scholarly journals Induction of Heparanase in Bovine Granulosa Cells by Luteinizing Hormone: Possible Role during the Ovulatory Process

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 413-421 ◽  
Author(s):  
Eyal Klipper ◽  
Ehud Tatz ◽  
Tatiana Kisliouk ◽  
Israel Vlodavsky ◽  
Uzi Moallem ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Luteinizing Hormone ◽  
Granulosa Cells ◽  
Prostaglandin F2α ◽  
Follicular Development ◽  
Mrna Levels ◽  
Ru 486 ◽  
Protein Levels ◽  
Gonadotropin Surge ◽  
Ovulatory Process

Follicular development, follicular rupture, and corpus luteum (CL) formation are accompanied by extensive tissue remodeling. We examined whether heparanase (HPSE), which cleaves heparan sulfate glycosaminoglycans, is induced during these processes. Prostaglandin F2α injection, which initiated luteolysis and the development of a preovulatory follicle, moderately increased HPSE mRNA in bovine granulosa cells (GCs). GnRH, used to induce gonadotropin surge, markedly augmented HPSE mRNA levels 12 h after its injection. The temporal pattern of HPSE gene expression in follicular-luteal transition was further examined in follicles collected before, and 4, 10, 20, 25, and 60 h after GnRH injection. HPSE mRNA increased transiently 10–20 h after GnRH injection to levels 10-fold higher than in untreated heifers. HPSE protein levels were similarly elevated 20 h after GnRH injection in GCs, but not in the theca layer. Cyclooxygenase-2 (PTGS2) mRNA peaked before ovulation when HPSE levels returned to baseline levels. HPSE mRNA abundance also remained low in the CLs. The antiprogesterone, RU-486, elevated HPSE levels in GC culture, suggesting that progesterone secreted by CLs may inhibit HPSE. HPSE immunostaining was more abundant in GCs than thecae. In cultured GCs, LH induced a transient increase in HPSE mRNA 3–6 h after its addition, but not at 24 h. However, PTGS2 mRNA was clearly induced at this time. These findings suggest that: 1) HPSE may play a role in ovulation but much less so during CL development, and 2) GC-derived HSPE may be a novel member of the LH-induced extracellular matrix-degrading enzyme family and may contribute to follicular rupture. Granulosa-derived heparanase is a novel member of the luteinizing hormone-induced extracellular matrix-degrading enzymes contributing to follicular rupture and ovulation.

scholarly journals Two pathways for prostaglandin F2α synthesis by the primate periovulatory follicle

Reproduction ◽  
2008 ◽  
Vol 136 (1) ◽  
pp. 53-63 ◽  
Author(s):  
Brandy L Dozier ◽  
Kikuko Watanabe ◽  
Diane M Duffy
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Prostaglandin F2α ◽  
Follicular Development ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Cell Conversion ◽  
Gonadotropin Surge ◽  
Cell Expression

Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2α levels in primate periovulatory follicles. To better understand the role of PGF2α in ovulation, pathways utilized for PGF2α synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40 h periovulatory interval. Human granulosa cells were also obtained (typically 34–36 h after hCG) from in vitro fertilization patients. PGF2α can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24–36 h after hCG administration. Human granulosa cells converted PGD2 into 11β-PGF2α, confirming that these cells possess AKR1C3 activity. PGF2α can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0–12 h, peaked at 24 h, and returned to low levels by 36 h after hCG administration. Human granulosa cell conversion of [3H]PGE2 into [3H]PGF2α was reduced by an AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2α can be synthesized via two pathways during the periovulatory interval.

scholarly journals ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells

2017 ◽  
Vol 312 (3) ◽  
pp. C341-C353 ◽  
Author(s):  
Yongjie Xiong ◽  
Huatao Chen ◽  
Pengfei Lin ◽  
Aihua Wang ◽  
Lei Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Er Stress ◽  
Granulosa Cells ◽  
Physiological Function ◽  
Steroid Hormone ◽  
Follicular Development ◽  
S Phase ◽  
Mrna Levels ◽  
Hormone Production ◽  
Protein Levels

Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 ( Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein 4 ( Igfbp4) expression and decreased hyaluronan synthase 2 ( Has2), prostaglandin-endoperoxide synthase 2 ( Ptgs2), and prostaglandin F receptor ( Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.

