Secretion of 17α-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

1987 ◽  
Vol 65 (9) ◽  
pp. 1951-1956 ◽  
Author(s):  
Benjamin K. Tsang ◽  
Arezoo Taheri ◽  
Louis Ainsworth ◽  
Bruce R. Downey
Keyword(s):  
Granulosa Cells ◽  
Cell Types ◽  
High Yield ◽  
Aromatase Activity ◽  
Steroid Substrate ◽  
Preovulatory Follicles ◽  
Serum Gonadotropin ◽  
Exogenous Progesterone ◽  
Theca Interna ◽  
Estradiol 17Β

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10−8 to 10−5 M progesterone, 17α-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 μM, an inhibitor of 3β-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17α-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17α-hydroxyprogesterone and androstenedione, and exogenous 17α-hydroxyprogesterone to androstenedione and estradiol-17β in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17β than did granulosa cells in the absence of aromatizable substrate, but estradiol-17β secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17α-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.

scholarly journals A Novel Pathway Involving Progesterone Receptor, Endothelin-2, and Endothelin Receptor B Controls Ovulation in Mice

2006 ◽  
Vol 20 (11) ◽  
pp. 2784-2795 ◽  
Author(s):  
Gopinath S. Palanisamy ◽  
Yong-Pil Cheon ◽  
Jaeyeon Kim ◽  
Athilakshmi Kannan ◽  
Quanxi Li ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Critical Role ◽  
Cell Types ◽  
Downstream Target ◽  
Preovulatory Follicles ◽  
Dependent Protein ◽  
Multiple Cell ◽  
B Mice ◽  
Theca Interna

Abstract The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.

Expression and function of brain-derived neurotrophin factor and its receptor, TrkB, in ovarian follicles from the domestic hen (Gallus gallus domesticus)

2001 ◽  
Vol 204 (12) ◽  
pp. 2087-2095 ◽  
Author(s):  
T. Jensen ◽  
A. L. Johnson
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Layer ◽  
Preliminary Evidence ◽  
Neurotrophin Receptor ◽  
Bdnf Mrna ◽  
Preovulatory Follicles ◽  
Layer Increase ◽  
Theca Interna ◽  
And Function

SUMMARY This report summarizes patterns of mRNA expression for the brain-derived neurotrophic factor (BDNF) together with its high-affinity neurotrophin receptor trkB within the hen ovary during follicle development, describes hormonal mechanisms for the regulation of trkB gene expression and provides preliminary evidence for a novel function for BDNF-mediated TrkB signaling within the granulosa layer. Levels of BDNF mRNA in the thecal layer and of trkB mRNA within the granulosa cell layer increase coincident with entrance of the follicle into the preovulatory hierarchy. Localization of the BDNF mRNA transcript correlates with expression of BDNF protein within the theca interna of preovulatory follicles, while localization of trkB mRNA and protein occurs extensively within the granulosa cell layer of preovulatory follicles. This pattern of expression suggests a paracrine relationship between theca and granulosa cells for BDNF signaling via TrkB. Vasoactive intestinal peptide and gonadotropin treatments stimulate increases in levels of trkB mRNA within cultured granulosa cells derived from both prehierarchal and preovulatory follicles, and this response is increased by co-treatment with 3-isobutyl-1-methylxanthine. Finally, BDNF treatment of cultured granulosa cells from preovulatory follicles results in a modest, but significant, reduction in basal progesterone production, whereas this effect was reversed by k252a, an inhibitor of Trk kinase activity. These results support the proposals that BDNF functions as a paracrine signal in hen granulosa cells and that its physiological functions may include the modulation of steroidogenesis.

Tokishakuyakusan Stimulates Progesterone and Estradiol-17 β Production by Rat Granulosa Cells and Progesterone, Testosterone and Estradiol-17 β by the Residual Portion of the Follicle in Vitro

1991 ◽  
Vol 19 (02) ◽  
pp. 155-161 ◽  
Author(s):  
Satoshi Usuki
Keyword(s):  
Granulosa Cells ◽  
Cell Level ◽  
Testosterone Production ◽  
Progesterone Production ◽  
Preovulatory Follicles ◽  
Immature Rats ◽  
Serum Gonadotropin ◽  
Tissue Compartments ◽  
Stimulation Of

