Bovine Follicles Research Articles

169 INVASION OF MURINE ANNEXIN A1 IN BOVINE OVARIAN CORTEX TISSUE DURING SHORT-TIME XENOTRANSPLANTATION IN CONVENTIONAL AND IMMUNE DEFICIENT MICE

In 2012, 6.7 million women worldwide were diagnosed with cancer, including young women and children. Almost 85% of the latter survive the disease. However, the necessary cytotoxic treatment reduces their reproductive potential by damaging the stock of, basically preantral, ovarian follicles. Current fertility preservation techniques try to overcome this problem. Still, the success rate is low due to a lack of optimization and standardization. Nowadays, culturing preantral follicles in vitro is not yet routinely possible. Therefore, we used an in vivo model based on xenotransplantation of bovine ovarian cortex tissue in recipient mice, as an alternative to studying human reproduction. To further characterise this model, we assessed the influence from the host on the graft at the protein level. Visualising murine annexin A1, an anti-inflammatory protein, in the transplanted bovine cortex tissue should help to elucidate the process of immunological rejection. Hereto its distribution around the present bovine follicles is measured. In total, 12 mice (6 conventional fluorescent C57BL/6-eGFP and 6 immune deficient Balb/c-Nu) were used as graft recipients. All mice were anesthetized with an intraperitoneal injection and subsequently sterilized. Small pieces (max. 9 mm3) of adult bovine ovarian cortex, retrieved from slaughterhouse ovaries, were grafted retroperitoneal. After a transplantation period of 14 (3 mice/mice strain) or 28 (3 mice/mice strain) days, the mice were killed and all grafts were successfully recovered. These were fixed (4% formaldehyde) and processed into histological paraffin slides. Murine annexin A1 (Sigma-Aldrich HPA011271) was localised using immunofluorescence through a rhodamine label (RITC filter). We measured the shortest distances between 10 annexin fluorescent signals and the basal membrane of each follicle, found in the graft. We calculated the average of these measurements, resulting in one data point per follicle. The dataset was fitted in a linear mixed model with annexin as dependent variable, and transplantation period and mice strain as fixed effects. Fluorescent signals of murine annexin A1 were found in all grafts form both mice strains, surrounding bovine follicles. Data show no interaction between mice strain and the duration of the transplantation period. The influence of the mice strain showed a trend towards significance (P = 0.08), possibly due to the immunological state of the host. To our knowledge, this is the first time an attempt has been made to characterise the host/donor interaction in xenografting procedures. We can conclude that annexin A1 from the murine host (whether immunodeficient or immunocompetent) invades the bovine graft tissue in a short period of time.

167 THE EFFECT OF ELEVATED TEMPERATURE ON THE HEAT SHOCK PROTEIN 70 (HSP70) EXPRESSION IN BOVINE CUMULUS–OOCYTE COMPLEXES AFTER IN VITRO MATURATION

