceptible to mycotoxins due to the limited internal exposure resulting from rumen microbiota activity. However, the significant amounts of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) frequently detected in bovine follicular fluid samples suggest that they could affect ovarian function. Both mycotoxins trigger several patterns of cell death and activate the NLRP3 inflammasome in the intestine. In vitro studies have reported a number of adverse effects on bovine oocytes. However, the biological relevance of such findings with regard to realistic concentrations of DON and ZEN in bovine follicular fluid is still not clear. Hence, it is important to better characterize the effects of dietary exposure to DON and ZEN on the bovine ovary. Using bovine primary theca cells, this study investigated the effects of real-life patterns for bovine ovary exposure to DON and ZEN, but also DON metabolite DOM-1, on cell death and NLRP3 inflammasome activation. Exposure to DON starting from 0.1 μM significantly decreased theca cell viability. The kinetics of phosphatidylserine translocation and loss of membrane integrity showed that ZEN and DON, but not DOM-1, induce an apoptotic phenotype. qPCR analysis of the expression of NLRP3, PYCARD, IL-1β, IL-18, and GSDMD in primary theca cells at concentrations of mycotoxin previously reported in cow follicular fluid clearly indicated that DON and DOM-1 individually and in mixture, but not ZEN, activate NLRP3 inflammasome. Altogether, these results suggest that real-life dietary exposure of cattle to DON may induce inflammatory disorders in the ovary., (© 2023. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

Extracellular vesicles such as exosomes contain several types of transcripts, including mRNAs and micro RNAs (miRNAs), and have emerged as important mediators of cell-to-cell communication. Exosome-like vesicles were identified in the ovarian follicles of several mammalian species. Although the miRNA contents have been extensively characterized, the detailed investigation of their mRNA profiles is lacking. Here, we characterize the mRNA profiles of exosome-like vesicles in ovarian follicles in a pig model. The mRNA contents of the exosome-like vesicles isolated from porcine follicular fluid were analyzed and compared with those from mural granulosa cells (MGCs) using the Illumina HiSeq platform. Bioinformatics studies suggested that the exosomal mRNAs are enriched in those encoding proteins involved in metabolic, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) -protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) pathways. While the mRNA profile of the exosome-like vesicles resembled that of MGCs, the vesicles contained mRNAs barely detectable in MGCs. Thus, while the majority of the vesicles are likely to be secreted from MGCs, some may originate from other cell types, including theca cells and oocytes, as well as the cells of non-ovarian organs/tissues. Therefore, the mRNA profiles unveiled several novel characteristics of the exosome-like vesicles in ovarian follicles., Competing Interests: The authors have declared that no competing interests exist.

Background: Ovarian retinoid homeostasis plays an important role in the physiological function of the ovary. Retinol-binding protein 4 (RBP4) acts as the mediator for the systemic and intercellular transport of retinol and is heavily involved in cellular retinol influx, efflux, and exchange. However, the expression patterns and regulatory mechanisms of Rbp4 in the ovary remain unclear., Methods: The expression pattern of ovarian Rbp4 was examined in immature mice during different developmental stages and in adult mice during different stages of the estrous cycle. The potential regulation and mechanisms of ovarian Rbp4 expression by estrogen and related gonadotropins in mouse ovaries were also investigated., Results: The present study demonstrated that the ovarian expression of Rbp4 remained constant before puberty and increased significantly in the peripubertal period. In adult female mice, the expression of Rbp4 increased at proestrus and peaked at estrus at both the mRNA and protein levels. The protein distribution of RBP4 was mainly localized in the granulosa cell and theca cell layer in follicles. In addition, the expression of Rbp4 was significantly induced by follicle-stimulating hormone (FSH) or FSH + luteinizing hormone (LH) in combination in immature mouse (3 weeks old) ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels. In contrast, treatment with LH or 17β-estradiol did not exhibit any observable effects on ovarian Rbp4 expression. Transcription factors high-mobility group AT-hook 1 (HMGA1), steroidogenic factor 1 (SF-1), and liver receptor homolog 1 (LRH-1) (which have been previously shown to be involved in activation of Rbp4 transcription), also responded to FSH stimulation. In addition, H-89, an inhibitor of protein kinase A (PKA), and the depletion of HMGA1, SF-1, and LRH-1 by small interfering RNAs (siRNAs), resulted in a dramatic loss of the induction of Rbp4 expression by FSH at both the mRNA and protein levels., Conclusions: These data indicate that the dynamic expression of Rbp4 is mainly regulated by FSH through the cAMP-PKA pathway, involving transcriptional factors HMGA1, SF-1, and LRH-1, in the mouse ovary during different stages of development and the estrous cycle.

This study aimed to investigate leptin immuno-staining of the porcine ovary in different reproductive stages. Ovaries from 21 gilts were collected from slaughterhouses. The ovarian tissue sections were incubated with a polyclonal anti-leptin as a primary antibody. The immuno-staining in ovarian tissue compartments was calculated using imaging software. Leptin immuno-staining was found in primordial, primary, preantral and antral follicles. Leptin immuno-staining was expressed in the oocyte and granulosa and theca interna layers in both preantral and antral follicles. In the corpora lutea, leptin immuno-staining was found in the cytoplasm of the luteal cells. The leptin immuno-staining in the granulosa cell layer of preantral follicles did not differ compared to antral follicles (90.7 and 91.3%, respectively, P > 0.05). However, the leptin immuno-staining in the theca interna layer of preantral follicles was lower than antral follicles (49.4 and 74.3%, respectively, P < 0.001). There was no difference in leptin immuno-staining in the granulosa cell layer between follicular and luteal phases (92.4 and 89.7%, respectively, P > 0.05). However, the leptin immuno-staining in the theca interna layer of follicular phase was greater than that in the luteal phase (72.7 and 51.0%, respectively, P < 0.001). These findings indicated that leptin exists in different compartments of the porcine ovary, including the oocyte, granulosa cells, theca interna cells, corpus luteum, blood vessel and smooth muscles. Therefore, this morphological study confirmed a close relationship between leptin and ovarian function in the pig., (© 2017 Blackwell Verlag GmbH.)

Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte-cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1-4) were determined. The obtained results confirmed the follicle-type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1-4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action., (© 2016 Blackwell Verlag GmbH.)

In a previous microarray study, we identified a subset of micro RNAS (miRNAs), which expression was distinctly higher in atretic than healthy follicles of cattle. In the present study, we investigated the involvement of those miRNAs in granulosa and theca cells during atresia. Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) confirmed that miR-21-5p/-3p, miR-150, miR-409a, miR-142-5p, miR-378, miR-222, miR-155, and miR-199a-5p were expressed at higher levels in atretic than healthy follicles (9-17 mm, classified based on steroidogenic capacity). All miRNAs except miR-21-3p and miR-378 were expressed at higher levels in theca than granulosa cells. The expression of 13 predicted miRNA targets was determined in follicular cells by RT-qPCR, revealing downregulation of HIF1A, ETS1, JAG1, VEGFA, and MSH2 in either or both cell types during atresia. Based on increases in miRNA levels simultaneous with decreases in target levels in follicular cells, several predicted miRNA target interactions were confirmed that are putatively involved in follicular atresia, namely miR-199a-5p/miR-155-HIF1A in granulosa cells, miR-155/miR-222-ETS1 in theca cells, miR-199a-5p-JAG1 in theca cells, miR-199a-5p/miR-150/miR-378-VEGFA in granulosa and theca cells, and miR-155-MSH2 in theca cells. These results offer novel insight on the involvement of miRNAs in follicle development by identifying a miRNA target network that is putatively involved in follicle atresia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In the ovary, the development of new capillaries from pre-existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietin (ANPT) and hypoxia-inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5-5, 5-20, 20-180 and >180 ng/ml FF). The corresponding sizes of follicles were 5-7, 8-10, 10-13, 12-14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12 13-16 and >18 of the oestrous cycle and months 1-2, 3-4, 6-7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT-qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF-A, FGF-2, IGF-1 and IGF-2, ANPT-2/ANPT-1 and HIF-1-alpha was found during final follicle maturation and in CL during the early luteal phase (days 1-4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function., (© 2015 Blackwell Verlag GmbH.)

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tissue inhibitor of metalloproteinase 1 (TIMP1) belongs to a group of endogenous inhibitors that control the activity of matrix metalloproteinases and other metalloproteinases. TIMP1 is ubiquitously expressed and implicated in many physiological and pathologic processes. In this study, the full-length complementary DNA of goat (Capra hircus) Timp1 was cloned from adult goat ovary for the first time to better understand the regulatory role of TIMP1. The putative TIMP1 protein shared a high amino acid sequence identity with other species. Real-time polymerase chain reaction results showed that Timp1 was widely expressed in adult goat tissues, and messenger RNA expression was higher in the ovary than in other tissues; meanwhile, increasing expression of Timp1 was also discovered during the process of follicle growth and corpus luteum. We then investigated Timp1 expression patterns in different types of ovarian follicular cells from goats. In small or large antral follicles, Timp1 expression was higher (P < 0.05) in theca cells than in granulosa cells, cumulus cells, and oocytes. Increasing expression of Timp1 in theca and granulosa cells was observed as the variation of the follicle size. Immunohistochemical analyses further revealed the presence of the TIMP1 proteins in follicles at all antral stages of development. The most intense staining for TIMP1 was observed in the theca cells and granulosa cells of large antral follicles and corpus luteum. Timp1 was highly (P < 0.05) induced in granulosa cells in vitro after treatment with the luteinizing hormone agonist, human chorionic gonadotropin. Treatments with forskolin, phorbol 12-myristate 13-acetate, or phorbol 12-myristate 13-acetate + forskolin could also stimulate Timp1 messenger RNA expression. The effects of human chorionic gonadotropin were reduced (P < 0.05) by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase, indicating that Timp1 expression could be adjusted by luteinizing hormone-initiated activation of these signaling mediators. Our results suggested that TIMP1 may be involved in regulating ovarian follicle development and ovulation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-β were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-β (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-β were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Most of the previous studies on ovarian hyaluronan (HA) have focused on mature antral follicles or corpora lutea, but scarcely on small preantral follicles. Moreover, the origin of follicular HA is unknown. To clarify the localization of HA and its synthases in small growing follicles, involvement of HA in follicle growth, and gonadotropin regulation of HA synthase (Has) gene expression, in this study, perinatal, immature, and adult ovaries of Wistar-Imamichi rats were examined histologically and biochemically and by in vitro follicle culture. HA was detected in the extracellular matrix of granulosa and theca cell layers of primary follicles and more advanced follicles. Ovarian HA accumulation ontogenetically started in the sex cords of perinatal rats, and its primary site shifted to the intrafollicular region of primary follicles within 5 days of birth. The Has1-3 mRNAs were expressed in the ovaries of perinatal, prepubertal, and adult rats, and the expression levels of Has1 and Has2 genes were modulated during the estrous cycle in adult rats and following administration of exogenous gonadotropins in immature acyclic rats. The Has1 and Has2 mRNAs were predominantly localized in the theca and granulosa cell layers of growing follicles respectively. Treatments with chemicals known to reduce ovarian HA synthesis induced follicular atresia. More directly, the addition of Streptomyces hyaluronidase, which specifically degrades HA, induced the arrest of follicle growth in an in vitro culture system. These results indicate that gonadotropin-regulated HA synthesis is involved in normal follicle growth.