To examine the possible effects of Tokishakuyakusan (TS) on steroidogenesis by preovulatory follicles at the cell level, the expressed granulosa cells and remaining portion of follicles from pregnant mare's serum gonadotropin (PMS)-treated immature rats were incubated in vitro with increasing concentrations of TS for 3 h. TS significantly stimulated progesterone and estradiol-17 b production, with a predominant stimulation of progesterone, by the expressed granulosa cells, while testosterone production was not stimulated. In the remaining portion of the follicle, TS also significantly stimulated progesterone, testosterone and estradiol-17 b production. Similar to the effect produced by granulosa cells, the stimulatory effect of TS was stronger on progesterone than on testosterone and estradiol-17 b production. These results suggest that TS has a potent, direct stimulatory effect on steroidogenesis, especially progesterone production, by constituent tissue compartments of rat preovulatory follicles in vitro.

scholarly journals Human Chorionic Gonadotropin-Dependent Up-Regulation of Genes Responsible for Estrogen Sulfoconjugation and Export in Granulosa Cells of Luteinizing Preovulatory Follicles

Endocrinology ◽  
2006 ◽  
Vol 147 (9) ◽  
pp. 4222-4233 ◽  
Author(s):  
Kristy A. Brown ◽  
Monique Doré ◽  
Jacques G. Lussier ◽  
Jean Sirois
Keyword(s):  
Chorionic Gonadotropin ◽  
Granulosa Cells ◽  
Human Chorionic Gonadotropin ◽  
Cell Layer ◽  
Rt Pcr ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Steady State Levels ◽  
17Β Estradiol ◽  
Theca Interna

Estrogen sulfotransferase (EST) is responsible for the sulfoconjugation of estrogens, thereby changing their physical properties and preventing their action via the estrogen receptors. These sulfoconjugated steroids no longer diffuse freely across the lipid bilayer; instead, they are exported by members of the ATP-binding cassette family, such as ABCC1. The objective of this study was to investigate the regulation of EST and ABCC1 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The transcripts for EST and ABCC1 were cloned by RT-PCR, and the regulation of their mRNAs was studied in preovulatory follicles obtained during estrus at 0, 12, 24, 30, 33, 36, and 39 h after hCG. Results obtained from RT-PCR/Southern blot analyses showed significant changes in steady-state levels of both EST and ABCC1 mRNA after hCG treatment (P < 0.05). In granulosa cells, a significant increase in EST transcript was observed 30–39 h after hCG. Similarly, ABCC1 transcript levels were induced in granulosa cells 12–39 h after hCG. In contrast, no significant changes in either EST or ABCC1 were detected in theca interna samples after hCG. The increase in EST and ABCC1 transcripts observed in granulosa cells was reflected in preparations of intact follicle walls, suggesting that the granulosa cell layer contributes the majority of EST and ABCC1 expression in preovulatory follicles. The present study demonstrates that follicular luteinization is accompanied not only by a decrease in 17β-estradiol biosynthesis but also by an increase in expression of genes responsible for estrogen inactivation and elimination from granulosa cells, such as EST and ABCC1, respectively.

scholarly journals Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles

2003 ◽  
Vol 31 (3) ◽  
pp. 473-485 ◽  
Author(s):  
D Boerboom ◽  
DL Russell ◽  
JS Richards ◽  
J Sirois
Keyword(s):  
Progesterone Receptor ◽  
Chorionic Gonadotropin ◽  
Granulosa Cells ◽  
Human Chorionic Gonadotropin ◽  
Reproductive Tract ◽  
Pcr Analysis ◽  
Preovulatory Follicles ◽  
Theca Interna ◽  
A Disintegrin And Metalloproteinase ◽  
Ovulatory Process

One member of a new family of metalloproteinases, a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS-1), has been found to be expressed and hormonally induced in granulosa cells of ovulating rodent follicles. Furthermore, the targeted disruption of the ADAMTS-1 gene resulted in ovarian defects associated with severely impaired fertility. While these data demonstrate the importance of ADAMTS-1 in rodent ovarian physiology, the potential role of ADAMTS-1 in the ovulatory process of monoovulatory species remains unknown. The objectives of this study were to clone the equine ADAMTS-1 primary transcript and to study its regulation during human chorionic gonadotropin (hCG)-induced ovulation. A 3573 bp follicular cDNA library clone was isolated and found to encode a nearly complete, highly conserved ADAMTS-1 homologue. Real-time RT-PCR analysis detected this transcript in diverse tIssues, including previously unreported sites of ADAMTS-1 expression such as the male reproductive tract, the follicular theca interna and the mature corpus luteum. The tIssue distribution of the progesterone receptor (PR), a known regulator of ADAMTS-1 expression in rodent preovulatory follicles, was found to overlap that of ADAMTS-1 in some tIssues. A study of the regulation of follicular ADAMTS-1 and PR mRNAs during the hCG-induced ovulatory process revealed distinct patterns of regulation in granulosa cells and in theca interna. In granulosa cells, ADAMTS-1 mRNA was found to be induced at 12 h post-hCG (P<0.05), followed by a return to basal levels by 30 h and a re-increase at 33-39 h (P<0.05). A concomitant increase in PR mRNA (P<0.05) was observed at 12 h post-hCG. In theca interna, abundant ADAMTS-1 mRNA was detected at all timepoints, and levels increased transiently at 33 h post-hCG (P<0.05), whereas no significant change was observed in PR mRNA. Together, these data demonstrate for the first time the hormonally regulated ovarian expression of ADAMTS-1 in a monoovulatory species, and identify a novel biphasic regulation of ADAMTS-1 in granulosa cells and a regulated expression in theca interna that were not previously observed in rodents.

