scholarly journals Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 641-649 ◽  
Author(s):  
Inger B Carlsson ◽  
Mika P E Laitinen ◽  
Jennifer E Scott ◽  
Henna Louhio ◽  
Louiza Velentzis ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Culture Media ◽  
Follicular Development ◽  
Ovarian Follicles ◽  
Kit Ligand ◽  
Kit Receptor ◽  
Ovarian Follicular Development

The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit usingin situhybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian folliclesin vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development.

scholarly journals The conflict between hierarchical ovarian follicular development and superovulation treatment

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 287-294 ◽  
Author(s):  
Kenneth P McNatty ◽  
Derek A Heath ◽  
Norma L Hudson ◽  
Karen L Reader ◽  
Laurel Quirke ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Follicular Phase ◽  
Ovulation Rate ◽  
Cell Populations ◽  
Antral Follicles ◽  
Functional States ◽  
Ovarian Follicular Development

In mammals with a low ovulation rate phenotype, ovarian follicular development is thought to be hierarchical with few, if any, antral follicles at similar stages of development. The hypothesis being tested herein was that if most follicles are in a functionally different state, then the application of exogenous hormones to increase ovulation rate will not overcome the hierarchical nature of follicular development. Using sheep as the experimental model, the functional states of all non-atretic antral follicles ≥2 mm diameter were assessed in individual ewes (N=10/group) during anoestrus with or without pregnant mare's serum gonadotrophin (PMSG) treatment, or after a standard superovulation regimen, or during the follicular phase of the oestrous cycle. The functional states of these follicles were assessed by measuring the FSH- or human chorionic gonadotrophin (hCG)-induced cAMP responses of granulosa cellsin vitro. There were significant overall effects across the treatment groups on the responses of granulosa cells to either FSH or LH (bothP<0.001). It was concluded that for anoestrous ewes with or without PMSG treatment, and ewes during the follicular phase, granulosa cell populations of many follicles (≥2 mm diameter) did not share a similar cAMP response to FSH (∼50% of follicles) or hCG (>90% of follicles) either on a per cell or total cell basis. After superovulation, ≤30 and 10% respectively of the granulosa cell populations shared similar responses to FSH and LH with regard to follicular diameter and cAMP output. Thus, exogenous hormone treatments used routinely for increasing oocyte yield do not effectively override the hierarchical pattern of ovarian follicular development during the follicular phase.

In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex

2018 ◽  
Vol 30 (2) ◽  
pp. 359 ◽  
Author(s):  
Érica S. S. Leal ◽  
Luis A. Vieira ◽  
Naíza A. R. Sá ◽  
Gerlane M. Silva ◽  
Franciele O. Lunardi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Culture Media ◽  
Follicular Development ◽  
Vascular Endothelial ◽  
Fresh Tissue ◽  
Ovarian Cortex ◽  
Agnor Staining ◽  
Vitro Development ◽  
Endothelial Growth Factor

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P < 0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P > 0.05). The average overall and daily follicular growth was highest (P < 0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P < 0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.

scholarly journals Alterations in ovarian function of mice with reduced amounts of KIT receptor

Reproduction ◽  
2001 ◽  
pp. 229-237 ◽  
Author(s):  
K Reynaud ◽  
R Cortvrindt ◽  
J Smitz ◽  
F Bernex ◽  
JJ Panthier ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Ovarian Follicle ◽  
Mutant Mice ◽  
Wild Type ◽  
Oocyte Growth ◽  
Preantral Follicles ◽  
Kit Ligand ◽  
Kit Receptor

The KIT receptor, present on oocyte and theca cells in ovarian follicles, and its ligand, KIT LIGAND, produced by granulosa cells, are encoded at the Kit gene and the Mgf gene, respectively. Both Kit and Mgf mutations affect oogenesis and folliculogenesis. In this study, the ovarian function of heterozygous mice with a mutation Kit(W-lacZ) was examined. Firstly, the amounts of KIT and KIT LIGAND proteins in the ovaries of mice at different ages were determined. Secondly, in vivo and in vitro folliculogenesis of wild type and heterozygous mice were compared. Western blotting showed that the amounts of both KIT and KIT LIGAND proteins were decreased in mutant mice. Ovarian follicle populations were counted and more type 5a follicles and fewer type 5b (preantral follicles) were present in ovaries from Kit(W-lacZ/+) ovaries. Furthermore, the relationships between oocyte size and follicle size differed between wild type and heterozygous mice. This finding may be a consequence of altered proliferation of granulosa cells or of altered oocyte growth in mutant mice. Other features of folliculogenesis, such as initiation of follicular growth, total follicle population and follicular atresia, were not affected by the mutation. Analysis of in vitro folliculogenesis did not reveal other differences between wild type and mutant mice. It is concluded that the Kit(W-lacZ) mutation affects the expression of KIT and KIT LIGAND proteins, resulting in alterations in granulosa cell proliferation and/or oocyte growth in preantral follicles.

scholarly journals rno-miR-128-3p promotes apoptosis in rat granulosa cells (GCs) induced by norepinephrine through Wilms tumor 1 (WT1)