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To determine the relationships among vasculature, mitotic activity, and expression of endothelial nitric oxide synthase (eNOS) of antral follicles in Bos indicus, bovine ovaries were obtained on day 6 of the estrous cycle from 10 crossbred (Brahman to Thai native cows) after a synchronized estrus with prostaglandin F2α analogue. Ovaries were fixed, paraffin-embedded, and used for immunofluorescence detection of factor VIII (a marker of endothelial cells). Immunostaining of eNOS and proliferating cell nuclear antigen (PCNA) were performed with specific monoclonal antibodies. Vasculature and positive staining of eNOS and PCNA were quantitatively evaluated with the image analysis. Follicles were classified by size (small, medium, and large) and by structure as healthy and atretic follicles (n = 82). The expression of factor VIII and eNOS were detected greater in the blood vessels of the theca layers of the healthy follicles than those in atretic follicles. The labeling indices (LIs) in granulosa and theca cells were greater (P < 0.05) in the healthy small and medium follicles than in the healthy large follicles. Vasculature, capillary area density, and capillary number density were positively correlated with eNOS expression and the LIs of granulosa and theca cells but were negatively correlated with the healthy follicle size. During the growing phase of antral follicle in Bos indicus, relationships among vasculature, mitotic activity, and eNOS were observed predominantly in healthy antral follicles. Thus, these data highlight the importance of vasculature, cell proliferation, and eNOS expression of growing and atretic follicles in the first follicular wave., (Copyright © 2013 Elsevier Inc. All rights reserved.)

As stage progresses in the cystic follicle, granulosa cells are lost. We hypothesized that the granulosa and theca interna layers are detached in association with weakened expression of cell adhesion molecules such as cadherin (cell-cell adhesion) and integrin (cell-extracellular matrix adhesion) in cystic follicles. To elucidate this hypothesis, we immunolocalized these molecules in the granulosa and theca interna and compared them between cystic and small healthy follicles. Sections were immunostained with cadherin and integrin β1 antibodies and their localizations were compared. Cadherin-positive reaction was seen in the cytoplasma of all granulosa cells. No increase in the frequency of cadherin-positive area in the granulosa layers and the intensity of cadherin immunoreaction in the theca interna was detected in cystic follicles compared with healthy ones. A dense immunoreaction product of integrin β1 was detected in the theca interna in both cystic and healthy follicles. Intensity of integrin β1 -immuno reaction in the granulosa layers and integrin β1 -positive area in the theca interna was significantly lower in the cystic follicle than in the healthy follicles. These results suggest that granulosa and theca interna cells are detached while maintaining the cell-cell adhesion, resulting in the consequent loss of these layers from the cystic follicle., (© 2012 Japanese Society of Animal Science.)

The aims of this study were to investigate the expression levels of mRNA for platelet-derived growth factor (PDGF) receptors (PDGFR-α and -β) in caprine follicles at different developmental stages and to evaluate the influence of PDGF on the in vitro development of pre-antral follicles. For this, goat primordial, primary and secondary follicles, as well as small (1-3 mm) and large (3-6 mm) antral follicles, were obtained, and PDGFR-α and -β mRNA levels were quantified by real-time PCR. Furthermore, pre-antral follicles (≥ 200 μm) were isolated from goat ovaries and cultured for 18 days in α- minimum essential medium supplemented with PDGF at 50 or 100 ng/ml, containing or not FSH. Real-time PCR showed highest PDGFR-α mRNA levels in secondary follicles, while PDGFR-β mRNA levels were highest in primary follicles onwards. Both receptors showed higher mRNA levels in granulosa/theca cells from small and large antral follicles than in their corresponding cumulus-oocyte complexes. In culture, the percentage of antrum formation was significantly higher in 100 ng/ml PDGF compared with the same PDGF concentration associated with FSH. After 18 days, PDGF in both concentrations associated with FSH promoted follicular growth significantly higher than the control. Moreover, the addition of FSH to 50 ng/ml PDGF positively influenced the follicular growth when compared with the same PDGF concentration in the absence of FSH. In conclusion, PDGF is important for early goat folliculogenesis, because the presence of PDGFR-α and -β mRNA was detected in all follicular categories, and PDGF associated with FSH stimulated the growth of goat pre-antral follicles isolated and cultured in vitro., (© 2011 Blackwell Verlag GmbH.)

The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development.

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows., (© 2011 Blackwell Verlag GmbH.)

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n=5) or before (n=5), during (n=4), and after (n=4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP-labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles >or=2mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5mm) and medium (2.5 to 4.0mm) follicles, but large follicles (>4.0mm) expressed higher mRNA and protein levels (all P<0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size., (Copyright 2010 Elsevier Inc. All rights reserved.)

The present study was performed to determine how the development of cystic ovarian disease (COD) affecting the ovarian expression of heat shock proteins (HSP) in cows were expressing extrous cycles. HSP27, HSP60, HSP70 and HSP90 were evaluated in different ovarian components by Western blot and semiquantitative immunohistochemical analysis. Greater expression of the HSP27 gene was detected in the granulosa and theca cells of primary, secondary, tertiary and cystic follicles, with decreasing amount in atretic follicles. HSP60, HSP70 and HSP90 showed a similar pattern of immunostaining, with moderate gene expression in primary and secondary follicles, increased expression in tertiary and atretic follicles with the greatest gene expression in cystic follicles. HSP were also localized in the corpus luteum, corpus albicans, interstitial tissue and tunica albuginea. The relative amount of protein in the follicular wall of small and large healthy follicles and cystic follicles as analysed by Western immunoblot was consistent with the immunohistochemical data. We speculate that altered expression of HSP genes decreases apoptosis in the follicular wall and leads to the delayed regression of cystic follicles. This study supports earlier observations suggesting that aberrant HSP gene expression, observed in cells of the cystic follicles, is probably associated with the intra-ovarian component of COD pathogenesis., (Copyright 2009 Elsevier B.V. All rights reserved.)

