Granulosa Cell Layer Research Articles

Di-(2-ethylhexyl) phthalate induced developmental abnormalities of the ovary in quail (Coturnix japonica) via disruption of the hypothalamic-pituitary-ovarian axis

An increasing number of epidemiologic studies show that women have a special exposure profile to phthalates, and the exposures have attracted attention regarding their potential health hazards. Here, we developed a model for studying the ovarian action of di-(2-ethylhexyl) phthalate (DEHP) and its major metabolite monoethylhexyl phthalate (MEHP). In vivo, treatment with DEHP (250, 500, and 1000 mg kg^-1) induced decreased thickness of ovarian granulosa cell layer and mitochondrial damage in quail, caused oxidative stress, interfered with the transcription of hypothalamic-pituitary-ovarian axis (HPOA) steroid hormone-related factors (increased transcription of StAR, 3β-HSD, P450scc, and LH and decreased transcription of 17β-HSD, P450arom, FSH, and ERβ), and blocked the secretion of steroid hormones (decreased FSH, E2, and T levels and increased LH, P, and PRL levels). In vitro, granulosa cells were cultured with MEHP (50, 100, and 200 μM), activator of PPARγ (rosiglitazone, 50 μM), or antagonist of PPARγ (GW9662, 10 μM) for 24 h and gene and protein expression were analyzed by real time RT-PCR and western blot. Rosiglitazone, like MEHP, significantly decreased mRNA and protein levels of P450arom. Antagonist GW9662 partially blocked the suppression of P450arom by MEHP, suggesting that MEHP acts through PPARγ, but not exclusively. Our model shows that MEHP acts on granulosa cells in quail by stimulating PPARs, which leads to decreased gene and protein expression of P450arom. Therefore, the environmental endocrine disruptor DEHP and its major metabolite MEHP act through a receptor-mediated signaling pathway to inhibit the production of estradiol, interfere with the modulation of HPOA, suppress the synthesis of sex hormones, and cause sex hormone secretion disorders, resulting in severe toxicity in the female reproductive system. A framework for an adverse outcome pathway of DEHP/MEHP-induced ovarian toxicity was constructed, which can facilitate an improved understanding of the mechanism of female reproductive toxicity.

Inactivation of Wt1 causes pre-granulosa cell to steroidogenic cell transformation and defect of ovary development†.

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1-/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3β-HSD-positive steroidogenic cells were detected in ovaries of Wt1-/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3β-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1-/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1-/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.

HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed β-subunit (HIF1B) and a regulated α-subunit (HIF1A). In the present study, we analyzed the HIF1 driven transcriptional activity in bovine granulosa cells (GC). Treatment of GC with FSH (follicle stimulating hormone) and IGF1 (insulin-like growth factor 1) resulted in the upregulation of HIF1A mRNA expression under normoxia. Immunohistochemistry of bovine ovarian sections showed distinct staining of HIF1A in the GC layer of different staged ovarian follicles. Suppression of HIF1 using echinomycin and gene knockdown procedures revealed that HIF1 transcriptionally regulates the genes associated with steroidogenesis (STAR, HSD3B and CYP19A1) and proliferation (CCND2 and PCNA) of GC. Further, our data suggest that CYP19A1, the key gene of estradiol production, is one of the plausible downstream targets of HIF1 in bovine GC as shown by gene expression, radioimmunoassay, and chromatin precipitation analysis. Based on these results, we propose that HIF1 driven transcriptional activity plays a crucial role in GC functionality, especially steroidogenesis and proliferation in developing bovine ovarian follicles.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number.

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.

Investigation of the effects of vitamin D treatment on the ovarian AMH receptors in a polycystic ovary syndrome experimental model: an ultrastructural and immunohistochemical study

The aim of this study was to demonstrate the effects of vitamin D treatment on ultrastructural changes and AMHR2 expression in the ovary in PCOS rat model. A total of 24 female prepubertal rats were divided into 3 groups. In group 1, sesame oil was injected and used as control group. In group 2, PCOS was created by the injection of 6 mg/kg/day DHEA. In group 3, PCOS was created and 120 ng/100 g 1,25 (OH)2D3 treatment was performed. At the end of the 28th day, the blood samples were collected. The ovarian tissues were obtained for electron microscopic and immunohistochemical examinations.Serum AMH, testosterone, FSH, LH levels and LH/FSH ratios were higher in the PCOS group compared to the control group and decreased in the treatment group compared to the PCOS group. AMHR2 expression was increased in atretic and premature luteinizate antral follicles in the PCOS group compared to the control group, and decreased in the treatment group compared to the PCOS group. PCOS group electron micrographs showed degenerative changes in developing follicles, cystic follicles characterised with granulosa cell layer attenuation and thickening of the theca cell layer, and lipid accumulation in the interstitial cells. Structural changes observed in the PCOS group were improved with vitamin D treatment.As a result, there is an interaction between PCOS, AMH serum levels and AMHR2 in the ovarian follicles. Vitamin D has a positive effect on hormonal and structural changes in the PCOS group. We concluded that vitamin D supplementation may be beneficial in PCOS patients.

