Expression of anti-Müllerian hormone mRNA during gonadal and follicular development in the brushtail possum (Trichosurus vulpecula)

2002 ◽  
Vol 14 (6) ◽  
pp. 345 ◽  
Author(s):  
Jennifer L. Juengel ◽  
Lisa J. Whale ◽  
Katherine A. Wylde ◽  
Penny Greenwood ◽  
Kenneth P. McNatty ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Sexual Differentiation ◽  
Cell Function ◽  
Mammalian Species ◽  
Follicular Development ◽  
Surface Epithelium ◽  
Brushtail Possum ◽  
Postnatal Life ◽  
Preovulatory Follicles

The ontogeny of anti-Müllerian hormone (AMH) gene expression in the brushtail possum during formation of the ovary and growth of follicles was examined using in situ hybridization. For comparative purposes, the expression pattern of AMH was also examined in the developing testis. In the female, AMH mRNA was observed in the ovary of 50% (3/6) of pouch young collected around the time of sexual differentiation of the gonad (Days 1–5): the signal was predominately localized to the inner-cortical and outer-medullary region of the ovary. Thereafter, AMH mRNA was not observed in the developing ovary until Days 78–113 of postnatal life when follicles first formed at the cortical–medullary boundary. At this time, AMH mRNA was observed in the cuboidal granulosa cells of some early growing (i.e. transitional) follicles and in the granulosa cells of primary follicles. Thereafter, AMH mRNA was present in granulosa cells at all subsequent stages of follicular growth (i.e. primary through antral), but not in preovulatory follicles. In all cases, once follicles had formed, AMH mRNA was limited to the granulosa cells and was not observed in the surface epithelium, stromal cells, oocytes, theca, corpus luteum, medullary cords, rete or interstitial glands. In the possum testis, Sertoli cells strongly expressed AMH around the time of sexual differentiation of the gonad, but expression decreased to very low levels in adults, suggesting that AMH plays a similar role in brushtail possums to that observed in other mammalian species. In conclusion, localization of mRNA for AMH exclusively to granulosa cells of growing follicles in the brushtail possum is consistent with a central role for this hormone in control of granulosa cell function in marsupials. In addition, expression of AMH in the developing ovary around the time of morphological sexual differentiation raises intriguing questions regarding the possible role of AMH at this time.

scholarly journals The role of IGFs in the regulation of ovarian follicular growth in the brushtail possum (Trichosurus vulpecula)

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Jennifer L Juengel ◽  
Lisa J Haydon ◽  
Brigitta Mester ◽  
Brian P Thomson ◽  
Michael Beaumont ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Function ◽  
Antral Follicle ◽  
Brushtail Possum ◽  
Follicular Growth ◽  
Theca Cells ◽  
Ovarian Follicular Growth

IGFs are known to be key regulators of ovarian follicular growth in eutherian mammals, but little is known regarding their role in marsupials. To better understand the potential role of IGFs in the regulation of follicular growth in marsupials, expression of mRNAs encoding IGF1, IGF2, IGF1R, IGF-binding protein 2 (IGFBP2), IGFBP4 and IGFBP5 was localized by in situ hybridization in developing ovarian follicles of the brushtail possum. In addition, the effects of IGF1 and IGF2 on granulosa cell function were tested in vitro. Both granulosa and theca cells synthesize IGF mRNAs, with the theca expressing IGF1 mRNA and granulosa cell expressing IGF2 mRNA. Oocytes and granulosa cells express IGF1R. Granulosa and theca cells expressed IGFBP mRNAs, although the pattern of expression differed between the BPs. IGFBP5 mRNA was differentially expressed as the follicles developed with granulosa cells of antral follicles no longer expressing IGFBP5 mRNA, suggesting an increased IGF bioavailability in the antral follicle. The IGFBP protease, PAPPA mRNA, was also expressed in granulosa cells of growing follicles. Both IGF1 and IGF2 stimulated thymidine incorporation but had no effect on progesterone production. Thus, IGF may be an important regulator of ovarian follicular development in marsupials as has been shown in eutherian mammals.