2021 ◽  
Author(s):  
Ming Li ◽  
Ling Xue ◽  
Weibin Xu ◽  
Pingping Liu ◽  
Feng Li
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Wilms Tumor ◽  
Sympathetic Innervation ◽  
Follicular Development ◽  
Wilms Tumor 1 ◽  
Ovarian Follicular Development ◽  
Induced Apoptosis

AbstractThe mechanism related to ovarian follicular is complex, which has not been fully elucidated. Abundant reports have confirmed that the ovarian function development is closely related to sympathetic innervation. As one of the major neurotransmitters, norepinephrine (NE) is considered an effective regulator of ovarian functions like granulosa cell (GC) apoptosis. However, the mechanism between NE and GC apoptosis in rat is still unclear. In our study, GCs were isolated and cultured in vitro with NE treatment. The apoptosis of GCs was facilitated by NE. Wilms tumor 1 (WT1) was found to be significantly downregulated in GCs after NE treatment, and overexpression of WT1 repressed apoptosis in rat GCs induced by NE. rno-miR-128-3p was found to be significantly enhanced by NE in GCs, and inhibition of rno-miR-128-3p repressed apoptosis in rat GCs induced by NE. Mechanistically, rno-miR-128-3p interacted with WT1 and repressed its expression. In summary, inhibition of rno-miR-128-3p may enhance WT1 expression, and then repress NE-induced apoptosis in rat GCs. Our research may provide a new insight for the improvement of ovarian follicular development.

Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†

2020 ◽  
Vol 102 (6) ◽  
pp. 1177-1190
Author(s):  
Aly Warma ◽  
Kalidou Ndiaye
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Developmental Stages ◽  
Polyclonal Antibodies ◽  
Differentially Expressed Gene ◽  
Follicular Development ◽  
Mapk Signaling ◽  
Tissue Growth ◽  
Ovulatory Follicles

Abstract Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.

scholarly journals Dynamic characteristics of lipid metabolism in cultured granulosa cells from geese follicles at different developmental stages

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Shanyan Gao ◽  
Xiang Gan ◽  
Hua He ◽  
Shenqiang Hu ◽  
Yan Deng ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Granulosa Cells ◽  
Dynamic Characteristics ◽  
Developmental Stages ◽  
Lipid Synthesis ◽  
Follicular Development ◽  
Vital Role ◽  
Intracellular Lipids ◽  
Expression Of Genes

Abstract Previous studies have shown that lipid metabolism in granulosa cells (GCs) plays a vital role during mammalian ovarian follicular development. However, little research has been done on lipid metabolism in avian follicular GCs. The goal of the present study was to investigate the dynamic characteristics of lipid metabolism in GCs from geese pre-hierarchical (6–10 mm) and hierarchical (F4-F2 and F1) follicles during a 6-day period of in vitro culture. Oil red O staining showed that with the increasing incubation time, the amount of lipids accumulated in three cohorts of GCs increased gradually, reached the maxima after 96 h of culture, and then decreased. Moreover, the lipid content varied among these three cohorts, with the highest in F1 GCs. The qPCR results showed genes related to lipid synthesis and oxidation were highest expressed in pre-hierarchical GCs, while those related to lipid transport and deposition were highest expressed in hierarchical GCs. These results suggested that the amount of intracellular lipids in GCs increases with both the follicular diameter and culture time, which is accompanied by significant changes in expression of genes related to lipid metabolism. Therefore, it is postulated that the lipid accumulation capacity of geese GCs depends on the stage of follicle development and is finely regulated by the differential expression of genes related to lipid metabolism.

Kit Ligand Expression in granulosa cells during follicular culture and associated maturation rate of oocytes in buffalo (Bubalus bubalis)

2015 ◽  
Author(s):  
Atul Mahajan ◽  
Parveen Kumar ◽  
Sachin Atole ◽  
B P Brahmkshtri ◽  
K. R. Tajane ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Culture Media ◽  
Pcr Amplification ◽  
Bubalus Bubalis ◽  
Ovarian Follicles ◽  
Kit Ligand ◽  
Tissue Culture Media ◽  
Maturation Rate ◽  
Granulose Cells

The objectives of the study to associate the expression of Kit Ligand gene under different gonadotropins and growth factors supplementation with the maturation rate of oocytes in buffalo ovarian follicular cells during whole follicular culture at subsequent intervals of 24 hours. Intact ovarian follicles were dissected out of buffalo ovaries and cultured in Tissue Culture Media-199 supplemented with gonadotropins and growth factors for 24 hours. A semi-quantitative RT-PCR amplification was obtained in granulose cells of buffalo ovarian follicles and its expression was found to be restricted during initial stage of follicle culture. KL expression persistency in granulosa cells beyond 4 hours during follicular culture was found to be negatively correlated with maturation rate of oocytes. It was concluded that expression of Kit Ligand gene in granulose cells could be predictive role for assessing oocytes maturation rate and possibly the oocytes competence in buffalo.

