Androgen receptor gene deficiency results in the reduction of steroidogenic potential in porcine luteal cells.

Luteal steroidogenesis is critical to implantation and pregnancy maintenance in mammals. The role of androgen receptors (AR) in the progesterone (P4) producing luteal cells of porcine corpus luteum (CL) remains unexplored. The aim of the present study was to establish AR gene knock out (KO) porcine luteal cell culture system model by CRISPR/Cas9 genome editing technology and to study the downstream effects of AR gene deficiency on steroidogenic potential and viability of luteal cells. For this purpose, genomic cleavage detection assay, microscopy, RT-qPCR, ELISA, annexin, MTT, and viability assay complemented by bioinformatics analysis were employed. There was significant downregulation (p < 0.05) in the relative mRNA expression of steroidogenic marker genes STAR, CYP11A1, HSD3B1 in AR KO luteal cells as compared to the control group, which was further validated by the significant (p < 0.05) decrease in the P4 production. Significant decrease (p < 0.05) in relative viability on third passage were also observed. The relative mRNA expression of hypoxia related gene HIF1A was significantly (p < 0.05) downregulated in AR KO luteal cells. Protein-protein interaction analysis mapped AR to signaling pathways associated with luteal cell functionality. These findings suggests that AR gene functionality is critical to luteal cell steroidogenesis in porcine.

Genome-wide methylation profiling in granulosa lutein cells of women with polycystic ovary syndrome (PCOS).

Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder amongst women of reproductive age, whose aetiology remains unclear. To improve our understanding of the molecular mechanisms underlying the disease, we conducted a genome-wide DNA methylation profiling in granulosa lutein cells collected from 16 women suffering from PCOS, in comparison to 16 healthy controls. Samples were collected by follicular aspiration during routine egg collection for IVF treatment. Study groups were matched for age and BMI, did not suffer from other disease and were not taking confounding medication. Comparing women with polycystic versus normal ovarian morphology, after correcting for multiple comparisons, we identified 106 differentially methylated CpG sites with p-values <5.8 × 10-8 that were associated with 88 genes, several of which are known to relate either to PCOS or to ovarian function. Replication and validation of the experiment was done using pyrosequencing to analyse six of the identified differentially methylated sites. Pathway analysis indicated potential disruption in canonical pathways and gene networks that are, amongst other, associated with cancer, cardiogenesis, Hedgehog signalling and immune response. In conclusion, these novel findings indicate that women with PCOS display epigenetic changes in ovarian granulosa cells that may be associated with the heterogeneity of the disorder.

FSH up-regulates angiogenic factors in luteal cells of buffaloes.

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4-6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.

The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle-luteal transition.

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.

The relationship between the production and the anti-gonadotrophic action of prostaglandin F 2 alpha in luteal cells from the marmoset monkey (Callithrix jacchus) in the early and mid-luteal phase.

To address the potential luteolytic role for prostaglandin F(2 alpha) (PGF(2 alpha)) in the corpus luteum of the common marmoset monkey (Callithrix jacchus), the ability of marmoset luteal cells, maintained in monolayer culture, to produce PGF(2 alpha) was determined in vitro in the presence and absence of human chorionic gonadotrophin (hCG) and other established pharmacological modulators of PGF(2 alpha) synthesis. We also assessed the effects of the PGF(2 alpha) analogue, cloprostenol, on progesterone output from luteal cells isolated in the early luteal phase versus the mid-luteal phase (days 3 and 14 post ovulation, respectively). Cloprostenol had no effect on progesterone output from luteal cells isolated on day 3 of the luteal phase, whereas it significantly inhibited both basal and hCG-stimulated progesterone synthesis by day 14 luteal cells during the culture period 48-72 h (P<0.001). Intra-luteal PGF(2 alpha) concentrations were 5-fold higher in luteal cells isolated in the early luteal phase than in mid-luteal phase cells (16.5+/-3.5 versus 3.5+/-0.6 pmol/10(5) cells). While PGF(2 alpha) production was unaffected by hCG in vitro, it was decreased by indomethacin (1000 ng/ml) (P<0.05) and stimulated by the calcium ionophore A23187 (10 micromol/l) (P<0.05) in luteal cells from both stages of the luteal phase. Phospholipase A(2) did not influence PGF(2 alpha) production by day 3 luteal cells whereas at 10 IU/ml, it significantly stimulated PGF(2 alpha) production by day 14 luteal cells (P<0.05). Hence, the timing of luteolysis in the common marmoset monkey appears to involve changes in both the luteal cell response to and production of PGF(2 alpha).