128 Study of preantral ovarian follicular population in fetal alpaca (Vicugna pacos) ovaries

In mammals, folliculogenesis begins at the fetal stage and is a complex, dynamic process that involves follicular quiescence, activation, growth, follicular migration, and cell interactions. At birth, the preantral ovarian follicular population, drastically reduced, constitutes &amp;gt;90% of all ovarian follicles, representing the ovarian reserve that will be used throughout the reproductive life. In alpacas, changes in follicular wave growth patterns and ovulation are different from other species; thus, it is important to know the ovarian reserve at fetal stage, as a starting point for future studies. Therefore, the aim of the present study was to establish the morphological characterisation and estimate the population of alpaca preantral follicles in the fetal stage. Ovaries from alpacas (n=5) in the fetal stage (fetus during the last third of gestation) were collected at a local slaughterhouse. Whole ovaries were individually fixed in 4% paraformaldehyde phosphate-buffered saline overnight at room temperature for routine histology. Ovaries were dehydrated in alcohol, cleared with xylene, and embedded in paraffin, and all tissue was serially sectioned at 7μm with a rotating microtome (Leica). The histological sections were mounted and stained with periodic acid-Schiff and hematoxylin. Preantral follicles were classified according to their developmental stage: primordial (one layer of flattened granulosa cells surrounding the oocyte), primary follicle transition (flattened cuboidal granulosa cells surrounding the oocyte), primary (single layer of cuboidal granulosa cells around the oocyte), or secondary (oocyte surrounded by more than one complete layer of cuboidal granulosa cells). We estimated the number of preantral follicles by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n=40 per group), oocyte and follicle diameters were measured using an ocular micrometer. The variable means were compared using unpaired Student's t-test analysis, with significance set at P ≤ 0.05. Estimation of preantral follicular population (mean±standard deviation) was 80 516±14 575 in the ovaries of alpaca fetuses. Most of the follicles found belong to the primordial (49.2%) or primary follicle transition (39.2%) categories, followed by primary (10.8%) and secondary (0.8%) stages. Follicle and oocyte diameters of primordial (33.3±7.2; 21.5±4.6μm) and primary follicle transition stages (36.7±3.0; 23.4±2.6μm) were significantly (P&amp;lt;0.05) smaller than those of primary-stage follicles (77.9±15.8; 50.02±11.1μm). Finally, preantral follicles classified with normal morphological integrity appearance for each developmental stage were 98.2% (primordial), 96.7% (primary follicle transition), 91.5% (primary), and 88.1% (secondary), respectively. In conclusion, this study shows for the first time an estimation of the population of preantral follicles in alpaca fetal ovaries and establishes follicle and oocyte diameters and their normal morphological integrity.

Molecular analysis of the effects of steroid hormones on mouse meiotic prophase I progression

BackgroundInfertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis.MethodsFetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique.ResultsMSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage.ConclusionsWe set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.

Abundances and localizations of Claudin-1 and Claudin-5 in the domestic cat (Felis catus) ovary during the estrous cycle

Claudins (CLDNs) are major Ca2+-independent cell adhesion molecules functioning at tight junctions (TJ). The presence and localization of cell adhesion molecules are important for understanding the mechanisms associated with follicular and luteal development in the ovary. In this study, there was an examination of whether CLDN-1 and CLDN-5 are present in a cell- and stage-specific manner during follicular and luteal development in the domestic cat ovary using immunohistochemistry and Western blot analysis. While results from immunoblot analyses revealed there were relatively similar abundances of CLDN-5 protein in three phases of the ovarian cycle, the abundance of CLDN-1 in the luteal phase was greater than those measured in the follicular and anestrous phases (P < 0.01). Results with immunohistochemistry indicate CLDN-1 and -5 are mainly localized in the cell nuclei and cytoplasm of all tissues of the cat ovary. In follicles, throughout the development from primordial to large antral follicles, CLDN-1 and -5 were present in oocytes, and the granulosa and theca cell layers. In follicles at all stages of atresia, there were cell-type and stage-specific protein distributions with immunostaining present in granulosa, thecal interstitial, and fibroblast-like cells. In corpora lutea, both small and large luteal cells stained positively for both claudins. In conclusion, the specific presence and localization patterns of CLDN-1 and -5 in the cat ovary is suggestive that these TJ proteins could have local functions in the regulation of most ovarian functions such as follicle development and atresia, ovulation, and corpus luteum formation and regression.

Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates.

Angiogenesis is essential to both ovulation and the formation of the corpus luteum. The thrombospondin (THBS) family of glycoproteins plays diverse roles in regulation of angiogenesis, but the role of these vascular regulators in ovulation and luteinization remain to be elucidated. Using the cynomolgus macaque as a model for human ovulation, we demonstrated that levels of THBS1 mRNA and protein in preovulatory follicle granulosa cells increased after the ovulatory gonadotropin surge, with peak levels just before the expected time of ovulation. THBS1 treatment of monkey ovarian microvascular endothelial cells in vitro stimulated migration, proliferation, and capillary sprout formation, consistent with a pro-angiogenic action of THBS1. Injection of an anti-THBS1 antibody into monkey preovulatory follicles reduced rates of follicle rupture and oocyte release in response to an ovulatory gonadotropin stimulus when compared with control IgG-injected follicles. Interestingly, two of three oocytes from anti-THBS1 antibody injected follicles were germinal vesicle intact, indicating that meiosis failed to resume as anticipated. Follicles injected with anti-THBS1 antibody also showed reduced granulosa cell layer expansion, endothelial cell invasion, and capillary formation when compared to control IgG-injected follicles. Overall, these findings support a critical role for THBS1 in follicular angiogenesis, with implications for both successful ovulation and corpus luteum formation.

Toll-like receptors and high mobility group box 1 in granulosa cells during bovine follicle maturation.

Toll-like receptors (TLRs) are present in the ovaries and reproductive tract of various mammals. The biological function of TLR during ovulation is one of the main contents in the research of reproductive immunology. In this study, we found that messenger RNA levels of TLR1-TLR10 in granulosa cells were different, and TLRs and high mobility group box 1 (HMGB1) in granulosa cells of large follicles were significantly higher than those of small and middle follicles. Coimmunoprecipitation results showed that HMGB1 interacts with TLR2 in granulosa cells, especially large follicles. The result of immunohistochemistry showed that TLRs and HMGB1 were present in granulosa cell layer of ovarian follicles. We also found 25 mIU/ml follicle-stimulating hormone (FSH) significantly upregulated the expression of TLRs and HMGB1. These results suggest that TLR2/4 and HMGB1 in granulosa cells may be involved in the ovarian innate immune and ovarian follicular maturation, regulated by FSH. However, further research of the function and mechanisms of TLRs and HMGB1 in granulosa cells are needed.

Dietary Glycotoxins, Advanced Glycation End Products, Inhibit Cell Proliferation and Progesterone Secretion in Ovarian Granulosa Cells and Mimic PCOS-like Symptoms.

Women with polycystic ovary syndrome (PCOS) have been reported to have an elevated serum advanced glycation end product (AGE) level. However, the effect of AGEs on the pathophysiological ovarian granulosa cells of PCOS is still unclear. In this study, five indented BSA-derived AGE products were used to evaluate their effect on the function of human granulosa cells. We found that the proliferation of both primary human ovarian granulosa (hGC) cells and human granulosa-like tumor (KGN) cells were inhibited by treatment with these five AGE products. The progesterone secretion level was also reduced in both hGC and KGN cells by treatment with these AGE products through downregulation of LH receptor/cAMP regulatory activity. The granulosa cell layer and serum progesterone level were reduced in rats by treatment with MG-BSA; moreover, an increased number of follicle cysts and an irregular estrous cycle were observed. MG-BSA treatment had a similar effect on the phenotypes of the DHEA-induced PCOS model. Additionally, the insulin resistance and hepatic lesions seen in the DHEA-induced PCOS model were observed in the MG-BSA treatment group. Taken together, we found that AGEs exert a toxic effect on ovarian granulosa cells, ovarian morphology, and the estrous cycle that mimics the DHEA-induced PCOS phenotypes.

