Cloning and Characterization of a Rat Ortholog of MMP-23 (Matrix Metalloproteinase-23), a Unique Type of Membrane-Anchored Matrix Metalloproteinase and Conditioned Switching of Its Expression during the Ovarian Follicular Development

Abstract In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.

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Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry

Reproduction ◽

10.1530/rep.0.1230363 ◽

2002 ◽

pp. 363-369 ◽

Cited By ~ 83

Author(s):

CJ Souza ◽

BK Campbell ◽

AS McNeilly ◽

DT Baird

Keyword(s):

Granulosa Cells ◽

Polyclonal Antibodies ◽

Follicular Development ◽

Ovarian Surface Epithelium ◽

Bone Morphogenetic Protein 2 ◽

Antigen Retrieval ◽

Surface Epithelium ◽

Inhibin A ◽

Bmp Receptors

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.

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Estrogen Receptor Alpha (ER-α) and Beta (ER-β) mRNAs in Normal Ovary, Ovarian Serous Cystadenocarcinoma and Ovarian Cancer Cell Lines: Down-Regulation of ER-β in Neoplastic Tissues

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.3.4788 ◽

1998 ◽

Vol 83 (3) ◽

pp. 1025-1028 ◽

Cited By ~ 4

Author(s):

Alfred W. Brandenberger ◽

Meng Kian Tee ◽

Roberts B. Jaffe

Keyword(s):

Ovarian Cancer ◽

Estrogen Receptor ◽

Granulosa Cells ◽

Ovarian Cancer Cell ◽

Ovarian Surface Epithelium ◽

Surface Epithelium ◽

Mrna Levels ◽

Rt Pcr ◽

Low Level ◽

Ovarian Cancer Cell Lines

The prognosis in ovarian carcinoma, the most lethal of the gynecologic neoplasms, is poor and has changed little in the last three decades. Only a small number respond to antiestrogen therapy, although the classic estrogen receptor, ER-α, has been identified in ovarian surface epithelium, from which approximately 90% of ovarian cancers originate. We have previously shown that ER-β mRNA is most abundant in human fetal ovaries, suggesting that it might play an important role in ovarian development. Therefore, we investigated the mRNA levels of both ERs in normal ovaries, ovarian serous cystadenocarcinomas, granulosa cells from patients undergoing in vitro fertilization (IVF), the ovarian surface epithelium cell line IOSE-Van, and the ovarian cancer cell lines SKOV3, HEY and OCC1. Northern blots of normal and neoplastic ovaries were hybridized with an ER-β riboprobe that spans the A/B domain. We detected two major hybridizing bands at approximately 8 and 10 kb. An RNase protection assay using the same probe revealed a single band of the expected size. Hybridizing the same blot with an ER-α riboprobe showed a strong hybridizing band at approximately 6.5 kb. In ovarian cancer samples, ER-β mRNA level was decreased when compared to normal ovaries. Using 25 cycles of RT-PCR followed by Southern blotting, we found equal amounts of ER-α and -β mRNAs in normal ovaries in all age groups from 33 to 75 years; however, in ovarian cancer tissue, the level of ER-α mRNA was similar or slightly higher, comparable to 103 to 104 copies of plasmid DNA, but ER-β mRNA levels were markedly decreased. Granulosa cells from IVF patients expressed high levels of ER-β mRNA. The OSE cell line expressed low level of ER-α, detectable after 40 cycles of RT-PCR and no ER-β mRNA. SKOV3, showed low level of ER-α and β mRNAs, whereas OCC1 showed low level of ER-β and relatively high level of ER-α. HEY did not contain detectable amounts of either ER after 40 cycles of RT-PCR. We found no evidence of differential splicing or major deletions in almost the entire coding region of ER-β in either normal ovaries or tumor samples.

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

Reproduction ◽

10.1530/rep.1.00502 ◽

2005 ◽

Vol 129 (4) ◽

pp. 463-472 ◽

Cited By ~ 39

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.

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Distribution of 125I-labelled follicle-stimulating hormone and human chorionic gonadotrophin in the gonads of hypogonadal (hpg) mice

Journal of Endocrinology ◽

10.1677/joe.0.0930247 ◽

1982 ◽

Vol 93 (2) ◽

pp. 247-NP ◽

Cited By ~ 9

Author(s):

H. M. Charlton ◽

Dilys Parry ◽

D. M. G. Halpin ◽

R. Webb

Keyword(s):