Stanniocalcin 1 (STC 1) is a glycoprotein involved in mineral homeostasis and was first identified in fish. Its mammalian homologue has been implicated in the regulation of various biological processes, including angiogenesis and steroidogenesis both of which are fundamental events in ovarian function. Interestingly, the highest level of STC 1 expression in mammals occurs in ovarian tissue but no information is available on swine species. Therefore, the present study was undertaken to investigate the expression and the immunolocalization of STC 1 in swine ovary. In addition, we evaluated whether swine granulosa cells synthesize STC 1 and its possible modulation by hypoxia, a physiological condition in ovarian follicle growth. Our data show STC 1 for the first time in swine ovary; moreover, we demonstrate STC 1 production by granulosa cells, both in basal condition and in response to oxygen deprivation. The latter is suggestive of a potential modulatory role for STC 1 in hypoxia-driven angiogenesis., ((c) 2009 Elsevier Inc. All rights reserved.)

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERalpha than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERalpha in the treated group. An increase in total ERalpha protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERbeta in animals with COD, and the total protein expression of ERbeta was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.

Theca-interstitial (T-I) cells of the ovary synthesize androgens in response to luteinizing hormone (LH). In pathological conditions such as polycystic ovarian syndrome, T-I cells are hyperactive in androgen production in response to LH and insulin. Because cholesterol is an essential substrate for androgen production, we examined the effect of human chorionic gonadotropin (hCG) and insulin on signaling pathways that are known to increase cholesterol accumulation in steroidogenic cells. Specifically, the effect of hCG and insulin on sterol regulatory element-binding transcription factor 1a (SREBF1a) required for cholesterol biosynthesis and uptake was examined. Primary cultures of T-I cells isolated from 25-day-old rat ovaries responded to hCG and insulin to increase the active/processed form of SREBF1a. The hCG and insulin significantly reduced insulin-induced gene 1 (INSIG1) protein, a negative regulator of SREBF processing. Furthermore, an increase in the expression of selected SREBF target genes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) and mevalonate kinase (Mvk), was also observed. Protein kinase A (PRKA) inhibitor completely abolished the hCG-induced increase in SREBF1a, while increasing INSIG1. Although the hCG-induced depletion of total and free cholesterol was abolished by aminoglutethimide, the stimulatory effect on SREBF1a was not totally suppressed. Treatment with 25-hydroxycholesterol abrogated the effect of hCG on SREBF1a. Inhibition of the phosphatidylinositol 3-kinase pathway did not block the insulin-induced increase in SREBF1a, whereas mitogen-activated protein kinase inhibition reduced the insulin response. These results suggest that the increased androgen biosynthesis by T-I cells in response to hCG and insulin is regulated, at least in part, by increasing the expression of sterol response element-responsive genes by increasing SREBF1a.

The biological effects of estrogens are largely mediated through estrogen receptors (ERs), which belong to the nuclear receptor gene family of transcription factors. ER-alpha36 has been recently identified as a new variant of ERalpha, but its expression and roles in female reproduction system remain unknown. Immunocytochemistry and confocal microscopy were employed to observe ER-alpha36 distribution in mouse ovary during postnatal development and in oocyte during meiotic maturation. ER-alpha36 was consistently present in the nuclei of oocytes regardless of follicular growth stage and mouse age until germinal vesicle breakdown (GVBD). Its immunosignal was smeared in granulosa cells. However, the ER-alpha36 signal is up-regulated and found in cytoplasm with little or no nuclear staining during corpus luteum development. ER-alpha36 was also found in theca cells. We showed by Western blot that ER-alpha36 was expressed in mouse oocytes at various maturation stages. When the function of nuclear ER-alpha36 was blocked by microinjecting anti-ER-alpha36 specific antibody into the germinal vesicle (GV) of mouse oocytes, the first polar body emission occurred earlier in a higher proportion of oocytes compared to the control. These results suggest that ER-alpha36 may play critical roles in mouse ovarian folliculogenesis and oocyte development.

The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle.

Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-beta (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells)., ((c) 2007 Wiley-Liss, Inc.)

Our study aimed at screening patients with primary Sjogren's syndrome (pSs) for the presence of antiovarian antibodies (AOAs). Detection of AOAs in patients' sera was achieved by ELISA, using bovine ovarian extract for coating. Western blot analysis and immunohistochemistry were used to characterize the antibody targets in the extract and to determine their locus on the bovine ovary, respectively. Specific AOAs were detected in 27% of 37 patients (two with premature menopause) and in none of the controls. Immunoreactivity mainly resided in five proteins of the extract with molecular masses 42, 49, 55, 64 and 72 kDa, and it might be attributed to their carbohydrate components. The antibody targets were mainly located in the granulosa and theca interna cells of the follicle, and in the endothelial cells and fibroblasts of corpus luteum. The detection, for the first time, of AOAs in a significant percentage of patients with pSs may suggest autoimmune oophoritis, clinical or subclinical.

The aim of the present study was to examine the frequencies of cell proliferation and death of granulosa and theca interna layers during development of cystic follicles in order to understand the mechanisms of cystic follicle formation. Paraffin sections of cystic follicles were immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in order to observe proliferating and apoptotic cells, respectively. The concentrations of estradiol-17beta and progesterone in the follicular fluid of these follicles were measured by ELISA. The granulosa and theca interna layers contained both PCNA- and caspase-3-positive cells, although their numbers were limited. There was significant negative correlation between the estradiol-17beta and progesterone concentrations in the follicular fluid. Regression analysis revealed no significant correlation, except for that between the PCNA-positive cells in the theca interna and the caspase-3-positive cells in the granulosa layer. These results indicate that the granulosa and theca interna cells of the cystic follicle show weak proliferative activity and low apoptotic frequency; this implies that the cystic follicle grows slowly and then maintains a static condition without degeneration, which leads to long-term persistence of the follicle.