Elevated temperature during in vitro maturation negatively affects the oocyte's developmental competence, leading to disturbances in nuclear and cytoplasmic maturation. In previous studies, the role of cumulus granulosa cells (CGC) in cumulus-oocyte complexes' (COC) response to elevated temperature was underestimated. However, CGC play an essential role in folliculogenesis, supporting the oocyte's metabolism as well as meiotic progression. Thus, CGC may be engaged in COC response to heat shock. Heat shock protein 70 (HSP70) is the major protein engaged in cellular response to heat stress. The aim of this study was to determine the HSP70 expression in both CGC and oocytes in response to elevated temperature after COC in vitro maturation. COC were collected from bovine ovarian follicles (diameter 2–6 mm) from slaughtered cows and divided into two treatment groups: I (control) – COC were in vitro matured in control temperature (38.5°C); II (experimental) – COC were in vitro matured in elevated temperature (41°C). After in vitro maturation they were mechanically separated into CGC and oocyte. In vitro maturation was conducted in TCM199 25 mM HEPES medium supplemented with 10% FBS, 0.02 IU mL–1 NIH-pFSH, 1 μg mL–1 17β-oestradiol, 22 μg mL–1 Na-pyruvate and 10 μg mL–1 gentamicin, adjusted to pH 7.4 in 5% CO2. The HSP70 expression in CGC and oocytes was performed by real-time PCR and normalized to S18/H2A and S18 gene expression, respectively. In addition, the immunocytofluorescent analysis of HSP70 expression in CGC and oocyte's were performed. The HSP70 was stained using primary monoclonal mouse antibodies raised against bovine HSP70 and secondary antibody raised against mouse conjugated with Alexa 488. Nuclei were stained with Hoechst 33342. Slides were analysed under laser confocal microscope (FV-500, Olympus, Center Valley, PA, USA). The HSP70 expression in CGC was measured as total optical density of HSP70 and normalized to total optical density of nuclei. Statistical analyses were performed by Portable Statgraphics 5.0 Centurion. Mean values of HSP70 expression in CGC and oocytes in real-time PCR and immunofluorescent analysis in CGC were compared using Tukey's HSD test (α = 0.01). After in vitro maturation, the expression of HSP70 in CGC was higher (0.13 ± 0.052 a.u., n = 35) in experimental temperature compared to the control (0.058 ± 0.008 a.u., n = 35) (P < 0.01). In oocytes, HSP70 expression in experimental temperature (32.5 ± 5.2 a.u., n = 12) was similar to control (29.8 ± 5.6 a.u., n = 12). The immunofluorescent analysis confirmed data from real-time PCR analysis, indicating that the HSP70 expression in CGCs in experimental temperature was higher (0.46 ± 0.07 a.u., n = 10) compared to the control (0.12 ± 0.02 a.u., n = 10; P < 0.01). After COC in vitro maturation in stress conditions, the HSP70 expression was up-regulated in CGC, but not in oocytes. That may indicate that CGCs play the major role in COC response to elevated temperature; however, the analysis of other heat shock proteins expression in CGC and oocytes need to be conducted. Research was supported by 505-10-023300-K00169-99.

Expression and molecular consequences of inhibition of estrogen receptors in granulosa cells of bovine follicles

BackgroundEstradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells.MethodsGranulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes.ResultsESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes.ConclusionsOur findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.

Expression and regulation of regulator of G-protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells.

The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropin (hCG) injection. Results showed that RGS2 mRNA levels were very low at 0 h but markedly increased 12-39 h post-hCG (P < 0.05). In the bovine species, results revealed that RGS2 mRNA levels were low in small and dominant follicles and in ovulatory follicles obtained at 0 h, but markedly increased in ovulatory follicles 6-24 h post-hCG (P < 0.05). To study the molecular control of RGS2 expression, primary cultures of bovine granulosa cells were used. Stimulation with forskolin induced an up-regulation of RGS2 mRNA in vitro. Studies using 5'-deletion mutants identified a minimal region containing full-length basal and forskolin-inducible RGS2 promoter activities. Site-directed mutagenesis indicated that these activities were dependent on CRE and ETS1 cis-elements. Electrophoretic mobility shift assays confirmed the involvement of these elements and revealed their interactions with CREB1 and ETS1 proteins. Chromatin immunoprecipitation assays confirmed endogenous interactions of these proteins with the RGS2 promoter in granulosa cells. Forskolin-inducible RGS2 promoter activity and mRNA expression were markedly decreased by PKA and ERK1/2 inhibitors, and treatment with an antagonist of PGR (RU486) and inhibitors of PTGS2 (NS398) and EGFR (PD153035) blocked the forskolin-dependent RGS2 transcript expression, suggesting the importance of RGS2 in ovulation. Collectively, this study reports for the first time the gonadotropin-dependent up-regulation of RGS2 in equine and bovine preovulatory follicles and presents some of the regulatory controls involved in RGS2 gene expression in granulosa cells.

Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess.

Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

Expression and function of lysophosphatidic acid in theca cells of the bovine ovarian follicle

Expression and function of lysophosphatidic acid in theca cells of the bovine ovarian follicle

Transcriptome profiling of the theca interna from bovine ovarian follicles during atresia.

The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.

Characterization of microRNAs differentially expressed during bovine follicle development.

