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 Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation

2006 ◽  
Vol 37 (2) ◽  
pp. 239-250 ◽  
Author(s):  
Khampoune Sayasith ◽  
Kristy A Brown ◽  
Jacques G Lussier ◽  
Monique Doré ◽  
Jean Sirois
Keyword(s):  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Growth Response ◽  
Cell Types ◽  
Early Growth ◽  
Response Factor ◽  
Coding Region ◽  
Rt Pcr ◽  
Early Growth Response ◽  
Preovulatory Follicles

Early growth response factor-1 (EGR-1) is a transcription factor that is involved in the transactivation of several genes. The objective of this study was to characterize gonadotropin-dependent regulation of bovine EGR-1 in preovulatory follicles prior to ovulation. Bovine EGR-1 cDNA was obtained by RT-PCR, 5′- and 3′-RACE, its open reading frame composed of 1623 bp, and its coding region encodes a 540-amino acid protein that displays 62–93% identity to known mammalian homologs. The regulation of EGR-1 mRNA was studied in bovine preovulatory follicles which were isolated 0–24 h post-hCG using semiquantitative RT-PCR/Southern blot. Results revealed that the levels of EGR-1 mRNA were very low in follicles at 0 h, markedly increased at 6 h (P < 0.05) when compared to 0 h, and decreased between 12 and 24 h post-hCG. High levels of the EGR-1 mRNA were also observed in corpus luteum, uterus, kidney, pituitary, and spleen, moderate and low in other bovine tissues tested. Analyses performed on isolated preparations of granulosa and theca cells indicated that EGR-1 mRNA was regulated in both cell types, with a predominant expression in granulosa cells. Immunohistochemistry on sections of preovulatory follicles isolated before and after hCG confirmed its protein expression in granulosa cells, 24 h post-hCG. Studies of EGR-1 regulation in primary granulosa cells cultured with forskolin showed that levels of EGR-1 mRNA were low at 0 h, highly increased at 6 h post-forskolin (P < 0.05), and declined to steady state thereafter. Immunoblotting confirmed forskolin-induced EGR-1 protein in cultures. Interestingly, overexpression of EGR-1 increased the levels of mRNA for prostaglandin (PG) G/H synthase-2 (PGHS-2), PG E synthase (PGES), PG E2 receptor (EP2), LH receptor (LH-R), but not for cytochrome P450-side chain cleavage (P450scc), and cytochrome P450 aromatase (P450arom) in granulosa cultures. Thus, this study reports for the first time, a gonadotropin-dependent induction of follicular EGR-1 prior to ovulation in large monoovulatory species and its stimulating effect on the expression of genes known to be involved in prostaglandin biosynthesis pathway, thereby suggesting its potential involvement in the regulation of preovulatory events in cattle.

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 &lt; 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.

A second expressed kininogen gene in mice

2006 ◽  
Vol 26 (2) ◽  
pp. 152-157 ◽  
Author(s):  
Edward G. Shesely ◽  
Chun-Bo Hu ◽  
François Alhenc-Gelas ◽  
Pierre Meneton ◽  
Oscar A. Carretero
Keyword(s):  
Molecular Weight ◽  
Open Reading Frame ◽  
Low Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Rt Pcr ◽  
Reading Frame ◽  
Rna Ligase ◽  
Mouse Tissues ◽  
Rapid Amplification ◽  
Race Pcr

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5′ ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

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 Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 969-981 ◽  
Author(s):  
Padmasana Singh ◽  
Amitabh Krishna ◽  
Rajagopala Sridaran
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Cell Types ◽  
Physiological Significance ◽  
Rt Pcr ◽  
Releasing Hormone ◽  
Theca Cells ◽  
Gonadotrophin Releasing Hormone