Endothelin-1, endothelin converting enzyme-1 and endothelin receptors in the porcine corpus luteum.

Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2alpha (PGF-2alpha) (ie, luteolytic sensitivity [LS]) until about day 12-13 of the estrous cycle. Although little is known of the control of LS in any species, endothelin-1 (EDN1) is believed to play a role in LS control in ruminants. Therefore, we measured mRNA and protein expression and examined the cellular localization of EDN1 precursor (pre-pro EDN1, or ppEDN1), EDN-converting enzyme-1 (ECE1), and EDN receptors (A, EDNRA and B, EDNRB) in porcine CLs collected on days 4, 7, 10, 13, and 15 of the estrous cycle to look for differences between CLs displaying (days 13-15) versus those lacking (days 4-10) LS. Abundance of ppEDN1 mRNA was greatest (and significant vs all other days) on day 7 of the cycle, whereas EDN1 protein expression did not vary during the cycle and was localized exclusively to endothelial cells (EC). Abundance of ECE1 mRNA was also greatest on day 7 (vs all other days), but ECE1 protein was significantly elevated on day 10 (vs day 4) and was immunolocalized to ECs and large luteal cells (LLC). Abundance of EDNRA mRNA was also maximal on day 7 (vs all other days) of the cycle, whereas EDNRA protein expression was not significantly changed during the cycle and was observed in LLCs, ECs, and small luteal cells (SLC). On day 13, EDNRB mRNA was significantly decreased (versus day 7). Expression of EDNRB protein was decreased on day 10 (versus all other days), and on days 13-15 (vs day 4), and was primarily localized to ECs. In conclusion, the observed elevation in ECE1 protein concentrations on day 10 and the presence of EDNRA on LLC suggests a possible role for EDN1 (resulting from the actions of ECE1) acting via EDNRA in the control of LS in the pig.

Progesterone inhibits oxytocin- and prostaglandin F2alpha-stimulated increases in intracellular calcium concentrations in small and large ovine luteal cells.

There is increasing evidence that the corpus luteum has an important role in regulating its own demise. A series of experiments was performed to study the effects of luteal concentrations of progesterone on the functions of steroidogenic luteal cells. In the first experiment, steroidogenic small luteal cells (SLCs) were separated from endothelial cells, and it was determined that it was the SLCs that contained receptors for oxytocin. Treatment with progesterone (95 muM) for as little as 1 h decreased (P < 0.05) the percentage of SLCs responding to oxytocin (10 muM) with an increase in intracellular concentrations of calcium, and this effect continued for the duration of the experiment. In a second experiment, the response to oxytocin was increased (P < 0.05) by 3 h (but not 1 h) following progesterone removal, with a further increase by 16 h. The ability of 1 muM prostaglandin F(2 alpha) (PGF(2 alpha)) to increase intracellular concentrations of calcium was also decreased (P < 0.05) by progesterone treatment. By 3 h following removal of progesterone, the percentage of steroidogenic large luteal cells (LLCs) responding to PGF(2 alpha) was increased and not different from that observed in cells 16 h after progesterone removal. Finally, cyclodextrins (methyl-beta cyclodextrin [M beta CD]) were used to remove cholesterol from the plasma membrane of luteal cells, and M beta CD loaded with cholesterol was used to put cholesterol back into the plasma membrane of progesterone-treated cells. Treatment with M beta CD reduced (P < 0.05) the responsiveness of SLCs to oxytocin and LLCs to PGF(2 alpha). Use of cholesterol-loaded M beta CD returned the responsiveness of both SLCs and LLCs treated with progesterone to that observed in vehicle (no progesterone)-treated controls. In summary, intraluteal concentrations of progesterone inhibit the ability of oxytocin to increase intracellular concentrations of calcium in SLCs and the ability of PGF(2 alpha) to increase intracellular concentrations of calcium in LLCs. The highest concentration of progesterone appears to act by influencing cholesterol content of the luteal cell membranes.