scholarly journals Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 455-466 ◽  
Author(s):  
Khampoune Sayasith ◽  
Nadine Bouchard ◽  
Monique Doré ◽  
Jean Sirois
Keyword(s):  
Granulosa Cells ◽  
Cell Types ◽  
Primate Species ◽  
Rt Pcr ◽  
Reading Frame ◽  
Preovulatory Follicles ◽  
Prostaglandin Dehydrogenase ◽  
Rapid Amplification ◽  
Species Specific ◽  
Ovulatory Process

The mammalian ovulatory process is accompanied by a gonadotropin-dependent increase in follicular levels of prostaglandin E2 (PGE2) and PGF2α, which are metabolized by 15-hydroxy prostaglandin dehydrogenase (PGDH). Little is known about ovarian PGDH regulation in non-primate species. The objectives of this study were to characterize the structure of equine PGDH and its regulation in follicles during human chorionic gonadotropin (hCG)-induced ovulation. The full-length equine PGDH was obtained by RT-PCR, 5′- and 3′-rapid amplification of cDNA ends (RACE). Its open reading frame encodes a 266-amino acid protein that is 72–95% homologous to other species. Semi-quantitative RT-PCR/Southern blot were used to study PGDH regulation in follicles isolated 0–39 h post-hCG. Results showed that PGDH mRNA expression was low in follicles obtained at 0 h, increased at 12 and 24 h (P< 0.05), and decreased at 36-h post-hCG. This induction of expression was biphasic, with elevated abundance of transcripts at 12 and 33 h post-hCG (P< 0.05) in mural granulosa and theca cells. Immunohistochemistry and immunoblotting confirmed regulated expression of PGHD protein in both cell types of preovulatory follicles after hCG. High levels of PGDH mRNA were observed in corpus luteum and other non-ovarian tissues tested, except kidney, muscle, brain, and heart. Thus, this study is the first to report the gonadotropin-dependent regulation of PGDH during ovulation in a non-primate species. PGDH induction was biphasic in theca and mural granulosa cells differing from primates in which this induction was monophasic and limited to granulosa cells, suggesting species-specific differences in follicular control of PGDH expression during ovulation.

scholarly journals Gonadotropin-dependent regulation of bovine pituitary adenylate cyclase-activating polypeptide in ovarian follicles prior to ovulation

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 441-453 ◽  
Author(s):  
Khampoune Sayasith ◽  
Kristy A Brown ◽  
Jean Sirois
Keyword(s):  
Adenylate Cyclase ◽  
Granulosa Cells ◽  
Cell Types ◽  
Rt Pcr ◽  
Reading Frame ◽  
Chain Cleavage ◽  
Acid Protein ◽  
Pituitary Adenylate Cyclase ◽  
Preovulatory Follicles ◽  
Ep2 Receptor

To study the regulation of bovine pituitary adenylate cyclase-activating polypeptide (PACAP) in preovulatory follicles prior to ovulation, PACAP cDNA was isolated by RT-PCR. Its open reading frame (ORF) is composed of 531 bp, and encodes for a 176-amino acid protein that bears 76–90% identity with other PACAP homologs. Using bovine preovulatory follicles obtained between 0 and 24 h after human chorionic gonadotropin (hCG) and semiquantitative RT-PCR/Southern blot, we demonstrate that levels of PACAP mRNA were low at 0 h, markedly increased at 6 and 12 h (P<0.05), and declined 18 and 24 h after hCG. Levels of PACAP mRNA were high in the bovine pituitary, testis, intestine and uterus, but moderate to low in other tissues. Analyses performed on isolated preparations of granulosa and theca cells showed a significant increase of PACAP transcripts in both cell types after hCG, whereas primary granulosa cell cultures revealed high levels of PACAP as well as its receptors PAC-1 and VPAC-2 mRNA after forskolin treatment. Overexpression of the catalytic subunit of protein kinase A (PKA) in granulosa cells stimulated, but treatment with H89 or PKA inhibitor protein inhibited PACAP mRNA expression, whereas PACAP overexpression stimulated an increase in abundance of transcripts for PGHS-2, PGES, EP2 receptor, progesterone receptor, and ADAMTS-1, but not for P450-side chain cleavage and P450 aromatase. Thus, this study demonstrates the gonadotropin-dependent regulation of PACAP mRNA in bovine preovulatory follicles, the importance of PKA activation in the expression of PACAP in granulosa cells, and stimulating effect of PACAP on gene expression during the ovulatory process.