The preovulatory rise in gonadotropins causes an expansion of the cumulus-oocyte complex, a process requiring the induction of several genes. The objectives of this study were to clone the equine tumor necrosis factor alpha-induced protein 6 (TNFAIP6), and investigate its regulation in equine follicles during human chorionic gonadotropin (hCG)-induced ovulation. The isolation of the equine TNFAIP6 cDNA revealed that it contains an open reading frame of 834 bp (including the stop codon), encoding a predicted 277 amino acid protein that is highly similar (91-93% identity) to known mammalian homologs. The regulation of TNFAIP6 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h post-hCG and in corpora lutea (CL) obtained on day 8 of the estrous cycle. Results from semi-quantitative RT-PCR/Southern blot showed that levels of TNFAIP6 mRNA were low in follicles obtained at 0 h, increased at 12 h, returned to basal levels at 24 h, and increased again at 36 h post-hCG (P<0.05). Levels of TNFAIP6 transcripts were relatively moderate in CL, but low in non-ovarian tissues tested. Analyses performed with isolated preparations of theca and granulosa cells indicated that TNFAIP6 mRNA was regulated in both layers, with a maximal induction obtained 33-36 h post-hCG (P<0.05). Immunohistochemical staining of sections of equine follicles isolated at 0 and 33 h post-hCG confirmed the induction of TNFAIP6 protein in both cell types after hCG treatment. Thus, the present study describes for the first time the gonadotropin-dependent regulation of follicular TNFAIP6 during the ovulation in a monoovulatory species. The biphasic induction of TNFAIP6 in equine theca and granulosa cells differs from the pattern observed in rodents, suggesting a distinct control of gene expression in this monoovulatory species.

SOX9 is an essential activating transcription factor that plays a critical role in Sertoli cell differentiation and subsequent testis cord formation. Cytoplasmic SOX9 is present in both sexes during early gonadal embryogenesis. While in males the protein is later translocated into the nucleus of pre-Sertoli cells, its expression is rapidly turned off in females. In mammalian male gonads, SOX9 activates the expression of anti-Müllerian hormone (AMH), a male hormone that initiates Müllerian ducts regression and that is also expressed in postnatal ovarian follicles. Here, we confirm that the SOX9 protein is not present in the immature ovary but also show that SOX9 is transiently expressed in the mature ovary depending on the follicular cycle. Indeed, SOX9 protein was found in the nuclear compartment of the inner cells of the theca interna cell layer which surrounds the pre-antral/antral follicles. In contrast, no expression was detected in the AMH expressing granulosa cells. While these findings exclude the possibility that SOX9 regulates AMH expression in the ovary, they show that SOX9 could nevertheless play a role in the developing follicle.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide transiently expressed in preovulatory follicles. PACAP acts by interacting with three types of PACAP receptors. PACAP type I receptor (PAC(1)-R), which binds specifically to both PACAPs and vasoactive intestinal polypeptide (VIP), although with lower affinity, and two VIP receptors, VPAC(1)-R and VPAC(2)-R, which bind to PACAP and VIP with equal affinity. In the present study, we showed the expression of all three receptors in whole ovaries obtained from juvenile and gonadotropin-treated immature rats. A more detailed analysis on cells from preovulatory follicles showed that PAC(1)-R and VPAC(2)-R were expressed in granulosa cells, whereas only VIP receptors were expressed in theca/interstitial (TI) cells and fully grown oocytes presented only PAC(1)-R. The distribution of the VIP receptors was confirmed by immunofluorescence. HCG treatment induced stimulation of PAC(1)-R in granulosa cells and VPAC(2)-R in TI cells. The presence of functional PACAP/VIP receptors was also supported by metabolic studies. We further evaluated the presence of PACAP and VIP receptors by testing the effect of these peptides on apoptosis in granulosa cells cultured, isolated or in whole follicles. Treatment of follicles with PACAP and VIP dose-dependently inhibited apoptosis, while only PACAP significantly inhibited isolated granulosa cells. These results demonstrate a different expression of PACAP/VIP receptors in the various follicle compartments and suggest a possible role for PACAP and VIP on granulosa and TI cells, both during follicle development and ovulation.

The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.

Tissue inhibitors of metalloproteinases (TIMPs) are potential regulators of tissue remodeling in the ovary. The aim of the present study was to examine the localization and temporal regulation of TIMP-4 protein in the mouse ovary. An induced superovulation model (eCG/hCG) was employed in immature mice to evaluate TIMP-4 protein expression profiles in ovaries collected during the follicular phase, the pre ovulatory period, and the luteal lifespan. Immunofluorescence results indicated that TIMP-4 protein was localized to theca of both antral and preovulatory follicles and adjacent ovarian stroma. After the initiation of luteinization with hCG, TIMP-4 was observed within the luteinizing granulosa cells and persisted throughout the lifespan of the corpus luteum. In the cycling ovary, TIMP-4 signaling localized to corpus luteum from previous estrous cycles, the theca of preovulatory follicles, and appeared to be lower in newly forming corpus luteum. Western analysis further showed that the levels of TIMP-4 increased significantly during the luteinization process of granulosa cells, but no significant change was found among all corpus luteum stages. A putative regulatory mechanism of TIMP-4 expression was identified utilizing an in vitro model. Treatment of cultured granulosa cells with hCG significantly augmented TIMP-4 protein expression levels. Together our data indicate that the luteinization process of granulosa cells is associated with up-regulation of TIMP-4 and that TIMP-4 might play an essential role in maintenance of the luteal function during the whole lifespan of corpus luteum.

A first step to elucidating the roles that steroids may play in the processes of ovarian development and early follicular growth is to identify the cell types that are likely to be receptive to steroids. Thus, cell types expressing receptors for oestrogen (alpha and beta form; ERalpha and ERbeta respectively), androgen (AR) and progesterone (PR) were determined by in situ hybridisation and immunohistochemistry in ovine ovarian tissues collected during ovarian development and follicular formation (days 26-75 of fetal life) as well as during the early stages of follicular growth. Expression of ERbeta was observed early during ovarian development and continued to be expressed throughout follicular formation and also during the early stages of follicular growth. ERbeta was identified in germ cells as well as in the granulosa cells. At the large preantral stage of follicular growth, expression of ERalpha was also consistently observed in granulosa cells. AR was first consistently observed at day 55 of fetal life in stroma cells throughout the ovary. Within the follicle, expression was observed in granulosa and thecal cells from the type-2 to -3 stage of follicular growth. PR mRNA did not appear to be expressed during ovarian development (days 26-75 of gestation). However, PR (mRNA and protein) was observed in the theca of type-3 (small preantral) and larger follicles, with mRNA -- but not protein -- observed in granulosa cells of some type-4 and 5 follicles. Expression of ERbeta, ERalpha and AR, as well as PR, was also observed in the surface epithelium and ovarian stroma of the fetal, neonatal and adult ovary. Thus, in sheep, steroid hormones have the potential to regulate the function of a number of different ovarian cell types during development, follicular formation and early follicular growth.