KIT receptor-positive cells in the bovine corpus luteum are primarily theca-derived small luteal cells.

The tyrosine kinase KIT receptor, the protooncogene CD117, plays a key role in growth and maturation of oocytes and follicles. Relevant data are sparse for the corpus luteum (CL). We first confirmed the presence of KIT mRNA and KIT protein in bovine CL homogenates. We then localized KIT-positive (KIT+) cells in CL sections by immunohistochemistry. At the CL stage of early development, the former theca transforming into capsule/septa showed a strong band-like KIT+ immunoresponse. In addition, CD45+ leukocytes in septa included subpopulations of CD45+/KIT+ and CD14+/KIT+ leukocytes as validated by double immunofluorescence localization. At the early secretory stage, KIT+ cells appeared within the septa/capsule region and in the periphery of the CL parenchyma, there forming a complex network. This was separate from the capillary bed as determined by double staining for CD117 and FVIII-related endothelial cell antigen (FVIIIr). The KIT+ network coincided with cells positive for cytochrome P450 17alpha-hydroxylase, a thecal cell-specific enzyme. The late secretory stage was defined by an advanced manifestation of the KIT+ network in the CL periphery. At the stage of regression, the KIT+ network was absent. The CL of pregnancy expressed high levels of KIT mRNA and KIT protein uniformly throughout pregnancy. The KIT+ immunolocalization revealed small fibroblast-like cells, luteal cells with granules, and clusters of large luteal cells with staining of the cell membrane. We conclude that a majority of KIT+ cells in the bovine CL are primarily theca-derived small luteal cells, and that a minority represent KIT+ leukocytes, in some cases KIT+ monocytes.

11beta-Hydroxysteroid dehydrogenase expression and activities in bovine granulosa cells and corpora lutea implicate corticosteroids in bovine ovarian physiology.

Cortisol-cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD1) enzyme and the oxidative NAD(+)-dependent type 2 11betaHSD (11betaHSD2). This study related the expression of 11betaHSD1 and 11betaHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11beta-dehydrogenase (11beta-DH) activities in bovine follicular granulosa and luteal cells. Granulosa cells were isolated from follicles of < 4, 4-8, > 8 and > 12 mm in diameter in either the follicular or luteal phase of the ovarian cycle. Luteal cells were obtained from corpora lutea (CL) in the early non-pregnant luteal phase. Enzyme expression was assessed by reverse transcription-PCR and western blotting, while enzyme activities were measured over 1 h in cell homogenates using radiometric conversion assays with 100 nM [(3)H]cortisone or [(3)H]cortisol and pyridine dinucleotide cofactors. Irrespective of follicle diameter, the expression of 11betaHSD2 and NAD(+)-dependent oxidation of cortisol predominated in granulosa cells harvested in the follicular phase. In contrast, in granulosa cells obtained from luteal phase follicles and in bovine luteal cells, expression of 11betaHSD1 exceeded that of 11betaHSD2 and the major enzyme activity was NADP(+)-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11betaHSD2 and 11betaHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11betaHSD1 migrated with a molecular mass of 34 kDa, whereas in the dominant follicle, CL and all luteal phase granulosa cells, a second protein band of 68 kDa was consistently detected. In all samples, 11betaHSD2 had a molecular mass of 48 kDa, but in large antral follicles (> 8 mm), there was an additional immunoreactive band at 50 kDa. We conclude that 11betaHSD2 is the predominant functional 11betaHSD enzyme expressed in follicular phase granulosa cells from growing bovine antral follicles. In contrast, in bovine granulosa cells from dominant or luteal phase follicles, and in bovine luteal cells from early non-pregnant CL, 11betaHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP(+)- and NAD(+)-dependent 11beta-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

Constitutive steroidogenesis in ovine large luteal cells may be mediated by tonically active protein kinase A.