Granulosa Cells ◽

Follicular Development ◽

Interstitial Cells ◽

Chorionic Gonadotrophin ◽

Human Chorionic Gonadotrophin ◽

Testicular Development ◽

Similar Response ◽

Thecal Cells ◽

Lh Rh ◽

Lh Releasing Hormone

Hypogonadal mice are deficient in LH releasing hormone (LH-RH), the releasing factor for LH and FSH, with a consequent failure of postnatal ovarian and testicular development. After intravenous injection of hypogonadal females with 125I-labelled human chorionic gonadotrophin (hCG), followed by autoradiography of semi-thin (1 μm) slices of the ovary, labelled hCG was found to be associated with interstitial cells and thecal cells with little or no labelling of granulosa cells. Labelled human FSH was associated solely with granulosa cells. Hypogonadal females, implanted for 5 days with a silicone elastomer capsule of oestrogen, showed a similar response to that of normal females with hCG labelling of the granulosa cells of the larger follicles as well as of the thecal cell layer. Furthermore, subcutaneous injection of hypogonadal females with LH-RH (50 ng), 12 times daily for 5 days, increased uterine weight and stimulated ovarian development with some large follicles binding hCG to both thecal and granulosa cells. Therefore stimulation of follicular development may possibly be associated with increased oestradiol concentrations. In the male, after injection of 125I-labelled hCG, silver grains were associated with the interstitial cells alone in both hypogonadal and normal mice. Labelled human FSH was undetectable in semi-thin testicular sections, but the mode of injection (intravenous) may not have allowed enough labelled hormone to reach the testis in order to resolve the question as to whether the hypogonadal or normal testis can bind FSH.

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

Reproduction Fertility and Development ◽

10.1071/srb09abs509 ◽

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.

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Role of Gonadotropin-Releasing Hormone as an Autocrine Growth Factor in Human Ovarian Surface Epithelium1

Endocrinology ◽

10.1210/endo.141.1.7250 ◽

2000 ◽

Vol 141 (1) ◽

pp. 72-80 ◽

Cited By ~ 44

Author(s):

Sung Keun Kang ◽

Kyung-Chul Choi ◽

Kwai Wa Cheng ◽

Parimal S. Nathwani ◽

Nelly Auersperg ◽

...

Keyword(s):

Gnrh Agonist ◽

Messenger Rna ◽

Ovarian Surface Epithelium ◽

Inhibitory Effect ◽

Surface Epithelium ◽

Mrna Levels ◽

Dependent Manner ◽

Rt Pcr ◽

Ovarian Surface ◽

Ovarian Surface Epithelial

Abstract Epithelial ovarian cancer, which accounts for 80–90% of all ovarian cancers, is the most common cause of death from gynecological malignancies and is believed to originate from the ovarian surface epithelium. In the present study we investigated the expression of GnRH and its receptor in human ovarian surface epithelial (hOSE) cells and provided novel evidence that GnRH may have antiproliferative effects in this tissue. Using RT-PCR and Southern blot analysis, we cloned the GnRH and GnRH receptor (GnRHR) in hOSE cells. Sequence analysis revealed that GnRH and its receptor have sequences identical to those found in the hypothalamus and pituitary, respectively. To address whether GnRH regulates its own and receptor messenger RNA (mRNA), the cells were treated with different concentrations of the GnRH agonist (d-Ala6)-GnRH. Expression levels of GnRH and its receptor were investigated using quantitative and competitive RT-PCR, respectively. Interestingly, a biphasic effect was observed for the GnRH and GnRHR mRNA levels. High concentrations of the GnRH agonist (10−7 and 10−9m) decreased GnRH and GnRHR mRNA levels, whereas a low concentration (10−11m) resulted in up-regulation of GnRH and receptor mRNA levels. Treatment with the GnRH antagonist, antide, prevented the biphasic effects of the GnRH agonist in hOSE cells, confirming the specificity of the response. Furthermore, to investigate the physiological significance, we studied receptor-mediated growth regulatory effects of GnRH in human ovarian surface epithelial cells. The cells were treated with GnRH analogs, and the proliferative index of cells was measured using a [3H]thymidine incorporation assay. (d-Ala6)-GnRH had a direct inhibitory effect on the growth of hOSE cells in a time- and dose-dependent manner. This antiproliferative effect of the GnRH agonist was receptor mediated, as cotreatment of hOSE cells with antide abolished the growth inhibitory effects of the GnRH agonist. The results strongly suggest that GnRH can act as an autocrine/paracrine regulator in hOSE cells.

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Spatiotemporal changes in cytokeratin expression in the neonatal rat ovary

Biochemistry and Cell Biology ◽

10.1139/o98-002 ◽

1998 ◽

Vol 76 (1) ◽

pp. 27-35 ◽

Cited By ~ 10

Author(s):

Jie Pan ◽

Nelly Auersperg

Keyword(s):