Expression of mRNAs encoding oestrogen receptor (ER) α and ERβ, androgen receptor and progesterone receptor during gonadal and follicular development in the marsupial brushtail possum (Trichosurus vulpecula)

2008 ◽  
Vol 20 (3) ◽  
pp. 335 ◽  
Author(s):  
Lisa J. Haydon ◽  
Jennifer L. Juengel ◽  
Brian P. Thomson ◽  
Douglas C. Eckery
Keyword(s):  
Granulosa Cells ◽  
Oestrogen Receptor ◽  
Follicular Development ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Antral Follicles ◽  
Ovarian Cells ◽  
Preovulatory Follicles ◽  
Specific Manner ◽  
Medullary Cords

The objective of the present study was to determine which ovarian cells express mRNAs for oestrogen (ERα and ERβ), androgen (AR) and progesterone (PR) receptors during ovarian and follicular development in the brushtail possum. Expression of ERα and/or ERβ mRNA was observed from birth, initially in cells of the blastema, then in the medullary cords from Day 20. ERα was expressed in the oocytes and granulosa cells of secondary and antral follicles. Preovulatory follicles did not express ERα mRNA, although their oocytes were not examined for any gene. ERβ mRNA was observed in oocytes at all follicular stages examined, but was not consistently observed in granulosa or theca cells. Expression of AR mRNA before Day 40 was very faint; thereafter, expression was observed in the medullary cords, peaking between Days 60 and 120. Oocytes, granulosa cells and theca of secondary and antral, but not preovulatory, follicles expressed AR mRNA. PR mRNA was expressed throughout the gonad by Day 20. Granulosa cells of some secondary and antral follicles and theca of antral follicles expressed PR mRNA. Thus, the expression of mRNAs encoding steroidogenic receptors in a time- and cell-specific manner supports a role for steroids in the process of ovarian follicular formation and growth.

Fibronectin production by chicken granulosa cells in vitro: effect of follicular development

1992 ◽  
Vol 127 (5) ◽  
pp. 466-470 ◽  
Author(s):  
Elikplimi K Asem ◽  
Jacqueline A Carnegie ◽  
Benjamin K Tsang
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Ovarian Granulosa Cells ◽  
Preovulatory Follicles ◽  
Vitro Effect ◽  
Dose Dependent

In vitro studies were conducted to investigate the role of chicken ovarian granulosa cells in the production of fibronectin, a component of the basal lamina of ovarian follicles. Collagenase dispersed granulosa cells obtained from the first (F1; about 35 mm in diameter) and third (F3; 15–20 mm in diameter) largest preovulatory follicles, as well as from a pool of small yellow follicles (SF; 6–10 mm in diameter), were incubated in serum-free medium-199 for 24 to 96 h in the absence and presence of luteinizing hormone (LH) or forskolin. Fibronectin secreted in the medium was quantitated by enzyme linked immunosorbent assay. Basal fibronectin production (which increased with the duration of incubation) was significantly greater (p<0.001) in granulosa cells derived from mature follicle (F1) than in F3 or SF cells. Both LH and forskolin stimulated fibronectin production in SF and F3 cells in a dose-dependent manner; however, they were without effect in F1 cells. The magnitude of increase in fibronectin production elicited by LH or forskolin was greater in SF cells than in F3 cells. The cytoplasm of cultured granulosa cells taken at all stages of follicular development stained positively for fibronectin. These findings indicate that chicken granulosa cells produce fibronectin. This ability is acquired early in follicular development and the stimulatory effect of the gonadotropin (LH) diminished as the follicle approached ovulation.

scholarly journals MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

2017 ◽  
Vol 2017 ◽  
pp. 1-18 ◽  
Author(s):  
Baoyun Zhang ◽  
Long Chen ◽  
Guangde Feng ◽  
Wei Xiang ◽  
Ke Zhang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Granulosa Cells ◽  
Messenger Rna ◽  
Expression Patterns ◽  
Follicular Development ◽  
Functional Networks ◽  
Signal Pathways ◽  
Differentially Expressed Mirnas ◽  
Selection Of