scholarly journals Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Reproduction ◽  
2004 ◽  
Vol 128 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Rachel C Hirst ◽  
Margaret H Abel ◽  
Vivienne Wilkins ◽  
Christine Simpson ◽  
Phil G Knight ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Inhibin B ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Α Subunit ◽  
Lh Receptor ◽  
Protein Levels ◽  
Subunit Mrna ◽  
Major Cell Type

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHβ knockout (FSHβKO) females where disruption of the gene encoding FSHβ results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin α subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the βA and βB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the βA and βB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin βA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin α and βB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

scholarly journals BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

2013 ◽  
Vol 27 (12) ◽  
pp. 2093-2104 ◽  
Author(s):  
Hsun-Ming Chang ◽  
Jung-Chien Cheng ◽  
Christian Klausen ◽  
Peter C. K. Leung
Keyword(s):  
Granulosa Cells ◽  
Critical Role ◽  
Regulatory Protein ◽  
Granulosa Cell Tumor ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Granulosa Cells ◽  
Progesterone Production ◽  
Steroidogenic Acute Regulatory

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

scholarly journals Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 463-472 ◽  
Author(s):  
Takashi Shimizu ◽  
Izumi Ohshima ◽  
Manabu Ozawa ◽  
Satoko Takahashi ◽  
Atsushi Tajima ◽  
...  
Keyword(s):  
Heat Stress ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Receptor Expression ◽  
Female Rats ◽  
Aromatase Activity ◽  
Mrna Levels ◽  
Gonadotropin Receptor ◽  
Estradiol Levels

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 °C, 50% RH; heat stress: 35 °C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.

509. REGULATING MURINE FOLLICLE DEVELOPMENT BY STIMULATION OF THE JAK2/STAT3 SIGNAL TRANSDUCTION PATHWAY

2009 ◽  
Vol 21 (9) ◽  
pp. 108
Author(s):  
R. A. Keightley ◽  
B. Nixon ◽  
S. D. Roman ◽  
D. L. Russell ◽  
R. L. Robker ◽  
...  
Keyword(s):  
Significant Role ◽  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Expression Patterns ◽  
Follicular Development ◽  
Janus Kinase ◽  
Follicle Development ◽  
Mrna Levels ◽  
Primordial Follicles ◽  
Stat3 Signalling

Follicular development requires the recruitment of primordial follicles into the growing follicle pool following initiation of multiple cytokine signalling pathways. Suppression of follicular development is thought to be key to maintaining the population of primordial follicles and allowing for controlled release of these follicles throughout the reproductive lifespan of the female. However, little is known of the processes and signalling molecules that suppress primordial follicle activation and early follicle growth. Our group has identified significant upregulation of the Janus Kinase 2 (JAK2)/ Signal Transducer and Activator of Transcription 3 (STAT3) signalling pathway inhibitor the Suppressor of Cytokine Signalling 4 (SOCS4) that coincides with the initial wave of follicular activation in theneonatal mouse ovary. Further studies by our group have localised the SOCS4 protein to the granulosa cells of activating and growing follicles, suggesting SOCS4 expression may be linked to follicular activation. We have focused on examining protein localisation and gene expression patterns of the eight SOCS family members CIS and SOCS1-7. We have recently demonstrated that co-culture of neonatal ovaries with Kit Ligand (KL) for 2 days increases the mRNA levels of all SOCS genes. We also demonstrated the co-localisation of SOCS2 proteins with the KL receptor c-kit in the mural granulosa cells of antral, and large pre-antral follicles suggesting a significant role for SOCS2 in the later stages of follicular development. We have also shown that culturing ovaries with the potent JAK2 inhibitor AG490 substantially reduces mRNA levels of all SOCS and STAT genes that we have so far measured. We hypothesise a significant role for JAK2/STAT3 signalling in promoting the activation and early growth of ovarian follicles. Our investigations have identified significant roles for JAK2/STAT3 and the SOCS family in the regulation of ovarian follicle development.

scholarly journals Activin B is produced early in antral follicular development and suppresses thecal androgen production

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 637-650 ◽  
Author(s):  
J M Young ◽  
S Henderson ◽  
C Souza ◽  
H Ludlow ◽  
N Groome ◽  
...  
Keyword(s):  
Follicular Development ◽  
Activin A ◽  
Mrna Levels ◽  
Negative Effects ◽  
Inhibin A ◽  
Protein Levels ◽  
Follicle Diameter ◽  
Preovulatory Follicles ◽  
Follicle Size

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1–3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3–4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1–3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