Variation in follicle health and development in cultured cryopreserved ovarian cortical tissue: a study of ovarian tissue from patients undergoing fertility preservation

This study investigated how follicle health and development in human ovarian tissue cryopreserved for fertility preservation varied between patients before and after 6 d of in vitro culture. Ovarian tissue from 12 patients (9–25 years) was used. In 3 patients, a 1hr neutral red (NR) incubation was used to identify tissues with viable follicles. Tissues were fixed, sectioned and follicles staged and graded for health. Inter-patient differences were observed in the non-cultured tissue in the number of both healthy follicles (p = 0.005) and growing follicles (p = 0.005). After culture there was significant variation in the number of transitional, primary and secondary follicles between patients (p < 0.001). Asymmetric primary follicles with a single complete layer of granulosa cells plus two or more additional partial layers were 5.5 times more likely to be observed in cultured compared to non-cultured tissue (p = 0.0063). Non-cultured (p = 0.0125) and cultured (p < 0.001) tissue selected using NR had more healthy follicles compared to tissue not selected using NR. Non-cultured and cultured tissue selected using NR had more healthy follicles compared to tissue not selected using NR (p = 0.0125; p < 0.001). We demonstrate that inter-patient variation exists in the health and development of follicles before and after culture. Culture systems need to be optimized to support cryopreserved ovarian tissue and these findings should prompt researchers to consider patient variation when evaluating culture systems.

Development of the sheep ovary during fetal and early neonatal life and the effect of fecundity genes

In female sheep fetuses, the mesonephros and genital ridge can be identified at days 20 and 23 of gestation (term = 145 days), respectively. Moreover oogonia can be observed at the genital ridge from as early as day 23. Around day 55 of gestation, some germ cells enter meiosis coincident with the arrival of mesonephric-derived somatic cells (i.e. the rete ovarii). From days 75, 100, 120 and 135 of gestation, primordial (one layer of flattened granulosa cells), primary (one complete layer of cuboidal granulosa cells; early preantral), secondary (preantral) and tertiary (antral) follicles, respectively, develop within the innermost regions of the ovarian cortex. During the early neonatal period highly variable numbers of antral follicles may be present. After examination of Booroola fetuses from day 28 of gestation, it seems that the FecBB gene is associated with retarded development of the heart (day 28) mesonephros (days 30–40) and from day 30 to early neonatal life, the ovary. With respect to the ovary, fewer oogonia (days 30–40), primordial follicles (day 75–90) and growing follicles (day 120 to 6 weeks after birth) have been observed in females carrying the FecB gene. By contrast, the FecB gene is not associated with differences in plasma gonadotrophin or immunoreactive inhibin until early neonatal life. In Inverdale (I) fetuses heterozygous for the FecXI gene (I+), retarded germ cell development was observed at days 40 and 90 of gestation. In putative homozygous carriers (II) of the Inverdale gene, germ cell development appeared normal until day 100, but thereafter from day 120 normal secondary follicles were not observed, although many abnormal follicular-like structures were present. In both I+ and II fetuses no obvious differences in gonadotrophin concentrations have been noted. Collectively, the evidence suggests that the fecundity genes FecBB and FecXI, which affect ovulation rate in sexually mature females, are regulating organ differentiation or germ cell maturation or both processes during fetal life.

Lateral Inhibition in Cell Specification Mediated by Mechanical Signals Modulating TAZ Activity

Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz-/- follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.

The effects of human menstrual blood stem cells-derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure.

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy-induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty-four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF-induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI-labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI-labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.

126 Regulation of initiation of follicle growth and dynamics of early follicular development in the sheep.

Primordial follicles embedded in the ovarian cortex are the source of developing follicles. Follicle growth activation and development up to the small antral follicle stage are controlled by cell interactions. The sheep ovary offers an appropriate non-rodent model to study these interactions, thanks to the development of in vitro and in vivo experimental approaches, ex vivo molecular analyses and in silico mathematical modeling. Each primordial follicle, composed of an oocyte surrounded by a single layer of quiescent granulosa cells, relies on nutrients and growth factors supplied by the surrounding stroma of connective tissue. In vitro cultures of ovarian cortex have shown that primordial follicles are activated by the lifting of mechanisms maintaining quiescence, some of them involving AMH secreted by already growing follicles. Afterwards, follicle development is supported by a finely tuned molecular dialog between the growing oocyte and proliferating granulosa cells. The isolation of preantral follicles and their development in vitro as individual follicles perturb this dialog, leading to an acceleration of follicular maturation. In vivo, mutations in the oocyte factors BMP15, GDF9 or their receptor BMPR1B also impair this dialog, leading to an imbalance between oocyte growth and cell proliferation, which can be reproduced by models for cell dynamics. During the growth of preantral follicles, the recruitment and differentiation of theca cells from the ovarian stroma provide them with a structural and vascularized support. In vivo exposure of sheep fetal ovaries to testosterone imprints the stroma cells so that the expression of genes involved in extracellular matrix organization and cell-cell adhesion is affected in theca at adulthood; this leads to a lower ovarian tissue rigidity that can account for the accelerated follicle growth observed in androgenized ewes. A better knowledge of cell interactions during early follicular development will help to improve the biotechnology methods of fertility preservation.