Ovaries, which provide a place for follicular development and oocyte maturation, are important organs in female mammals. Follicular development is complicated physiological progress mediated by various regulatory factors including microRNAs (miRNAs). To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing. Furthermore, via bioinformatic analyses, we dissected the associated functional networks of the observed significant miRNAs, in terms of interacting with signal pathways and transcription factors. During the growth and selection of dominant follicles, 15 dysregulated miRNAs and 139 associated pathways were screened out. In comparison of different styles of follicles, 7 commonly abundant miRNAs and 195 pathways, as well as 10 differentially expressed miRNAs and 117 pathways in dominant follicles in comparison with subordinate follicles, were collected. Furthermore, SMAD2 was identified as a hub factor in regulating follicular development. The regulation of miR-26a/b onsmad2messenger RNA has been further testified by real time PCR. In conclusion, we established functional networks which play critical roles in follicular development including pivotal miRNAs, pathways, and transcription factors, which contributed to the further investigation about miRNAs associated with mammalian follicular development.

scholarly journals Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells

2002 ◽  
Vol 172 (1) ◽  
pp. 45-59 ◽  
Author(s):  
F Le Bellego ◽  
C Pisselet ◽  
C Huet ◽  
P Monget ◽  
D Monniaux
Keyword(s):  
Estrous Cycle ◽  
Granulosa Cells ◽  
Physiological Role ◽  
Follicular Development ◽  
Antral Follicles ◽  
Final Development ◽  
Beta 1 Integrin ◽  
Arginine Glycine Aspartic Acid

This study aimed to determine the physiological role of laminin (LN) and its receptor, alpha(6)beta(1) integrin, in controlling the functions of granulosa cells (GC) during follicular development in sheep ovary. Immunohistochemistry experiments showed the presence of increasing levels of LN (P<0.0001), and high levels of mature alpha(6)beta(1) integrin in GC layers of healthy antral follicles during the follicular and the preovulatory phases of the estrous cycle. In vitro, the addition of a function-blocking antibody raised against alpha(6) subunit (anti-alpha(6) IgG) to the medium of ovine GC cultured on LN impaired cell spreading (P<0.0001), decreased the proliferation rate (P<0.05) and increased the apoptosis rate (P<0.05). Furthermore, addition of anti-alpha(6) IgG enhanced estradiol (E2) secretion by GC in the presence or absence of follicle-stimulating hormone (FSH), luteinizing hormone or insulin-like growth factor-I in culture medium (P<0.0001), and inhibited progesterone (P4) secretion in basal conditions or in the presence of low (0.5 ng/ml) FSH concentrations only (P<0.0001). The anti-alpha(6) IgG effect was specific to an interaction of LN with alpha(6)beta(1) integrin since it was ineffective on GC cultured on heat-denatured LN, RGD (arginine-glycine-aspartic acid) peptides and non-coated substratum. Hence, this study established that alpha(6)beta(1) integrin 1) was expressed in GC of antral follicles, 2) mediated the actions of LN on survival, proliferation and steroidogenesis of GC, and 3) was able to dramatically modulate P4 and E2 secretion by GC in vitro. It is suggested that during the follicular and the preovulatory phases of the estrous cycle, the increasing levels of LN in GC of large antral follicles might support their final development to ovulation.

142 microRNA-17-92 CLUSTER REGULATES BOVINE GRANULOSA CELL FUNCTION BY TARGETING BMPR2 AND PTEN GENES

2016 ◽  
Vol 28 (2) ◽  
pp. 201
Author(s):  
E. Andreas ◽  
D. Salilew-Wondim ◽  
M. Hoelker ◽  
C. Neuhoff ◽  
E. Tholen ◽  
...  
Keyword(s):  
Target Genes ◽  
Cell Function ◽  
Follicular Development ◽  
Rna Isolation ◽  
Luciferase Reporter ◽  
Cell Diameter ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Method ◽  
Differentiated Cells