Several different miRNAs have been proposed to regulate ovarian follicle function; however, very limited information exists on the spatiotemporal patterns of miRNA expression during follicle development. The objective of this study was to identify, using microarray, miRNA profiles associated with growth and regression of dominant-size follicles in the bovine monovular ovary and to characterize their spatiotemporal distribution during development. The follicles were collected from abattoir ovaries and classified as small (4-8 mm) or large (12-17 mm); the latter were further classified as healthy or atretic based on estradiol and CYP19A1 levels. Six pools of small follicles and individual large healthy (n=6) and large atretic (n=5) follicles were analyzed using Exiqon's miRCURY LNA microRNA Array 6th gen, followed by qPCR validation. A total of 17 and 57 sequences were differentially expressed (greater than or equal to twofold; P<0.05) between large healthy and each of small and large atretic follicles respectively. Bovine miRNAs confirmed to be upregulated in large healthy follicles relative to small follicles (bta-miR-144, bta-miR-202, bta-miR-451, bta-miR-652, and bta-miR-873) were further characterized. Three of these miRNAs (bta-miR-144, bta-miR-202, and bta-miR-873) were also downregulated in large atretic follicles relative to large healthy follicles. Within the follicle, these miRNAs were predominantly expressed in mural granulosa cells. Further, body-wide screening revealed that bta-miR-202, but not other miRNAs, was expressed exclusively in the gonads. Finally, a total of 1359 predicted targets of the five miRNAs enriched in large healthy follicles were identified, which mapped to signaling pathways involved in follicular cell proliferation, steroidogenesis, prevention of premature luteinization, and oocyte maturation.

Transcriptome profiling of the theca interna in transition from small to large antral ovarian follicles.

The theca interna layer of the ovarian follicle forms during the antral stage of follicle development and lies adjacent to and directly outside the follicular basal lamina. It supplies androgens and communicates with the granulosa cells and the oocyte by extracellular signaling. To better understand developmental changes in the theca interna, we undertook transcriptome profiling of the theca interna from small (3–5 mm, n = 10) and large (9–12 mm, n = 5) healthy antral bovine follicles, representing a calculated >7-fold increase in the amount of thecal tissue. Principal Component Analysis and hierarchical classification of the signal intensity plots for the arrays showed no clustering of the theca interna samples into groups depending on follicle size or subcategories of small follicles. From the over 23,000 probe sets analysed, only 76 were differentially expressed between large and small healthy follicles. Some of the differentially expressed genes were associated with processes such as myoblast differentiation, protein ubiquitination, nitric oxide and transforming growth factor β signaling. The most significant pathway affected from our analyses was found to be Wnt signaling, which was suppressed in large follicles via down-regulation of WNT2B and up-regulation of the inhibitor FRZB. These changes in the transcriptional profile could have been due to changes in cellular function or alternatively since the theca interna is composed of a number of different cell types it could have been due to any systematic change in the volume density of any particular cell type. However, our study suggests that the transcriptional profile of the theca interna is relatively stable during antral follicle development unlike that of granulosa cells observed previously. Thus both the cellular composition and cellular behavior of the theca interna and its contribution to follicular development appear to be relatively constant throughout the follicle growth phase examined.

Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia

BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes

BackgroundAt later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow > 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease dividing and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small (< 5 mm; n = 10) and large (> 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets).ResultsPrincipal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT, IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues.ConclusionsOur data suggest that as follicles enlarge more genes and/or pathways are activated than are inactivated, and gene expression becomes more uniform. These findings could be interpreted that either the cells in large follicles are more uniform in their gene expression, or that follicles are more uniform or a combination of both and that additional factors, such as LH, are additionally controlling the granulosa cells.

Lipopolysaccharide (LPS) inhibits steroid production in theca cells of bovine follicles in vitro: distinct effect of LPS on theca cell function in pre- and post-selection follicles.

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation and leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS on steroid production in bovine theca cells at different stages of follicular development. Theca cells isolated from pre- and post-selection follicles (PRFs, <8.5 mm in diameter, and POFs, >8.5 mm in diameter, respectively) of bovine ovaries were exposed to LPS under luteinizing hormone (LH) conditions, estradiol (E2) conditions or both conditions in vitro. Bovine theca cells expressed the LPS receptor gene complex: Toll-like receptor 4 (TLR4), CD14 and MD2. LPS suppressed progesterone (P4) and androstenedione (A4) production with downregulation of steroidogenic enzyme transcripts when theca cells were stimulated with LH. By contrast, LPS did not affect P4 or A4 production when theca cells were stimulated with E2. P4 and A4 production in theca cells from PRFs was suppressed by LPS as early as at 48 h of culture, whereas the effect of LPS on theca cells from POFs was observed at 96 h of culture. The results demonstrate that LPS inhibits steroid production in theca cells under LH conditions. Moreover, theca cells from POFs showed a slower response to LPS compared with that of theca cells from PRFs, which might imply a distinct effect of LPS on follicles at different developmental stages. These findings suggest a possible mechanism of ovarian dysfunction and subsequent infertility in cows with endometritis.