Interaction between the expression of retinol binding protein 4 and gonadotropin receptors in follicular granulosa cells of pigs

Retinol binding protein 4 (RBP-4) transports retinol from the liver to peripheral tissues, and the function is thought to be regulated by gonadotropins in the reproductive tissues. This study was conducted to measure the expression of RBP-4 in porcine ovaries and further investigate the interaction between the expression of RBP-4 and gonadotropin receptors in cultured granulosa cells. The expression of RBP-4 was detected by the immunohistochemical analysis of ovarian tissues and by western blot and real-time PCR analysis of the granulosa cells isolated from follicular fluids. The results showed that RBP-4 was located in the granulosa cell layer and inner theca layer of porcine follicles. Moreover, real-time PCR and western blot revealed that the expression levels of RBP-4 mRNA and protein in the granulosa cells treated with exogenous follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were significantly higher than those in the controls (P < 0.05). Granulosa cells treated with RBP-4 also showed an increased expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). Therefore, we conclude that RBP-4 might regulate the ovarian function through its interaction with gonadotropins.

116 Differential expression of connexin 43 and 37 mRNA transcripts during the estrous cycle in canines

Gap junctions are intercellular channels that mediate cell-to-cell communication, allowing the passage of small signalling molecules. In the ovary, connexin 43 (Cx43) and connexin 37 (Cx37) are important gap junctional proteins expressed in the granulosa and cumulus cells or oocytes of several species. Gap junctions and connexins are required for the regulation of the oocytes meiotic resumption in preovulatory follicles after the surge of LH. However, unlike other species, canine oocytes do not resume meiosis before ovulation, which could be related to expression patterns of Cx43 and Cx37 during oocyte development and ovulation. Therefore, this study aimed to address the canine Cx37 and Cx43 gene expressions throughout the oestrous cycle, including the preovulatory period. The ovaries were obtained from bitches 1-6 years old (n=72) following ovariohysterectomy. The stage of the oestrous cycle was assessed according the ovarian structures and by measurements of serum progesterone (P4) levels obtained from blood samples on the day of surgery. Anestrus was &amp;lt;0.1 ng/mL P4 and absence of follicles or corpus luteum in the ovarian surface; proestrus was 0.2-2 ng/mL P4 and growing small to medium follicles on the surface of the ovaries; oestrus was 2-19 mg/mL P4 and large follicles on the surface of the ovaries; and diestrus was &amp;gt;20 ng/mL P4 and mainly predominant corpus luteum on the ovaries. For Cx43 analysis, follicular cells (granulosa and theca) were mechanically recovered from follicles (n=620) distributed into 4 types: prenatal (1 layer of granulosa cells up to the onset of antrum formation), small antral (~0.2-0.39mm), medium antral (~0.4-5.9mm), and large antral (~6-10mm). For Cx37 study, the cumulus-oocytes complexes (COC) from the same follicles were used. Total RNA extraction was performed, and the evaluation of gene expression levels was achieved by relative quantification quantitative PCR analysis in follicular cells and COC. The data from at least 3 independent experiments for each gene were evaluated by ANOVA. The gene expression of both Connexins were observed in all stages of follicular development; however, the mRNA levels varied over the oestrous cycle. Both Cx43 and Cx37 transcripts showed the highest (P&amp;lt;0.05) levels at anestrus when compared to other phases. The mRNA levels of both genes remained without changes in large follicles at oestrus stage, suggesting that, in contrast to other mammals where LH down-regulates connexins expression leading to the subsequent loss of intercellular coupling, the communication between the oocyte and follicular cells was maintained in canines. In conclusion, these 2 connexin genes were differentially expressed in canine follicular cells and COC during the follicular development. The maintenance of the gene expression of these connexins at the final follicular growth may be involved in the prolonged meiotic arrest in this species. Supported by Ga grant from FONDECYT (1171670).