Normal follicular development, especially from the preantral stage until ovulation, is the critical to ensure the release of a developmentally competent oocyte. We have previously shown that among several clusters of microRNAs, microRNA-17-92 cluster (miR-17-5p, miR-19a, miR-20a, and miR-92a) is differentially expressed between bovine granulosa cells (bGC) derived from preovulatory dominant and subordinate follicles. Here, we aimed to investigate the regulatory role of microRNA-17-92 cluster in bGC function. Among the target genes predicted by the miRWalk database, BMPR2 and PTEN genes were experimentally validated using the pmirGLO Dual Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). The bGC were aspirated from ovaries obtained from a local slaughterhouse. After determining cell viability and concentration using the trypan blue exclusion method, a total 2.5 × 105 bGC per well were seeded into CytoOne 24-well plate in DMEM/F12-Ham medium (Sigma Aldrich Chemie GmbH, Munich, Germany) supplemented with 10% FBS (Gibco BRL USA, Grand Isalnd, NY, USA) and 1% penicillin/streptomycin (Gibco BRL USA). Then, the bGC were cultured at 37°C with 5% CO2 and O2. To investigate the role of microRNA-17-92 cluster in bGC function, 100 nM of individual and cluster of microRNA-17-92 mimic, inhibitor, and negative controls were transfected into subconfluent-cultured bGC. The bGC were harvested 48 h post-transfection and used for RNA isolation and subsequent cDNA synthesis and expression analysis of candidate genes using real-time qPCR. Data analysis was performed using the comparative cycle threshold (Ct) method. A cell proliferation assay was performed using CCK-8 kit (Dojindo EU GmbH, Munich, Germany). Based on the cell diameter measurement done using ImageJ 1.48v software (National Institutes for Health, Bethesda, MD, USA), those bGC with diameter >14 µm were categorized as differentiated cells, whereas those with diameter = 14 µm were considered as undifferentiated cells. MicroRNA-17-92 cluster overexpression on bGC reduced both mRNA and protein expression of BMPR2 and PTEN genes, whereas inhibition of microRNA-17-92 cluster increased their expression. Bovine GC transfected with microRNA-17-92 cluster mimic showed higher proliferation activity and decreased rate of differentiation. The opposite phenotype was observed in bGC transfected with microRNA-17-92 cluster inhibitor. Similarly, miRNA-17-92 cluster mimic transfection increased the expression of markers of proliferation, CCND2 and PCNA, and resulted in down-regulation of CYP11A1 and STAR genes as markers of differentiation. The opposite expression pattern was observed after transfection of miRNA-17-92 cluster inhibitors. In conclusion, the miRNA-17-92 cluster members coordinately regulate bGC proliferation and differentiation by targeting the expression of BMPR2 and PTEN genes.

Follistatin but not α or βA inhibin subunit mRNA is expressed in ovine fetal ovaries in late gestation

1994 ◽  
Vol 13 (1) ◽  
pp. 1-9 ◽  
Author(s):  
R Braw-Tal ◽  
D J Tisdall ◽  
N L Hudson ◽  
P Smith ◽  
K P McNatty
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Cell Function ◽  
Follicular Development ◽  
Primordial Follicle ◽  
Late Gestation ◽  
Follicle Growth ◽  
Primordial Follicles ◽  
Fetal Ovary ◽  
Ovine Fetal

ABSTRACT The aim of this study was to investigate the sites of follistatin and α and βA inhibin mRNA expression in the ovaries of female sheep fetuses at 90, 100, 120 and 135 days of gestation (term=day 147). At 90 and 100 days primordial follicles were formed, followed by the appearance of primary follicles at 100 days of gestation. At days 120 and 135, primordial, primary and preantral (i.e. secondary) follicles were present in the ovaries, but antral (i.e. tertiary) follicles were not observed at any of these gestational ages. Two Booroola genotypes were studied: homozygous carriers (BB) and non-carriers (++) of the fecundity gene (FecB). Irrespective of genotype no specific hybridization of the α and βA inhibin riboprobes was detected in any ovarian cells at days 90, 100, 120 or 135 of gestation. In control mature ovaries, on the other hand, strong hybridization in the granulosa cells of antral follicles was observed. In contrast to α and βA inhibin, follistatin antisense (but not sense) riboprobes hybridized specifically to the granulosa cells of preantral follicles with two or more layers of cells at days 120 and 135 of gestation. Moreover, hybridization was also evident in the cells of the ovarian rete at days 120 and 135, but not at 90 or 100 days. No follistatin mRNA expression was observed in the granulosa cells of primordial or primary follicles or in any other ovarian cell type at any of the gestational ages examined. No FecB-specific differences in follistatin expression were noted with respect to stage of preantral follicular development and there were no obvious differences in the intensity of expression. These results show that follistatin mRNA is expressed specifically in the granulosa cells and intraovarian rete. Expression of follistatin in rete cells was coincident with the increasing numbers of growing follicles within the fetal ovary, indicating that rete cell function may have a role in the ontogeny of early follicular growth. Our results suggest that follistatin and α and βA inhibin may not be important for the initiation of follicle growth in the sheep ovary, since these genes are not expressed during the transformation of a primordial follicle to a primary structure. However, the evidence for follistatin mRNA expression in the ovine fetal ovary implies that this hormone is likely to play a role during the early stages of follicle growth.