Leptin Supplementation Stimulates Synergism with Growth Factors and Hormones to Express Its Receptor in Cultured Preantral Follicles of Sheep

2020 ◽  
Author(s):  
K. Sravani Pragna ◽  
V. Praveen Chakravarthi ◽  
Deepa Pathipati ◽  
B. Rambabu Naik ◽  
L.S.S. Varaprasad Reddy ◽  
...  
Keyword(s):  
Growth Factors ◽  
Leptin Receptor ◽  
Culture Media ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
Antral Follicles ◽  
Receptor Mrna

Background: Leptin receptor is a transmembrane receptor that regulates reproduction at molecular level.Since for action of any hormone on target cell and to have local action on any tissue, expression of its own receptor is necessary and also it is not known whether such improvement in ovarian follicular development by Leptin is mediated through presence of its homologous receptors in the sheep ovaries. Therefore this study aimed on expression of Leptin receptor mRNA in cultured ovarian follicles of sheep by RT PCR.Methods: Leptin receptor mRNA expression in sheep was studied using qRT-PCR from: (i) In vivo grown preantral, early antral, antral, large antral follicles and cumulus oocyte complexes obtained from large antral follicles subjected to 24h of in vitro maturation and (ii) PFs’ exposed to three different culture media for 3min, two, four or six days and subsequently matured in vitro for 24h. Result: Leptin receptor was observed at all stages ovarian follicles in both cumulus cells and oocytes. Leptin supplementation along with other growth factors and hormones stimulated the expression of its receptor mRNA which is parallel to in vivo stages which could suggest synergistic action of growth factors and hormones with Leptin. 

scholarly journals Relaxin protein and gene expression in ovarian follicles of immature pigs

1998 ◽  
Vol 21 (2) ◽  
pp. 179-187 ◽  
Author(s):  
KM Ohleth ◽  
Q Zhang ◽  
CA Bagnell
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Ovarian Follicle ◽  
Follicular Development ◽  
Ovarian Follicles ◽  
In Vitro Growth ◽  
Follicle Size ◽  
Theca Interna

Relaxin production by the ovarian follicle of gonadotropin-primed, prepubertal gilts is well documented. As far as we are aware, a source of relaxin in pig follicles, independent of gonadotropins, has not yet been reported. Therefore, the objective of this study was to determine whether relaxin is produced in porcine follicles in the absence of exogenous or cyclic gonadotropins. In immature pigs, immunoreactive relaxin was detected in fluids from small (1-3 mm), medium (4-5 mm) and large (>6 mm) follicles and localized to the theca interna of large follicles. Relaxin levels in follicular fluid significantly increased with follicle size (P<0.05). Relaxin mRNA was detected in whole small- and medium-sized follicles. In large follicles, the relaxin gene was expressed in thecal layers, but not granulosa cells. The abundance of relaxin transcript did not change with follicle size. In summary, relaxin protein and mRNA were detected in porcine follicles from immature animals, indicating that relaxin is produced in the porcine follicle in the absence of exogenous or cyclic gonadotropins. Relaxin's in vitro growth effects on porcine granulosa and theca cells support this follicular relaxin as a growth modulator during porcine follicular development.

scholarly journals Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1533
Author(s):  
Aly Warma ◽  
Jacques G. Lussier ◽  
Kalidou Ndiaye
Keyword(s):  
Signal Transduction ◽  
Granulosa Cells ◽  
Target Genes ◽  
Follicular Development ◽  
Signal Transduction Pathways ◽  
Inositol Polyphosphate ◽  
Control Of Gene Expression ◽  
Binding Partners ◽  
Ovarian Follicular Development

Members of the Tribbles (TRIB) family of pseudokinases are critical components of intracellular signal transduction pathways in physiological and pathological processes. TRIBs, including TRIB2, have been previously shown as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling network associated with TRIB2 and its binding partners in granulosa cells during ovarian follicular development is not fully defined. We previously reported that TRIB2 is differentially expressed in growing dominant follicles while downregulated in ovulatory follicles following the luteinizing hormone (LH) surge or human chorionic gonadotropin (hCG) injection. In the present study, we used the yeast two-hybrid screening system and in vitro coimmunoprecipitation assays to identify and confirm TRIB2 interactions in granulosa cells (GCs) of dominant ovarian follicles (DFs), which yielded individual candidate binding partners including calmodulin 1 (CALM1), inhibin subunit beta A (INHBA), inositol polyphosphate phosphatase-like 1 (INPPL1), 5′-nucleotidase ecto (NT5E), stearoyl-CoA desaturase (SCD), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and Ras-associated protein 14 (RAB14). Further analyses showed that all TRIB2 binding partners are expressed in GCs of dominant follicles but are differentially regulated throughout the different stages of follicular development. CRISPR/Cas9-driven inhibition along with pQE-driven overexpression of TRIB2 showed that TRIB2 differently regulates expression of binding partners, which reveals the importance of TRIB2 in the control of gene expression linked to various biological processes such as proliferation, differentiation, cell migration, apoptosis, calcium signaling and metabolism. These data provide a larger view of potential TRIB2-regulated signal transduction pathways in GCs and provide strong evidence that TRIB2 may act as a regulator of target genes during ovarian follicular development.

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