The mechanisms responsible for the increased basal rates of progesterone secretion from large steroidogenic luteal cells (LLC) relative to small steroidogenic luteal cells (SLC) have not been clearly defined. To determine if protein kinase A (PKA) is tonically active in LLC, the adenylate cyclase activator forskolin and a specific PKA inhibitor (PKI) were utilized in a 2 x 2 factorial treatment with each steroidogenic cell type. Progesterone and cAMP production were quantified after the different treatments. In addition, the effects of the treatments on the concentrations and relative phosphorylation status of the steroidogenic acute regulatory (STAR) protein in the two cell types were determined as a measure of PKA activity. Treatment with PKI blocked forskolin-induced increases in progesterone secretion by SLC without affecting the production of cAMP. The treatment of LLC with PKI significantly decreased basal progesterone secretion in the presence or absence of forskolin, indicating that the high level of steroidogenesis in this cell type requires PKA activity. There were no differences in the steady-state concentrations of STAR protein in either cell type after treatment. However, the percentage of relative STAR phosphorylation was higher in the LLC than in SLC, and PKI treatment significantly decreased the phosphorylation of STAR in the LLC. The relative phosphorylation status of STAR and the concentrations of progesterone in the media were significantly correlated with the treatments in both cell types. The amount of progesterone secreted per picogram of cAMP was higher in the LLC than in the SLC, and this was accompanied by a significant increase in the ratio of relative STAR phosphorylation to the steady-state concentration of STAR protein. These data are compatible with the theory that LLC are constitutively steroidogenic, partly because they have tonically active PKA. In addition, the phosphorylation of STAR appears to be a primary activity of PKA in both types of ovine steroidogenic luteal cells.

Localization and temporal regulation of tissue inhibitor of metalloproteinases-4 in mouse ovary.

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 quantitative study of blood capillary formation (angiogenesis) concomitant with parenchymal tissue differentiation.

Examination of the rat corpus luteum (CL) provided quantitative data supporting adaptation of the developing vasculature to maximise efficient acceptance of steroids secreted from the luteal cells. Numbers of endothelial cells (ECs) significantly increased during the initial formation of the CL, followed by a further significant proliferation from day 10 to day 16 when there was maximal growth of the CL. As a consequence, there was significant growth of the vascular compartment during this time interval. The final phase of expanding endothelium (days 10 to 16) was a result of increased ECs volume with elongation of the EC in the direction of growth. Continued increase in capillary surface area and a corresponding marked reduction in diffusion distance between LC and ECs evidenced adaptation of the developing microvasculature to enable efficient endocrine function by day 16, when steroid secretion is maximal. Furthermore, from day 1 to day 3 there was close apposition of pericytes to the endothelium, suggesting the important role of pericytes in the initiation of angiogenesis. However, this degree of association was reduced from day 10 to day 16 and was a consequence of expansion of the EC cytoplasm to provide a greater surface area for transport of steroids.

The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro.

The human corpus luteum expresses genomic progesterone receptors (PRs) suggesting that progesterone may have an autocrine or paracrine role in luteal function. We hypothesised that the reduction in luteal PR reported in the late-luteal phase augmented progesterone withdrawal and had a role in luteolysis. We therefore tested the hypothesis that luteal rescue with human chorionic gonadotrophin (hCG) would maintain PR expression. PR was immunolocalised to different cell types in human corpora lutea (n = 35) from different stages of the luteal phase and after luteal rescue with exogenous hCG. There was no change in the staining intensity of theca-lutein cell or stromal cell PR throughout the luteal phase or after luteal rescue. In the late-luteal phase, granulosa-lutein cell PR immunostaining was reduced (P < 0.05) but the trend to reduction was also seen after luteal rescue with hCG (P = 0.055). To further investigate the effect of hCG on granulosa-lutein cell PR expression, an in vitro model system of cultured human luteinised granulosa cells was studied. Cells were cultured for 12-13 days exposed to different patterns of hCG and aminoglutethamide to manipulate progesterone secretion (P < 0.0001). Expression of PR A/B and PR B isoforms was examined by quantitative real-time RT-PCR. PR A/B mRNA was lower (P < 0.05) after 11-13 days of culture than after 7 days of culture. This reduction could not be prevented by hCG in the presence (P < 0.05) or absence (P < 0.05) of stimulated progesterone secretion. The expression of PR B mRNA showed a similar pattern (P = 0.054). Simulated early pregnancy in vivo and hCG treatment of luteinised granulosa cells in vitro did not appear to prevent the down-regulation of PR seen during luteolysis.