187 EXPRESSION AND REGULATION OF THE FIBROBLAST GROWTH FACTOR GENE FAMILY DURING MOUSE FOLLICULAR DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 243
Author(s):  
S. Furukawa ◽  
K. Naito ◽  
K. Sugiura
Keyword(s):  
Gene Family ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
Mouse Ovary ◽  
Expression Levels ◽  
Before And After ◽  
Equine Chorionic Gonadotropin

Recent studies have shown the critical roles of fibroblast growth factors (FGFs), including FGF8 produced by oocytes, in regulating follicular development. However, the expression and regulation of the FGF gene family, which consists of 22 ligands and 4 receptors, in the mouse ovary have not been well understood. The aim of the present study was to assess the expression and regulation of FGF ligands and receptors in the mouse ovary. Transcript levels of FGF ligands and receptors in immature (3-week-old) and adult (7- to 8-week-old) ovaries as well as other tissues of B6/DBA2F1 mice were analysed with RT-PCR. Furthermore, expression levels of FGF receptors in cumulus cells (CC) and mural granulosa cells (MG) before and after equine chorionic gonadotropin (eCG) treatment were determined with RT-quantitative PCR. Among 21 FGF ligands examined, 12 and 9 transcripts were detectable in immature and adult ovaries, respectively. More FGF ligands were detected in ovary, testis, heart, and brain compared to other tissues, including liver and spleen. Transcripts of all 4 FGF receptors (Fgfr1–4) were detectable in both immature and adult ovaries. Expression levels of Fgfr1 and Fgfr2 were significantly higher in MG compared with CC before and after the eCG treatment. Levels of Fgfr4 were comparable between MG and CC before the eCG treatment, but became significantly different with higher expression levels in MG after the eCG treatment. Fgfr3 transcripts were barely detectable in CC and MG. Overall levels of Fgfr1 in granulosa cells (CC and MG) were downregulated by eCG treatment, whereas those of Fgfr2 and Fgfr4 were upregulated. In summary, many FGF ligands are expressed, at least in mRNA levels, in mouse ovaries. Moreover, the expression levels of Fgfr transcripts in granulosa cells are dynamically regulated during follicular development.

226. Expression of matrix metalloproteinases in bovine thecal cells

2005 ◽  
Vol 17 (9) ◽  
pp. 88
Author(s):  
L. Harland ◽  
H. F. Irving-Rodgers ◽  
S. E. Morris ◽  
R. J. Rodgers
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteinases ◽  
Phorbol Ester ◽  
Granulosa Cells ◽  
Gelatin Zymography ◽  
Mrna Levels ◽  
Cell Culture Systems ◽  
Thecal Cells ◽  
Theca Interna ◽  
Free Media

As follicles grow the thecal layers expand. It is likely that extracellular matrix is remodelled in this process and possibly by matrix metalloproteinases (MMPs). A promising candidate to regulate MMPs is insulin-like factor 3 (INSL3). It is produced by thecal cells, its receptor, LGR8, is expressed in the theca interna (unpublished) and a related molecule, relaxin, regulates turnover of matrix in a number of tissues. For this reason we sort to examine the role of INSL3 in matrix turnover. However, in all thecal cell culture systems examined LGR8 receptors appear to be down regulated within 24 h. We therefore examined the effects of second messenger pathway activators. Thecal and granulosa cells were isolated and cultured and the levels of RNA for MMP2 and 9 quantitated by RT-PCR. MMP2 mRNA levels in thecal tissues were >10 fold higher than in granulosa cells (n = 19 follicles >10 mm). MMP2 levels were substantially greater than MMP9. At 12 h phorbol ester (100 nM phorbol 12,13-didecanoate) increased thecal expression of MMP 9 mRNA levels 11.5 fold (P < 0.001) and at 48 h MMP2 mRNA was increased 5 fold (P < 0.01). Pieces of whole follicle wall [follicles <5 mm in diameter, classified as healthy (n = 12) or atretic (n = 6)] were cultured in serum free media. Expression of the steroidogenic enzymes 17β HSD and P450scc but not 3β HSD were detected by immunohistochemistry even after 10 days. MMP activity on day 2 was analysed by gelatin zymography. Treatment with phorbol ester increased active MMP9 19 fold (P < 0.001). Treatment of thecal cells or follicle walls with 1 mM dibutyryl cAMP induced additional MMP activities at sizes of 110 and 122kDa. No effects on MMP2 activity were observed. In conclusion whilst we do not know the ligand inducers of the synthesis and activator of MMPs in thecal cells they can be regulated. Hence MMPs are candidates for remodelling the extracellular matrix of thecal layers.

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