Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 233-240
Author(s):  
Ahmed S.A. Sosa ◽  
Sally Ibrahim ◽  
Karima Gh. M. Mahmoud ◽  
Mohamed M. Ayoub ◽  
Mohamed S.S. Abdo ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Marker Gene ◽  
Follicular Development ◽  
Nuclear Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Suitable Model ◽  
Follicular Size

SummaryThis study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5–8 mm, third group 9–15 mm and fourth group 16–20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5–8 mm, 9–15 mm and 16–20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

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 Effects of Osthole on Progesterone Secretion in Chicken Preovulatory Follicles Granulosa Cells

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2027
Author(s):  
Na Sun ◽  
Yutong Zhang ◽  
Yaxin Hou ◽  
Yanyan Yi ◽  
Jianhua Guo ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Regulatory Protein ◽  
Adenosine Monophosphate ◽  
Nuclear Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Active Constituent ◽  
Progesterone Secretion ◽  
Corticosterone Secretion ◽  
Significant Difference ◽  
Preovulatory Follicles

Osthole (Ost) is an active constituent of Cnidium monnieri (L.) Cusson which possesses anti-inflammatory and anti-oxidative properties. It also has estrogen-like activity and can stimulate corticosterone secretion. The present study was aimed to check the role of Ost on progesterone (P4) secretion in cultured granulosa cells obtained from hen preovulatory follicles. Different concentrations (5, 2.5, and 1.25 µg/mL) of Ost was added to granulosa cells for 6, 12, 18, and 24 h to investigate the level of progesterone secretions using enzyme linked immunosorbent assay (ELISA). The results showed that progesterone secretion was significantly increased in cells treated with Ost at 2.5 μg/mL. Also, qRT-PCR showed that mRNA expression of steroidogenic acute regulatory protein (StAR) was significantly up-regulated by Ost at 2.5 μg/mL concentration. Cytochrome P450 side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly up-regulated by Ost. However, no significant differences were observed for the expression of proliferating cell nuclear antigen (PCNA). The protein expression of StAR, P450scc and 3β-HSD were significantly up-regulated by Ost treatment. The concentration of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in cell lysates showed no change with Ost treatment at 2.5 μg/mL by ELISA. An ROS kit showed non-significant difference in the level of reactive oxygen species (ROS). In conclusion, Ost treatment at a concentration of 2.5 μg/mL for 24 h had significantly up-regulated P4 secretion by elevating P450scc, 3β-HSD and StAR at both gene and protein level in granulosa cells obtained from hen preovulatory follicles.

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 Targeted Disruption of Mapk14 (p38MAPKα) in Granulosa Cells and Cumulus Cells Causes Cell-Specific Changes in Gene Expression Profiles that Rescue COC Expansion and Maintain Fertility

2010 ◽  
Vol 24 (9) ◽  
pp. 1794-1804 ◽  
Author(s):  
Zhilin Liu ◽  
Heng-Yu Fan ◽  
Yibin Wang ◽  
JoAnne S. Richards
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Knockout Mice ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Cumulus Cells ◽  
Mutant Mice ◽  
Prostaglandin E

Abstract MAPK14 (p38MAPKα) is critical for FSH and prostaglandin E (PGE)2 signaling cascades in granulosa cells (GCs) and cumulus cell-oocyte complexes (COCs) in culture, indicating that this kinase might impact follicular development and COC expansion in vivo. Because Mapk14 knockout mice are embryonic lethal, we generated GC specific Mapk14 knockout mice (Mapk14gc−/−) by mating Mapk14fl/fl and Cyp19-Cre mice. Unexpectedly, the Mapk14gc−/− female mice were fertile. Analyses of gene expression patterns showed that amphiregulin (Areg) and epiregulin (Ereg), two key regulators of ovulation and COC expansion, were up-regulated in the GCs but down-regulated in cumulus cells of the mutant mice in vivo. COCs from the mutant mice expanded and expressed matrix-related genes, if cultured with AREG, but not when cultured with forskolin or PGE2, the latter being a key factor regulating MAPK14 activity in cumulus cells. Conversely, when GCs from the Mapk14gc−/− mice were cultured with forskolin, they produced more Areg and Ereg mRNA than did wild-type GCs. These results indicate that disruption of Mapk14 selectively alters the expression of Areg and other genes in each cell type. Greater AREG and EREG produced by the GCs appears to by-pass and compensate for the critical need for MAPK14 signaling and induction of Areg/Ereg (and hence matrix genes) by PGE2 in cumulus cells of the mutant mice. In conclusion, although MAPK14 is not overtly essential for preovulatory follicle development or events associated with ovulation and luteinization in vivo, it does impact gene expression profiles.

scholarly journals HIF-1α Protects Granulosa Cells From Hypoxia-Induced Apoptosis During Follicular Development by Inducing Autophagy

2021 ◽  
Vol 9 ◽  
Author(s):  
Zonghao Tang ◽  
Renfeng Xu ◽  
Zhenghong Zhang ◽  
Congjian Shi ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Ovarian Follicle ◽  
Hypoxia Inducible Factor ◽  
Follicular Development ◽  
B Cell Lymphoma ◽  
Underlying Mechanism