Expression of sex hormone-binding globulin (SHBG) in human granulosa-lutein cells.

Sex hormone-binding globulin (SHBG) was classically thought to be a plasma steroid-carrying protein of hepatic origin, but recently, locally produced, membrane-bound SHBG has been shown to influence cell functions in several steroid-responsive tissues. In the ovary, SHBG is known to be present in the follicular fluid, but information about a possible intracellular presence of SHBG in this organ is still very scarce. In this study the presence of SHBG was assessed by immunohistochemistry in human granulosa-lutein cells (GLC) collected by follicle puncture for in vitro fertilization. SHBG was detected in the cytoplasm of GLC before and after in vitro culture for up to 96 h. The presence of full-length SHBG messenger RNA was demonstrated in GLC by reverse transcription-polymerase chain reaction (RT-PCR) in both cultured and uncultured cells. These results demonstrate a local synthesis of SHBG in GLC and raise the question of the physiological significance of these findings in follicular physiology.

Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary.

Steroidogenic factor-1 (SF-1) (NR5A1) is an orphan nuclear receptor that plays a premier role in ovarian organogenesis. Recent studies document mRNA expression of the structurally related factor NR5A2 (FTF, LRH-1, SF-2) in the adult ovary and more specifically in granulosa cells and luteal cells but not theca cells. Conversely, SF-1 was shown to be expressed at higher levels in theca/interstitial cells. These latter observations raised the possibility that FTF/LRH-1 may control target gene expression in granulosa cells of developing follicles. Using quantitative PCR our results show that FTF/LRH-1 message is expressed at higher levels in the ovary than in liver or other tissues analyzed. We show by in situ hybridization and LacZ expression in ovaries of transgenic mice bearing an FTF-promoter-LacZ fusion gene that FTF/LRH-1 is selectively expressed in granulosa cells of rat and mouse ovaries and is not present in theca cells or interstitial cells. However, by a variety of approaches, we showed that SF-1 mRNA and protein are expressed in greater amounts than FTF/LRH-1 in granulosa cells of follicles at all stages of development. Expression of SF-1 mRNA and protein in granulosa cells was verified by in situ hybridization, immunohistochemistry of ovarian sections, and immunocytochemistry of cultured rat granulosa cells. The significance of SF-1 in regulating target gene activation was supported by EMSA. An abundant granulosa cell protein binding to the SF-1-binding motif (CCAAGGTCA) present in the aromatase promoter and an FTF/LRH-1 motif (TGTCCTTGAACA) in the alpha-fetoprotein promoter was supershifted by two SF-1-specific antibodies but not by an FTF antibody. Conversely, with the same probes, a less abundant protein/DNA complex present in liver and ovarian cell extracts was shifted by an FTF antibody but not by the SF-1 antibodies. SF-1 and FTF/LRH-1 were differentially regulated in vivo by estradiol, FSH and prolactin. Collectively these data indicate that granulosa cells of small and preovulatory follicles express both SF-1 and FTF/LRH-1 and that each orphan receptor may regulate target gene expression in these cells.

Cell-specific distribution of progesterone receptors in the bovine ovary.

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non-pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down-regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.

Cyclic changes of granulocyte colony-stimulating factor (G-CSF) mRNA in the human follicle during the normal menstrual cycle and immunolocalization of G-CSF protein.