Oocyte-secreted factros regulate granulosa cell steroidogenesis

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 317-321 ◽  
Author(s):  
Barbara C. Vanderhyden
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Inhibitory Factor ◽  
Preovulatory Follicles ◽  
Loss Of Contact

Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellaset al., 1991; Huanget al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhydenet al., 1990, 1992) and cumulus expansion (Buccioneet al., 1990; Salustriet al., 1990; Vanderhydenet al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinisein vitro(Channinget al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisationin vitro(Ledwitz-Rigbyet al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.

scholarly journals Role of leptin receptors in granulosa cells during ovulation

Reproduction ◽  
2014 ◽  
Vol 147 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Lisa Dupuis ◽  
Yasmin Schuermann ◽  
Tamara Cohen ◽  
Dayananda Siddappa ◽  
Anitha Kalaiselvanraja ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Leptin Receptor ◽  
Critical Role ◽  
Follicular Development ◽  
Reproductive Function ◽  
Control Treatment ◽  
Maturation Stage ◽  
Leptin Receptors ◽  
Enhancer Binding Protein

Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein β (Cebpβ) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 μg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpβ. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

scholarly journals Characteristic of factors influencing the proper course of folliculogenesis in mammals

2018 ◽  
Vol 6 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Marta Rybska ◽  
Sandra Knap ◽  
Maurycy Jankowski ◽  
Michal Jeseta ◽  
Dorota Bukowska ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Follicular Development ◽  
Ovarian Follicles ◽  
Postnatal Life ◽  
Metabolic Hormones ◽  
Corona Radiata ◽  
Mural Cells ◽  
Thecal Cells ◽  
Reproductive Processes

AbstractFolliculogenesis is the process of ovarian follicle formation,, taking presence during foetal period. During the follicular development, oogoniums undergo meiosis and oocytes are formed. In the ovaries of new born sows, primary and secondary follicles are present and, 90 days after birth, tertiary follicles appear. During development in the ovarian follicles growth of granulosa cells and differentiation of the thecal cells can be observed. A cavity filled with follicular fluid appears. Granulosa cells are divided into: mural cells and corona radiata, which together with the oocyte form the cumulus oophorus. Corona radiata cells, mural layers and oolemma contact each other by a network of gap junctions. Secreted from the pituitary gland, FSH and LH gonadotropin hormones act on receptors located in granular and follicular cells. In the postnatal life tertiary follicles and Graafian follicles are formed. When the follicle reaches a diameter of 1 mm, further growth depends on the secretion of gonadotropins. Mature ovarian follicles produce: progestins, androgens and oestrogens. The growth, differentiation and steroidogenic activity of ovarian follicles, in addition to FSH and LH, is also affected by prolactin, oxytocin, steroid and protein hormones, numerous proteins from the cytokine and interleukin family, metabolic hormones like insulin, glucocorticoids, leptin, thyroid hormones and growth hormones. Despite numerous studies, many processes related to folliculogenesis have not been discovered Learning the mechanisms regulating reproductive processes would allow to easily distinguish pathological processes and discover more and more genes and mechanisms of their expression in cells that build ovarian follicles.

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