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

1 DIFFERENCES IN RESUMPTION OF OOCYTE MATURATION IN YOUNG AND OLD MARES

2008 ◽  
Vol 20 (1) ◽  
pp. 81 ◽  
Author(s):  
L. F. Campos-Chillon ◽  
C. M. Clay ◽  
J. L. Altermatt ◽  
G. J. Bouma ◽  
E. M. Carnevale
Keyword(s):  
Granulosa Cells ◽  
Expression Patterns ◽  
Cell Number ◽  
Specific Gene ◽  
Meiotic Arrest ◽  
Temporal Differences ◽  
Time Points ◽  
Follicular Maturation ◽  
Copy Numbers

The decline in fertility of aged mares is linked with declining oocyte quality. Oocyte viability is dependent on the ability of oocytes to remain in meiotic arrest until the initiation of maturation. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to determine temporal differences in the mRNA content of amphiregulin and epiregulin in granulosa cells; PDE4 in cumulus and granulosa cells; and PDE3A, GPR3, GDF9, and BMP15 in oocytes during in vivo maturation in young (3–12 years) v. old (>20 years) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Maturation was induced in estrous mares with a follicle >30 mm by injection of 750 �g of recombinant equine LH. Aspirations were attempted at 0, 6, 9, and 12 h after LH administration. Six oocytes and follicular cell samples from each age group and time point were collected and stored immediately after aspiration. Total RNA was isolated from single denuded oocytes and lysed cumulus and granulosa cells. A fraction of the total lysate was used to determine cell numbers from the DNA copy number of the equine CG� subunit gene. DsRED RNA was added to each RNA isolate to serve as an exogenous standard. Quantitative RT-PCR was performed from cDNA with equine primer pairs. Copy numbers were calculated with an intra assay standard curve of plasmid containing the specific gene and corrected with the exogenous DsRED RNA and cell number. For each gene, mean mRNA copy numbers for time points and age groups were compared by ANOVA and Tukey's HSD test. Expression of PDE4D in cumulus cells was similar between young and old mares and time points. However, PDE4D peaked (P < 0.05) at 6 h in granulosa cells from young, but not old mares. Amphiregulin expression in granulosa cells of young mares peaked (P < 0.05) at 9 h and did not increase in the old mares. Epiregulin expression in granulosa cells peaked (P < 0.05) at 9 h and 6 h in young and old mares, respectively. The pattern of expression of PDE3A for oocytes of young and old mares was similar with an increase (P < 0.05) at 9 h. There was an interaction (P < 0.05) in the expression of GPR3 for age � time. Expression peaked at 9 and 6 h in young and old mares, respectively. Pattern of expression of GDF9 was similar between young and old mares except for a decrease (P < 0.05) in expression in old mares at 9 h. There was an interaction (P < 0.05) in the expression of BMP15 for age � time. Expression in young mares peaked at 9 h while that in old mares peaked at 6 h and decreased at 9 h. These results suggest that key gene expression patterns involved in oocyte and follicular maturation cascades are asynchronous for young versus old mares and could explain some aspects of the age-associated decline in fertility.

Progesterone stimulates fibronectin production by chicken granulosa cells in vitro

1994 ◽  
Vol 130 (2) ◽  
pp. 159-165 ◽  
Author(s):  
Elikplimi K Asem ◽  
Michael D Conkright ◽  
Ruben P Novero
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Ru 486 ◽  
Progesterone Synthesis ◽  
Exogenous Progesterone ◽  
Chicken Ovary

Asem EK, Conkright MD, Novero RP. Progesterone stimulates fibronectin production by chicken granulosa cells in vitro. Eur J Endocrinol 1994;130:159–65. ISSN 0804–4643 Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F1: mature) and third-largest (F3: developing) preovulatory follicles as well as from a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. The amounts of basal fibronectin and progesterone produced by granulosa cells from F1, F3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F1 and F3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F1 and F3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F1 and F3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner. EK Asem, Department of Physiology and Pharmacology, School of Veterinary Medicine, Purdue University, 1246 Lynn Hall, West Lafayette, IN 47907-1246. USA

scholarly journals Spatio-Temporal Expression Patterns of Steroidogenic Acute Regulatory Protein (StAR) During Follicular Development in the Rat Ovary*

Endocrinology ◽  
1998 ◽  
Vol 139 (1) ◽  
pp. 303-315 ◽  
Author(s):  
Tamar Ronen-Fuhrmann ◽  
Rina Timberg ◽  
Steven R. King ◽  
Karen H. Hales ◽  
Dale B. Hales ◽  
...  
Keyword(s):  
Steroid Hormones ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Regulatory Protein ◽  
Follicular Development ◽  
Steroidogenic Acute Regulatory Protein ◽  
Interstitial Tissue ◽  
Immature Rats ◽  
Theca Interna ◽  
Steroidogenic Acute Regulatory

Abstract The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

scholarly journals Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus

Reproduction ◽  
2008 ◽  
Vol 135 (3) ◽  
pp. 335-342 ◽  
Author(s):  
Esther W Kabithe ◽  
Ned J Place
Keyword(s):  
Granulosa Cells ◽  
Expression Patterns ◽  
Follicular Development ◽  
Day Length ◽  
Phodopus Sungorus ◽  
Reproductive Aging ◽  
Primordial Follicles ◽  
Short Day ◽  
Siberian Hamsters ◽  
Secondary Stage

Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called ‘hypertrophied granulosa cells’ were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.

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