scholarly journals Cyclic Guanosine 5′-Monophosphate-Dependent Protein Kinase II Is Induced by Luteinizing Hormone and Progesterone Receptor-Dependent Mechanisms in Granulosa Cells and Cumulus Oocyte Complexes of Ovulating Follicles

2006 ◽  
Vol 20 (2) ◽  
pp. 348-361 ◽  
Author(s):  
Venkataraman Sriraman ◽  
Michael D. Rudd ◽  
Suzanne M. Lohmann ◽  
Sabine M. Mulders ◽  
JoAnne S. Richards
Keyword(s):  
Protein Kinase ◽  
Progesterone Receptor ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Dependent Protein Kinase ◽  
Pregnant Mare Serum Gonadotropin ◽  
Preovulatory Follicles ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Serum Gonadotropin

Abstract Cyclic GMP (cGMP)-dependent protein kinase II (Prkg2, cGK II) was identified as a potential target of the progesterone receptor (Nr3c3) in the mouse ovary based on microarray analyses. To document this further, the expression patterns of cGK II and other components of the cGMP signaling pathway were analyzed during follicular development and ovulation using the pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-primed immature mice. Levels of cGK II mRNA were low in ovaries of immature mice, increased 4-fold in response to pregnant mare serum gonadotropin and 5-fold more within 12 h after hCG, the time of ovulation. In situ hybridization localized cGK II mRNA to granulosa cells and cumulus oocyte complexes of periovulatory follicles. In progesterone receptor (PR) null mice, cGK II mRNA was reduced significantly at 12 h after hCG in contrast to heterozygous littermates. In primary granulosa cell cultures, cGK II mRNA was induced by phorbol 12-myristate 13-acetate enhanced by adenoviral expression of PR-A and blocked by RU486 and trilostane. PR-A in the absence of phorbol 12-myristate 13-acetate was insufficient to induce cGK II. Expression of cGK I (Prkg1) was restricted to the residual tissue and not regulated by hormones. Guanylate cyclase-A (Npr1; GC-A) mRNA expression increased 6-fold by 4 h after hCG treatment in contrast to pregnant mare serum gonadotropin alone and was localized to granulosa cells of preovulatory follicles. Collectively, these data show for the first time that cGK II (not cGK I) and GC-A are selectively induced in granulosa cells of preovulatory follicles by LH- and PR-dependent mechanisms, thereby providing a pathway for cGMP function during ovulation.

scholarly journals Proliferation of Rat Granulosa Cells during the Periovulatory Interval

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 414-422 ◽  
Author(s):  
Jennifer D. Cannon ◽  
Mary Cherian-Shaw ◽  
Charles L. Chaffin
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cyclin E ◽  
Ovarian Follicle ◽  
Terminal Differentiation ◽  
Cell Types ◽  
Cyclin D2 ◽  
Protein Levels ◽  
Serum Gonadotropin

Granulosa cell proliferation during luteinization and terminal differentiation has historically been assumed to decline rapidly after an ovulatory stimulus. In contrast, terminal differentiation in other cell types has recently been associated with a transient increase in proliferation, suggesting that this may occur in the ovarian follicle. The goal of the current study was to test the hypothesis that an ovulatory stimulus to rats results in additional granulosa cell proliferation before cell cycle arrest. Immature rats were given a single injection of pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) to initiate periovulatory events. The proportion of granulosa cells in S phase did not change until 12 h after hCG, although the majority of the post-hCG proliferation was localized to cumulus granulosa cells for up to 10 h after hCG. The expression of cyclin D2 mRNA did not decline until 12 h after hCG, although both cyclin-dependent kinase (Cdk)4 and Cdk6 mRNA increased at 6 h. Protein levels of cyclin D2 and Cdk4 did not change as a result of hCG, whereas cyclin E increased 6 h after hCG. Kinase activity of Cdk2 dropped markedly by 4 h after hCG, but a slight increase in activity was evident 6–8 h after hCG. These data suggest that cumulus granulosa cells continue to proliferate for up to 10 h after an ovulatory stimulus, possibly via cyclin E/Cdk2. It is concluded that proliferation is maintained in granulosa cells in the proximity of the oocyte during luteinization of the rat follicle.

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