Objective: In our previous papers, we demonstrated that TCDD and PCBs (both coplanar PCB 126 and non-coplanar PCB 153) caused the decreased estradiol secretion in cultured pig ovarian follicles. However, the mechanism of action is not clearly understood. Moreover, to our knowledge, the expression of AhR in pig follicular cells was not described yet., Methods: In order to elucidate if TCDD, PCB 126, and PCB 153 can disrupt ovarian steroidogenesis via AhR-dependent mechanism, granulosa and theca cells were isolated from pig ovarian small follicles and subsequently treated with 32 pg/ml of TCDD, 100 ng/ml of PCB 153, 100 pg/ml of PCB 126 in an in vitro culture. After 3-hour exposure to the chemicals, cells were prepared for immunohistochemical analysis or collected for immunobloting, and the effects of TCDD and PCBs on the levels of AhR protein were studied., Results: Under basal conditions, AhR staining showed a diffuse cytoplasmic pattern of distribution as well in granulosa and theca cells. However, immunocytochemical labeling of AhR in theca cells was less evident and only few cells displayed cytoplasmic staining. Immunoblotts prepared from granulosa and theca cell lysates detected a strong band of 120 kDa indicating AhR protein expression. The level of AhR expression detected by Western blot analysis was higher in granulosa cells than in theca cells. Intensive cytoplasmic and nuclear labeling corresponding to AhR protein in TCDD and PCB 126 treated granulosa cells was observed. In PCB 153 treated cells, the level of AhR staining was comparable with control. Western blot analysis showed a strong band in TCDD-treated grnulosa cells and only slightly differences compared with control in those exposed to PCB 126. The effect of both PCBs on immunocytochemical AhR staining in theca cells was less evident. Immunoblott analysis did not reveal any substantial differences between the control and TCDD- and PCBs-treated theca cells., Conclusions: This study has shown different AhR expression in porcine theca and granulosa cells, in respect of both its intracellular localization and cellular distribution. Both, dioxin and dioxin-like PCB activated AhRs in granulosa but not in theca cells. Differences in AhR-responsiveness of granulosa and theca cells to TCDD and dioxin-like PCB 126 are probably connected with the differences of these cells in estradiol secretion and different proliferative potential.

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.

One of the most prominent inflammatory reactions is the activation of the complement system. The activated complement system does not distinguish between pathogens and the host cell. In order to prevent autologous complement-mediated attack, host cells express a variety of both membrane-bound and fluid-phase complement regulatory proteins which control activity of the complement cascade by acting on convertase enzymes or the membrane-attack complex. Although the process of ovulation is facilitated by the inflammatory reaction, this reaction has the potential to cause serious damage to growing follicles, ovulated follicles, and other important ovarian tissues. This study was undertaken to characterize the expression and regulation of decay-accelerating factor (DAF), a complement regulator, as a potential mediator of ovarian tissue protection from ovulatory inflammation. DNA microarray and Northern blot analyses showed that an ovulatory gonadotropin stimulus dramatically yet transiently induced DAF mRNA expression in the immature rat ovary. Northern blot and PCR analyses revealed that of the three known DAF isoforms, glycosylphosphatidylinositol (GPI)-, soluble-, and transmembrane-(TM) DAF, GPI-DAF was the predominant form. In situ hybridization localized GPI-DAF mRNA expression in the theca-interstitial cells of the periovulatory ovary. Neither the anti-progestin RU486 nor the cyclooxygenase inhibitor indomethacin significantly inhibited human chorionic gonadotropin (hCG)-induced GPI-DAF mRNA expression in vivo. In vitro theca cell culture studies indicated that hCG induces GPI-DAF mRNA expression through the protein kinase A pathway. This study suggests that gonadotropin-induced GPI-DAF may be involved in the protection of ovarian tissues from the potential attack by the complement system activated by the inflammatory response associated with ovulation.

To elucidate changing patterns of inhibin/activin subunit mRNAs in the ovary of the golden hamster (Mesocricetus auratus) during the oestrous cycle, inhibin/activin subunit cDNAs of this species were cloned and ribonuclease protection assay and in situ hybridization were carried out. Inhibin alpha-subunit mRNA was localized in granulosa cells of primary, secondary, tertiary and atretic follicles throughout the 4-day oestrous cycle. It was also expressed in luteal cells on days 1 (oestrus), 2 (metoestrus) and 3 (dioestrus). betaA-subunit mRNA was localized in granulosa cells of large secondary (>200 microm) and tertiary follicles throughout the oestrous cycle. betaB-subunit mRNA was confined to granulosa cells of large secondary and tertiary follicles. Both alpha- and betaA-subunit mRNAs were also found in ovarian interstitial cells and theca interna cells of tertiary and atretic follicles in the evening of day 4 (pro-oestrus). A striking increase in betaA-subunit mRNA levels was also observed during the preovulatory period. The expression pattern of betaA-subunit mRNA during the preovulatory period is unique and not found in other species. An i.v. injection of anti-luteinizing hormone-releasing hormone (LHRH) serum before the LH surge abolished the expression of alpha- and betaA-subunit mRNAs in ovarian interstitial cells and theca interna cells. The treatment also abolished the preovulatory increase in betaA-subunit mRNA. Furthermore, administration of human chorionic gonadotrophin (hCG), which followed the injection of anti-LHRH serum, restored the expression patterns of alpha- and betaA-subunit mRNAs. The present study revealed that the golden hamster showed a unique expression pattern of betaA-subunit mRNA in response to the LH surge.