GnRH I and its receptors have been demonstrated in the ovaries of various vertebrates, but their physiological significance in reproductive cascade is fragmentary. Bradykinin is a potent GnRH stimulator in the hypothalamus. In the present study, the presence of GnRH I and its receptor, and bradykinin and its receptor in the ovaries of non-mammalian vertebrates were investigated to understand their physiological significance. GnRH I immunoreactivity in the ovaries of fish, frog, reptile and bird were mainly found in the oocyte of early growing follicles and granulosa cells and theca cells of previtellogenic follicles. Vitellogenic follicles showed mild GnRH immunoreactivity. GnRH I-receptor and bradykinin were localized in the same cell types of the ovaries of these vertebrates. The presence of GnRH I, GnRH I-receptor and bradykinin in the ovaries of these vertebrates was confirmed by immunoblotting. The presence of GnRH I mRNA was demonstrated in the ovary of vertebrates using RT-PCR. The ovaries of reptiles and birds showed significantly higher intensity of immunoreactivity for GnRH I-receptor as compared with the fish and amphibian. This may have a correlation with the higher yolk content in the ovary of reptile and bird. These results suggest the possibility of GnRH I and bradykinin as important regulators of follicular development and vitellogenesis in the vertebrate ovary.

scholarly journals Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 2782-2789 ◽  
Author(s):  
Ping Zhao ◽  
Ananya De ◽  
Zeng Hu ◽  
Jing Li ◽  
Sabine M. Mulders ◽  
...  
Keyword(s):  
Real Time ◽  
Granulosa Cells ◽  
Regulatory Protein ◽  
Rt Pcr ◽  
Paracrine Factors ◽  
Transcript Levels ◽  
Preovulatory Follicles ◽  
Major Increase ◽  
Steroidogenic Acute Regulatory ◽  
Stimulation Of

Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3β-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.

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 Peroxisome Proliferator-Activated Receptor γ Is a Target of Progesterone Regulation in the Preovulatory Follicles and Controls Ovulation in Mice

2008 ◽  
Vol 28 (5) ◽  
pp. 1770-1782 ◽  
Author(s):  
Jaeyeon Kim ◽  
Marcey Sato ◽  
Quanxi Li ◽  
John P. Lydon ◽  
Francesco J. DeMayo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Critical Role ◽  
Conditional Knockout ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Conditional Knockout Mouse ◽  
Preovulatory Follicles ◽  
Dependent Protein ◽  
Ovulatory Process

ABSTRACT The progesterone receptor (PR) plays a critical role during ovulation. Mice lacking the PR gene are anovulatory due to a failure in the rupture of the preovulatory follicles. The pathways that operate downstream of PR to control ovulation are poorly understood. Using gene expression profiling, we identified peroxisome proliferator-activated receptor γ (PPARγ) as a target of regulation by PR in the granulosa cells of the preovulatory follicles during the ovulatory process. To investigate the function of PPARγ during ovulation, we created a conditional knockout mouse in which this gene was deleted via Cre-Lox-mediated excision in granulosa cells. When these mutant mice were subjected to gonadotropin-induced superovulation, the preovulatory follicles failed to rupture and the number of eggs released from the mutant ovaries declined drastically. Gene expression analysis identified endothelin-2, interleukin-6, and cyclic GMP-dependent protein kinase II as novel targets of regulation by PPARγ in the ovary. Our studies also suggested that cycloxygenase 2-derived metabolites of long-chain fatty acids function as endogenous activating ligands of PPARγ in the preovulatory follicles. Collectively, these studies revealed that PPARγ is a key mediator of the biological actions of PR in the granulosa cells and activation of its downstream pathways critically controls ovulation.

scholarly journals Molecular characterization and regulation of the angiotensin-converting enzyme type 2/Angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle

2011 ◽  
Vol 13 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Joabel Tonellotto dos Santos ◽  
Rogério Ferreira ◽  
Bernardo Garziera Gasperin ◽  
Lucas Carvalho Siqueira ◽  
João Francisco de Oliveira ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Messenger Rna ◽  
Pcr Assay ◽  
Time Points ◽  
Mas Receptor ◽  
Preovulatory Follicles ◽  
Cytochrome P450 Aromatase ◽  
Luteinizing Hormone Surge ◽  
Ovulatory Process

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE2, NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE2, NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased ( p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE2, NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE2, NEP and PEP were differentially expressed after GnRH treatment ( p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE2, NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

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