BACKGROUND: Ovulation has several similarities with inflammation and is closely connected to the activity of leukocytes and inflammatory cytokines. Since granulocytes are one of the major leukocytes, we focused our attention on the presence and local production of granulocyte colony-stimulating factor (G-CSF) in the human ovary. METHODS: The presence of G-CSF protein in the follicular fluid and perifollicular tissues was examined by Western blot analysis (n = 5) and immunohistochemical staining (n = 10). The relative expression levels of G-CSF mRNA in relation to GAPDH in granulosa, theca and luteal cells during the menstrual cycle were measured by quantitative RT-PCR using TaqMan technology (n = 15). RESULTS: G-CSF protein was detected in all follicular fluid and located mainly in granulosa cells of the follicle and luteal cells. The expression level of G-CSF mRNA in the late follicular phase was 137.6 +/- 18.5, which was approximately 10-fold greater than other phases during the menstrual cycle (P < 0.05). CONCLUSIONS: These results demonstrate that G-CSF is produced in the human follicle shortly before the ovulatory phase and may play an important role in the mechanism of ovulation.

Angiogenesis in the corpus luteum of early pregnancy in the marmoset and the effects of vascular endothelial growth factor immunoneutralization on establishment of pregnancy.

This study investigated vascular and molecular changes in the corpus luteum (CL) of early pregnancy in the marmoset. Ovaries were studied on Days 21 (n = 6) and 28 (n = 6) of pregnancy and compared with corpora lutea from Day 21 (late luteal) of the nonconception cycle (n = 8). Endothelial cell proliferation was measured by immunocytochemical detection of incorporated bromodeoxyuridine. Endothelial cell and pericyte area were assessed by quantitative immunocytochemistry for CD31 and alpha-smooth muscle actin, respectively. Vascular endothelial growth factor (VEGF) and its receptors, kinase insert domain-containing region (KDR) and fms-like tyrosine kinase (Flt) mRNA, were localized and quantified in in situ hybridization. In addition, the effects of immunoneutralization of VEGF on establishment and maintenance of pregnancy were investigated by administering a VEGF neutralizing antibody on Days 0-10 of the luteal phase during potentially fertile cycles (n = 10) and compared with fertile controls (n = 6). No differences in the cellular or morphological parameters were found between pregnant and structurally intact nonpregnant corpora lutea. No major differences were found in expression of VEGF, Flt, or KDR in these CL. VEGF immunoneutralization markedly suppressed plasma progesterone secretion during treatment, but pregnancy rate was not significantly reduced. Thus, a role for VEGF in early pregnancy in the marmoset remains to be established. These results show that, by the late luteal phase in the marmoset, the corpus luteum has established a mature vascular system and the molecular capacity to synthesize VEGF and its receptors. A pregnancy-induced spurt of angiogenesis or gene expression does not appear to take place; rather, maintenance of the existing vasculature is all that is required for the establishment of pregnancy.

Cell-specific distribution of progesterone receptors in the bovine ovary.

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non-pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down-regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.

Distinct expression of endothelin receptor subtypes A and B in luteinized human granulosa cells.

Endothelins (ET) are potent vasoconstrictive peptides originally isolated from vascular endothelial cells. Their biological effects are mediated through two different receptors, the endothelin-1 (ET-1)-selective endothelin receptor subtype ETA and the non-selective receptor subtype ETB. ET-1 protein has been found in human ovarian follicular fluid and ET-1 mRNA expression has been demonstrated in ovarian tissue. These findings indicate that the endothelin-system participates in the modulation of ovarian function, probably acting in an autocrine/paracrine manner. In the current study we used freshly aspirated, luteinized human granulosa cells (hGC) representing an in vitro model of the early corpus luteum. By means of RT-PCR and immunocytochemistry we investigated whether luteinized human granulosa cells express ETA and ETB receptors. Specific amplification products of ETA transcripts were detected in all samples investigated. In contrast, only after using a three-fold amount of ETB reverse transcripts we were able to demonstrate specific, but weak amplification products. In addition, immunocytochemical staining for ETA but not for ETB was found in granulosa cell preparations. The present study provides clear evidence that human granulosa cells predominantly express ETA receptor subtype mRNA and protein hinting to its possible role in follicle maturation and corpus luteum formation.