Granulosa Cells ◽

Zona Pellucida ◽

Basal Membrane ◽

Ovarian Surface Epithelium ◽

Hydroxysteroid Dehydrogenase ◽

Collagen Iv ◽

Basement Membranes ◽

Neonatal Rat ◽

Surface Epithelium ◽

Collagen Type Iv

Ovarian granulosa cells are derived embryologically from two keratin-positive epithelia of mesodermal origin, the ovarian rete and the ovarian surface epithelium. In the rat, presumptive granulosa cells still express keratin at birth but as they acquire functions related to oocyte support and steroidogenesis in the maturing ovary they lose this epithelial differentiation marker. Using double-label immunofluorescence microscopy, we examined the distribution of keratin-expressing granulosa cells in rat ovaries on days 1-10 postpartum in relation to (i) laminin and collagen type IV in follicular basement membranes, (ii) the zona pellucida, and (iii) 3β-hydroxysteroid dehydrogenase activity. Keratin was present in most (pre)granulosa cells on days 1-3. As the cells became multilayered in growing follicles, keratin was retained by granulosa cells adjacent to follicular basement membranes but disappeared from cells that were displaced towards follicular centers. From day 7 on, large follicles lacked keratin altogether. Laminin was a consistent component of follicular basement membranes at all ages, while collagen IV varied and diminished in parallel with keratin. 3β-Hydroxysteroid dehydrogenase was demonstrable in stromal interstitial cells from day 7 on. Zona pellucida first appeared in primary follicles adjacent to keratin-positive cells and subsequently became surounded with keratin-negative granulosa cells in growing follicles. The results suggest different roles for laminin and collagen IV in follicular basement membranes and support the hypothesis that keratin expression by granulosa cells depends on paracrine interactions with the ovarian stroma. In early growing follicles, these interactions may be interrupted by physical removal from the vicinity of the basement membranes as the granulosa cells become multilayered. In the more mature follicles, the loss of keratin from all granulosa cells suggests that the required stromal signals cease, perhaps as the perifollicular stroma differentiates into the theca.Key words: ovary, differentiation, keratin, basal membrane, development.

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

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab187 ◽

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.

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107 Localization of Kisspeptin Immunoreactivity in the Cat Ovary on Different Reproductive Stages

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab107 ◽

2018 ◽

Vol 30 (1) ◽

pp. 193

Author(s):

P. Tanyapanyachon ◽

O. Amelkina ◽

K. Chatdarong

Keyword(s):

Corpus Luteum ◽

Granulosa Cells ◽

Ovarian Surface Epithelium ◽

Domestic Cat ◽

Primary Antibody ◽

Surface Epithelium ◽

Domestic Cats ◽

Luteal Cells ◽

Theca Cells ◽

Steroidogenic Cells

Kisspeptin (Kp) is considered one of the main regulators of the reproductive axis, exerting its effects via stimulating GnRH expression in the hypothalamus. Apart from its central localization in the hypothalamus, the presence of Kp has been reported in the ovary, with possible local function. To date, very little is known about the ovarian Kp in the domestic cat. Therefore, our aim was to investigate the presence and localization of Kp at different reproductive stages in domestic cat ovaries. Twenty ovaries were collected from free-ranging domestic cats (body weight 2.7–4.5 kg) after routine ovariohysterectomy. Reproductive stages were classified by ovarian gross morphology, vaginal cytology, and blood progesterone level. Ovarian samples were grouped into inactive (n = 6), follicular (n = 8), and luteal stages (n = 6). Tissues were fixed in 4% paraformaldehyde and processed routinely. Immunohistochemistry was performed using polyclonal rabbit Kp-10 primary antibody (AB9754; Millipore, Billerica, MA, USA) at 1:500 at 4°C overnight. Immunoreactive cells were identified by avidin-biotin-peroxidase system. Rat hypothalamic tissue was used as a positive control. Primary antibody was substituted with PBS and normal rabbit IgG as the negative and isotypic negative controls, respectively. In addition, primary antibody was incubated with metastin overnight and applied for preabsorption test. Negative, isotypic negative, and preabsorption tests showed no staining. Immunoreactive Kp was detected in the ovaries of all reproductive stages with no obvious changes in localization or intensity of staining between stages. Kisspeptin was present in the cytoplasm of oocytes, granulosa cells, and theca cells of preantral (primordial, primary, and secondary) follicles and antral follicles. Interestingly, in most follicles, Kp staining was more prominent in theca cells and oocytes compared with granulosa cells. In corpus luteum, Kp was localised in the cytoplasm of luteal cells, with more intense staining on the periphery of corpus luteum compared with the middle in 3 luteal samples, whereas the rest of the samples demonstrated homogeneous staining distribution. Apart from oocytes and steroidogenic cells, Kp was also present in the cytoplasm of cells of the ovarian surface epithelium. Our study for the first time demonstrated the presence and localization of Kp in the ovary of the domestic cats. The localization of Kp in the cat oocyte is similar to previous reports on hamsters and dogs, indicating a possible function in oocyte development. The staining in steroidogenic cells, mainly theca cells and luteal cells, is in good agreement with studies on hamsters, rats, humans, and marmosets, suggesting the possible local involvement of Kp in steroidogenesis. In addition, Kp staining in the ovarian surface epithelium suggests a possible role in the ovarian remodeling after ovulatory defects, as reported in humans and marmosets. This research was funded by the RGJ PhD program PHD/01882556; RG 7/2559.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00222.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. C341-C353 ◽

Cited By ~ 30

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.

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