Response of hen pre-recruitment ovarian follicles to follicle stimulating hormone, in vivo

In laying hens, pre-recruitment ovarian follicles (1–8 mm diameter) are arranged as a continuum of size and predicted maturity. Cyclic recruitment of a pre-recruitment follicle to the preovulatory stage begins, in part, by the ability of the granulosa cell (GC) layer to initiate responsiveness to follicle stimulating hormone- (FSH-) induced cyclic adenosine monophosphate. The objective of this study was to determine if increased circulating concentrations of FSH during the ovulatory cycle increase the number of recruited follicles, in a dose-dependent manner. Equine chorionic gonadotropin (eCG) was initially tested due to its FSH-like properties and long half-life. Laying hens were injected, i.m., with 0 or 100 IU eCG, and ovaries were collected 29 h later. Recruited follicles were initially identified based on incorporation of yellow yolk and a weight of 250–900 mg. Recruitment was subsequently confirmed by both incubating the GC layer for 3 h with recombinant human (rh) FSH to establish FSH-responsiveness and quantifying P450 side-chain cleavage enzyme (CYP11A1) mRNA. Additional hens were injected with 0, 30, 75, and 300 IU eCG to establish a dose–response. Because eCG exhibits some luteinizing hormone activity, FSH-induced recruitment was evaluated by injecting 0.1, 0.33, 0.66, 1 or 3.3 µg rhFSH. Ovaries were collected 29 h post-injection, and expression of CYP11A1 mRNA was quantitated in GCs from recruited and pre-recruitment follicles. One hundred IU eCG induced recruitment of 2–8 follicles compared to a single follicle in control hens. In contrast to pre-recruitment follicles, incubated GC from eCG-recruited follicles had initiated differentiation, indicated by increased CYP11A1 and rhFSH-induced STAR mRNA and progesterone. Equine CG and rhFSH each increased the number of recruited follicles in a dose-dependent manner. Further, CYP11A1 mRNA was significantly increased in GC layers from recruited, compared to non-recruited, follicles. We conclude that FSH-responsiveness within the GC layer of each pre-recruitment follicle increases with follicle size, and propose that this establishes the order of daily follicle recruitment.

Follicle dynamics and granulosa cell differentiation in the turkey hen ovary.

Similar to the domestic hen ovary, entry of a follicle into the preovulatory hierarchy in the turkey hen represents a process in which a single follicle initiates rapid growth and final maturation prior to ovulation. Published data derived from the laying hen support the proposal that differentiation of the follicle granulosa cell (GC) layer begins coincident with entry into the rapid growth phase and is characterized by the initial capacity for follicle stimulating hormone (FSH)-mediated cell signaling. The present studies were conducted with photostimulated B.U.T. Big 6 turkey hens to compare follicle dynamics and cellular mechanisms to those in the laying hen. The measurement and weights of turkey ovarian follicles greater than 1mm in diameter revealed a discrete size hierarchy that was maintained throughout follicle development. GC layers collected from the single follicle initiating rapid growth (at the 11 to 13mm stage of development) and incubated, in vitro, for 3h with recombinant human (rh) FSH (10ng/mL) responded with significantly increased steroidogenic acute regulatory protein (STAR) mRNA expression and progesterone production. The same treatment induced minimal STAR expression and no significant progesterone accumulation in GCs from 8 to 9mm follicles (prior to the rapid growth phase). By comparison, dispersed GCs from 8 to 9mm follicles pre-cultured for 18h followed by a 3h challenge with rhFSH resulted in significantly increased STAR expression plus progesterone production. Significantly, such cultured GCs pretreated for 15min with transforming growth factor alpha (TGFα; 10ng/mL) completely prevented both rhFSH-induced STAR expression and progesterone production. Culture of GCs from 8 to 9mm follicles for 21h with Bone Morphogenetic Protein 6 (BMP6) increased both cholesterol side-chain cleavage enzyme (CYP11A1) and FSH receptor mRNA (FSHR) expression. BMP6 also enhanced rhFSH-induced STAR expression, and this effect was blocked by TGFα. Collectively, these results support a conservation of mechanisms that maintain a hierarchy of follicles throughout development plus initiate FSH-responsiveness and GC differentiation as the recruited follicle enters the rapid growth phase in these closely related species.