We studied the protein and mRNA expression of activin-A, follistatin and activin receptors in goat ovaries to find evidence of their possible role in ovarian activity, particularly in the various stages of follicle development. Ovaries of cyclic goats were collected and then either fixed in paraformaldehyde for immunohistochemical localisation of activin-A, follistatin, activin receptors IIA/B (ActR-IIA/B) and IA (ActR-IA) proteins or used to obtain samples to demonstrate mRNA expression of activin-A (betaA subunit), follistatin, ActR-IIA, -IIB, -IA and -IB, using RT-PCR. For this latter goal, primordial, primary and secondary follicles were isolated mechanically, washed to remove the stromal cells and then used for RT-PCR. In addition, oocytes, cumulus, mural granulosa and theca cells from small (<3 mm) and large (3-6 mm) antral follicles, luteal cells and surface epithelium were collected to study mRNA expression. Activin-A and follistatin proteins were found in oocytes of all follicle classes, granulosa cells from the primary follicle stage onwards, theca cells of antral follicles, corpora lutea and ovarian surface epithelium. In antral follicles, these proteins were detected both in cumulus and mural granulosa cells. ActR-IIA/B protein was found at the same follicular sites, and also in granulosa cells of primordial follicles onward. The localisation of ActR-IA corresponded with that of ActR-IIA/B, but the former protein was absent in the theca of large antral follicles. The mRNAs for activin-A (betaA subunit), follistatin, and ActR-IIA, -IIB, -IA and -IB were detected at all follicular and cellular types studied, except that ActR-IIB was not found in follicles that had not developed an antrum yet. In conclusion, in goat ovaries, transcripts of activin-A (betaA subunit), its receptors and its binding protein follistatin are expressed and their proteins formed at all follicular stages and in corpora lutea. These findings indicate a role of activin-A in the local regulatory system during the entire follicular development and during luteal activity.

The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.

The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle.

The distribution of androgen receptor (AR) and cytochrome P450 aromatase was investigated in paraffin sections of pregnant pig ovary. Ovarian follicles and corpora lutea were isolated from ovaries obtained on Days 10, 18, 32, 71 and 90 post coitum (p.c.). Androgen receptor was localized in the nuclei of granulosa cells of follicles of various sizes. In addition, some follicles demonstrated staining in the nuclei of theca interna cells. Stroma cells also exhibited a positive immunostaining. At early and mid pregnancy (up to Day 71) AR was expressed in the nuclei of luteal cells. Corpora lutea of Day 71 showed mainly cytoplasmic staining while on Day 90 almost all luteal cells showed staining exclusively in the cytoplasm. Immuno-staining for the presence of cytochrome P450 aromatase was very faint in all investigated ovarian structures. The results could suggest that the process of androgen aromatization plays a negligible role in the ovary of the pregnant pig.

Studies comparing the regressing corpus luteum with the rescued corpus luteum have demonstrated that human chorionic gonadotrophin (hCG) has effects on cell types that do not express hCG receptors. As progesterone synthesis is hCG dependent and the corpus luteum has been shown to express genomic progesterone receptors, progesterone is a candidate molecule for these paracrine effects. This study aimed to define the cellular localisation of progesterone receptors in the human corpus luteum using dual-staining immunohistochemistry for genomic progesterone receptors and specific cellular markers. Well-characterised corpora lutea (n = 12) from different stages of the luteal phase were studied. The same distribution was observed in all corpora lutea examined. The steroidogenic cells (3beta-hydroxysteroid dehydrogenase positive) and both theca-lutein (17alpha-hydroxylase positive) and granulosa-lutein (aromatase positive) express progesterone receptors, as do stromal fibroblasts (vimentin positive, fibroblast antigen positive). Vascular endothelial cells (CD31 positive), pericytes (alpha-smooth muscle actin positive), macrophages (CD68 positive) and fibroblasts within the central clot do not express nuclear progesterone receptors. Progesterone is a candidate messenger molecule for the effects of hCG on the matrix metalloproteinase-producing stromal fibroblasts. Some of the effects of hCG on steroidogenic cells may be mediated by progesterone, but its effects on blood vessels and macrophages require alternate paracrine signalling mechanisms. In addition, there appears to be at least two fibroblast populations in the corpus luteum.

The bone morphogenetic protein (BMP) family is emerging as playing a crucial role in regulating normal follicle growth and determining ovulation rate. BMPs exert their effects via BMP receptors (BMPR-IA, -IB and -II). However, there is a paucity of information relating to the expression of the BMPRs within the ovary of large polyovular species such as the pig. Furthermore, there is a lack of information on the expression of BMPRs by fetal ovaries of any species. The purpose of this study was to investigate temporal and spatial expression of the BMPRs in the porcine ovary, at different developmental stages. Immunohistochemistry for BMPR-IA, BMPR-IB and BMPR-II was performed using sections from paraffin wax-embedded ovaries, obtained from fetal (n = 15), prepubertal (n = 3) and cycling postpubertal (n = 4) pigs. Results confirmed the presence of all three receptors in the fetal egg nests and in the granulosa cell layer of follicles ranging from primordial to late antral stages. Immunostaining was also observed in oocytes, theca layer, corpus luteum and ovarian surface epithelium. The expression of BMPRs by fetal ovaries may be related to follicle formation, whereas expression in pre- and post-pubertal animals indicates BMPs are involved in regulating porcine ovarian follicle growth.

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.