Activity of 3β-hydroxysteroid dehydrogenase associated with progesterone production in bovine granulosa cells cultured under different concentrations of serum, insulin-like growth factor I, and gonadotropin

Three-b-hydroxysteroid dehydrogenase (3b-HSD) is the enzyme responsible for progesterone production. This study aimed to determine whether 3b-HSD activity can be shown to reflect progesterone production by granulosa cells cultured under different serum conditions and follicle stimulating hormone (FSH), luteinising hormone (LH), and insulin-like growth factor I (IGF-I) concentrations. Large bovine follicles were dissected from abattoir ovaries to recover granulosa cells. Cells were washed, stained for viability, and plated for 48 h in basic medium with or without 5% foetal calf serum (FCS). Subsequently, cells were exposed to FSH (1 ng/mL), LH (10 or 100 ng/mL), or FSH (1 ng/mL) + IGF-I (1 or 10 ng/mL) in a serum-free medium for another 96 h to predict degree of luteinisation. Before and after incubation, granulosa cells were stained for 3b-HSD activity. The high dose of IGF-I (10 ng/mL) increased (P < 0.05) progesterone secretion over 2.5-fold compared with FSH alone or the low dose of IGF-I (1 ng/mL) in cells preincubated with FCS. This was clearly reflected by darker 3b-HSD staining than in cells exposed to FSH and low dose IGF-I.

Changes in granulosa cells gene expression associated with growth, plateau and atretic phases in medium bovine follicles.

BackgroundThe objective of this study was to build the transcriptomic profile of granulosa cells originating from follicles 6 to 9 mm in diameter in dairy cattle using microarrays.MethodsGranulosa cells originating from three different phases of antral follicle growth were compared: growing (G), plateau (P) and atresia (A), as categorized by flow cytometry profiles of DNA. The growing and atretic conditions were each hybridized against the plateau condition as a reference in order to understand the specific biological mechanisms modulated in this class of follicles.Results2,942 genes were differentially expressed (P < 0.05) in P vs. G and 1,974 in A vs. P. A clear segregation of the 3 phases was confirmed by between group analysis (BGA). The first characteristic of the plateau phase is the activation of the upstream regulators TP53 and PTEN which participate in the reduction of cell growth through MYC, FOS and E2F1-2-3. We also observed the down-regulation of steroidogenesis genes: CYP11A1 and CYP19A1, in the granulosa cells of the plateau phase relative to the growth phase. On the other hand, the A vs. P contrast showed up-regulation of multiple transcripts associated to apoptosis: CCT2, DAB2, DSG2 and TGM2.ConclusionsThis study offers multiple candidate genes to be further studied in order to elucidate their role in the modulation of follicular development and, ultimately, of oocyte quality.

Regulation of Granulosa Cell Cocaine and Amphetamine Regulated Transcript (CART) Binding and Effect of CART Signaling Inhibitor on Granulosa Cell Estradiol Production During Dominant Follicle Selection in Cattle1

We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.

Influence of lysophosphatidic acid on estradiol production and follicle stimulating hormone action in bovine granulosa cells

The objective of the study was to examine the effect of lysophosphatidic acid (LPA) on 17β-estradiol (E2) synthesis and follicle stimulating hormone (FSH) action in bovine granulosa cells. We found that granulosa cells in the bovine antral follicle, in addition to the uterus and the CL, are also the site of LPA synthesis and the target for LPA action in the bovine reproductive tract. Our findings suggest that LPA stimulates E2 synthesis, probably via increased expression of FSHR and 17β-HSD genes.

Expression, Regulation, and Promoter Activation of Vanin-2 (VNN2) in Bovine Follicles Prior to Ovulation1

Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2-4 mm in diameter), dominant follicles obtained on Day 5 of the estrous cycle, ovulatory follicles obtained 0-24 h after human chorionic gonadotropin (hCG), and corpora lutea on Day 5 of the cycle. The results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in the other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly up-regulated 12-24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA, and Ebox cis-elements. Electrophoretic mobility shift assays identified COUP-TF, GATA4, and upstream stimulating factor proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, the findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles and provide insights into some of the molecular bases of VNN2 gene expression in follicular cells.

Paraoxonase (PON) 1, 2 and 3 Expression in Granulosa Cells and PON1 Activity in Follicular Fluid of Dairy Cows

Normal metabolic activity in ovarian follicles may result in oxidative stress and damage to oocytes. The aim of this study was to evaluate expression of the natural anti-oxidants paraoxonase (PON) 1, 2 and 3 in granulosa cells and PON1 activity in follicular fluid (FF) and plasma of dairy cows. For the first experiment, ovaries were collected from cows at slaughter, after which follicles were dissected and classified as oestrogen active (EAF) or atretic (ATF). Expression of PON1, PON2 and PON3 mRNA was evaluated in granulosa cells, and activity of PON1 was measured in FF. PON1 mRNA was undetectable in granulosa cells, PON2 mRNA expression was not different between follicle types, and PON3 mRNA tended to be higher in EAF (p=0.11). The activity of PON1 in FF was higher (p=0.01) for EAF (82.6±8.0 kU/L) than ATF (53.9±6.8 kU/L), as were high-density lipoproteins (HDL), low-density lipoproteins (LDL) and total cholesterol concentrations. In the second experiment, we aimed to compare plasma and FF PON1 activity in early lactation Holstein cows (n=15) with pre-ovulatory EAF. Activity of PON1 was twofold higher (p<0.0001) in plasma (122.5±11.1 kU/L) than in FF (61.4±5.2 kU/L). Plasma concentrations were also higher (p<0.0001) for HDL, LDL and total cholesterol when compared to FF. In conclusion, FF concentrations of PON1, HDL, LDL and total cholesterol were higher in healthy oestrogen active bovine follicles than in atretic follicles. PON1 was not expressed by granulosa cells indicating that high PON1 activity in bovine FF is apparently derived by transfer from blood in association with HDL.

Molecular Characterization and Transcriptional Regulation of a Disintegrin and Metalloproteinase With Thrombospondin Motif 1 (ADAMTS1) in Bovine Preovulatory Follicles

The ovulatory process involves a complex remodeling of the extracellular matrix during which a desintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) is thought to play a key role, but its transcriptional regulation in bovine follicles remains largely unknown. The objectives of this study were to characterize the regulation of ADAMTS1 in bovine follicles before ovulation and to determine its transcriptional control in bovine granulosa cells. Regulation of ADAMTS1 was assessed using total RNA isolated from bovine preovulatory follicles obtained at various times after human chorionic gonadotropin treatment. Results from RT-PCR analyses showed that levels of ADAMTS1 mRNA were very low at 0 hours but increased at 6 to 24 hours after human chorionic gonadotropin in granulosa cells. To determine the regulatory mechanisms controlling ADAMTS1 gene expression in vitro, primary cultures of bovine granulosa cells were established, and treatment with forskolin up-regulated ADAMTS1 mRNA levels. Promoter activity assays, 5'-deletion, and site-directed mutagenesis identified a minimal region conferring full-length basal and forskolin-stimulated ADAMTS1 promoter activities, with both being dependent on Ebox cis-acting elements. EMSAs revealed upstream stimulating factor (USF) proteins as key trans-activating factors interacting with Ebox. Chromatin immunoprecipitation assays confirmed such interactions between USF and Ebox in vivo, and USF binding to Ebox elements was increased by forskolin treatment. ADAMTS1 promoter activity and mRNA expression were increased by forskolin and overexpression of the catalytic subunit of protein kinase A, but not by cotreatment with inhibitors of protein kinase A, ERK1/2, and epidermal growth factor receptor signaling pathways. Furthermore, treatment with a soluble epidermal growth factor induced ADAMTS1 mRNA expression in granulosa cells. Collectively, results from this study describe the gonadotropin/forskolin-dependent up-regulation of ADAMTS1 mRNA in granulosa cells of bovine preovulatory follicles in vivo and in vitro and identify for the first time some of the molecular mechanisms responsible for ADAMTS1 promoter activation in follicular cells of a large monoovulatory species.

Research Resource: Preovulatory LH Surge Effects on Follicular Theca and Granulosa Transcriptomes

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.