The renin-angiotensin system (RAS) has been found in mammalian ovarian tissue; however, its physiological role is unclear. This study examined the content of angiotensin II (Ang II) in porcine follicular fluid (pFF), Ang II localization and its receptors in ovary, and the effects of Ang II on porcine oocyte maturation. The concentrations of Ang II were 6951.82 +/- 1295.83, 3502.99 +/- 679.10, 3147.89 +/- 690.60, and 2545.92 +/- 407.01 pg/ml in pFF from small, medium, large, and extra-large follicles, respectively. In addition, Ang II was found on zona pellucidae (ZP) and granulosa cells by immunoreactive staining. The distribution of AT1, an Ang II receptor subtype, was in accordance with that of Ang II. However, AT2, another Ang II receptor, was mainly distributed in the stroma and thecal layers of follicles. When oocytes were cultured in media containing various concentrations of Ang II, a higher (P<0.05) proportion of oocytes reached metaphase II (MII) in the medium with 100 ng/ml (87.0%) than without Ang II (61%). When oocytes from different sizes of follicles were separately cultured in media containing 100 ng/ml Ang II, maturation rates were significantly higher in oocytes from small (61.5%) and medium (85.1%) follicles than that of their controls (45.1 and 72.6%, respectively). However, addition of Ang II inhibited nuclear maturation in oocytes from large follicles (77.8% versus 87.3%). Fertilization and male pronuclear (MPN) formation rates of oocytes matured in medium containing 100 or 1000 ng/ml of Ang II were higher (P<0.05) than that of oocytes matured in medium containing 0 or 10 ng/ml Ang II. Glutathione content in oocytes cultured for 44 h in medium containing 100 or 1000 ng/ml of Ang II was also higher (P<0.01) than that of oocytes cultured in medium containing 0 or 10 ng/ml Ang II. In conclusion, Ang II was present in porcine ovaries and may regulate follicle growth and oocyte maturation.

The aim of this study was to investigate the effect of fasting on both vascular endothelial growth factor (VEGF) production and VEGF mRNA expression in growing ovarian follicles (>5 mm in diameter) from gilts at 48 h after equine chorionic gonadotrophin (eCG) treatment. The concentrations of VEGF and albumin were measured in the follicular fluid of single follicles, and VEGF mRNA was determined in the follicle wall. Fasting resulted in a significant increase in VEGF concentrations in follicular fluid (20.64+/-0.72 versus 10.79+/-0.86 ng ml(-1), P<0.001), but it did not affect the total amount of VEGF mRNA in the follicle wall compared with that of fed animals. However, VEGF mRNA in the theca and granulosa compartments increased and decreased, respectively, compared with that of fed animals. The concentrations of albumin measured in follicular fluid as an index of vessel permeability were higher in fasted than in animals fed normally, most likely as a result of the increased VEGF production. Follicular steroidogenesis was impaired in fasted animals. Progesterone was the most abundant steroid in the follicular fluid and oestradiol was present in lower concentrations, thus indicating an alteration in the steroidogenic enzymatic cascade. In conclusion, fasting induces an increase in both VEGF production and vessel permeability. Such a reaction is unable under severe food deprivation to preserve follicle function, but may represent a mechanism that regulates blood vessel extension and distribution in relation to tissue requirements and availability of systemic nutrient.

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are now known to have important roles in mammalian ovarian folliculogenesis. This study determined the expression of the mRNA encoding for BMPs and their receptors in the chicken ovary and explored possible roles for them. The expression of the mRNA for BMP-2, -4, -6, -7, and BMPR-IA, -IB, and -II was determined and quantified by a semiquantitative RT-PCR. The mRNAs for all the BMPs and receptors determined were present in both the granulosa (G) and theca (T) cells of the F1, F2, and F3 follicles. All BMP mRNAs increased in G cells with follicular development, whereas only BMP-7 mRNA had this trend in the T cells. BMP-2, -4, and -6 mRNAs in T were similar between follicles. BMPR-IA mRNA was similar in F2G and F3G but lower in F1G. BMPR-IB mRNA was similar in G of all follicles, and BMPR-II mRNA increased with development. In the T, each receptor subtype showed equal distribution between follicles. mRNA levels for BMPR-IB and -II were higher in G than in T, suggesting that the G is a major target for BMPs. BMP-4 and -7 stimulated basal, IGF-I-, and gonadotropin-stimulated progesterone production by cultured G cells, with differential responses between cells from the F1 and F3/4. This suggests involvement in follicular differentiation. BMP-4 and -7 reversed the inhibitory effects of transforming growth factor (TGF)-alpha on basal and gonadotropin-stimulated G cell progesterone production, with greater effect in the F1 than in the F3/4. This effect suggests an important role for BMPs interacting with TGF-alpha in modulating the effects of gonadotropins and IGF-I on follicular differentiation. Finally, BMP-7 stimulated G cell proliferation, but BMP-4 inhibited TGF-alpha+ IGF-I- and/or FSH-stimulated G cell proliferation, suggesting a role in the control of follicular growth during development. These effects of BMP-4 and -7 on the G cell function showed relationships with the expression levels of the BMPs and the BMPR-II.

Steroid biosynthesis in ovary is enhanced by the orphan nuclear receptor, steroidogenic factor-1 (SF-1); however, we reported that liver receptor homolog-1 (LRH-1), a closely related receptor to SF-1, is also expressed in mouse ovary. To further investigate the role of LRH-1 in mouse ovary, we used in situ hybridization to identify the cell types that express LRH-1 versus SF-1, and carried out functional studies to determine the role of LRH-1 in the regulation of the human (h) ovary-specific CYP19 promoter. LRH-1 expression was found to be abundant and highly restricted to cells involved in estrogen biosynthesis-granulosa cells during the estrous cycle, and in corpora lutea (CL) of pregnancy. In contrast, SF-1 was expressed most highly in C(19)-steroid-producing theca cells and interstitium, and at low levels in granulosa and luteal cells. Transfection studies using granulosa cells demonstrated that LRH-1 is a potent regulator of both basal and forskolin-induced transcription of the ovary-specific hCYP19 promoter. This activity was dependent upon two nuclear receptor half-sites within the proximal hCYP19 promoter. Based on these findings, we propose that LRH-1 plays an important role as a competence factor in regulating aromatase, and thus estrogen biosynthesis, in ovary.