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 R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

2020 ◽  
Vol 27 (10) ◽  
pp. 2856-2871 ◽  
Author(s):  
Marie-Cécile De Cian ◽  
Elodie P. Gregoire ◽  
Morgane Le Rolle ◽  
Simon Lachambre ◽  
Magali Mondin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Cycle Progression ◽  
Ovarian Function ◽  
Follicular Growth ◽  
Oocyte Growth

Abstract R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/β-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility.

Influence of granulosa cells and of different somatic cell types on mammalian oocyte development in vitro

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 305-307 ◽  
Author(s):  
Sadra Cecconi ◽  
Rosella Colonna
Keyword(s):  
Gap Junctions ◽  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Cell Types ◽  
Paracrine Factors ◽  
Oocyte Growth ◽  
Major Route ◽  
High Concentrations ◽  
Development In Vitro

In mammals the ability of an oocyte to become fertilised is the result of a complex process occurring within the ovarian follicle which depends on the stagespecific expression of oocyte genes and the presence of granulosa cells (for a review see Buccioneet al., 1990a). The coordinated development of germinal and somatic components of the follicle is regulated by two principal systems of interaction, based on the presence of gap junctions and on the production of paracrine factors. Gap junctions link granulosa cells to each other and to the oocyte (Anderson & Albertini, 1976), and represent a major route for the transfer of small molecules involved in oocyte metabolism (for a review see Mangiaet al., 1992) and regulation of the arrest and resumption of meiosis (for a review see Eppig, 1993). The production of paracrine factors by granulosa cells has been suggested by the findings that these cells express the production of theSteellocus, the Steel factor (SLF) or kit ligand (KL; Motroet al., 1991; Manovaet al., 1993), and that this factor promotes oocyte growthin vitrowhen used at high concentrations (Packeret al., 1994). Since KL is too large to be transmitted through gap junctions, it must necessarily be released in the extracellular environment before binding to the c-kitreceptor present on oocyte membrane (Manovaet al., 1990; Horieet al., 1991).

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 Small RNA sequencing reveals distinct nuclear microRNAs in pig granulosa cells during ovarian follicle growth

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Derek Toms ◽  
Bo Pan ◽  
Yinshan Bai ◽  
Julang Li
Keyword(s):  
Granulosa Cells ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Ovarian Function ◽  
Ovarian Follicle ◽  
Small Rna Sequencing ◽  
Cell Fractionation ◽  
Steroid Synthesis ◽  
Non Coding Rna ◽  
Dna Binding Sites

AbstractNuclear small RNAs have emerged as an important subset of non-coding RNA species that are capable of regulating gene expression. A type of small RNA, microRNA (miRNA) have been shown to regulate development of the ovarian follicle via canonical targeting and translational repression. Little has been done to study these molecules at a subcellular level. Using cell fractionation and high throughput sequencing, we surveyed cytoplasmic and nuclear small RNA found in the granulosa cells of the pig ovarian antral preovulatory follicle. Bioinformatics analysis revealed a diverse network of small RNA that differ in their subcellular distribution and implied function. We identified predicted genomic DNA binding sites for nucleus-enriched miRNAs that may potentially be involved in transcriptional regulation. The small nucleolar RNA (snoRNA) SNORA73, known to be involved in steroid synthesis, was also found to be highly enriched in the cytoplasm, suggesting a role for snoRNA species in ovarian function. Taken together, these data provide an important resource to study the small RNAome in ovarian follicles and how they may impact fertility.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

scholarly journals A rapid and robust method for the cryopreservation of human granulosa cells

2021 ◽  
Author(s):  
Sarah Beschta ◽  
Katja Eubler ◽  
Nancy Bohne ◽  
Ignasi Forne ◽  
Dieter Berg ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Large Scale ◽  
Ovarian Function ◽  
Oocyte Retrieval ◽  
Cell Contact ◽  
Cellular Model ◽  
Preovulatory Follicle ◽  
Human Granulosa Cells ◽  
Ovarian Cells

AbstractHuman primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell–cell contact genes (GJA1) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Role of Melatonin as a Survival Factor for In vitro Development of Sheep Preantral Follicles

2021 ◽  
Author(s):  
C. Chetan Kumar ◽  
B. Rambabu Naik ◽  
A.V.N. Siva Kumar ◽  
A. Ravi ◽  
L.S.S. Varaprasad Reddy ◽  
...  
Keyword(s):  
Growth Factors ◽  
Ovarian Function ◽  
Follicular Development ◽  
Oocyte Quality ◽  
Free Radical Scavenger ◽  
Radical Scavenger ◽  
Optimum Level ◽  
Preantral Follicles ◽  
Positive Effects

Background: Melatonin, a powerful free radical scavenger and broad-spectrum antioxidant may directly affect ovarian function by regulating folliculogenesis, maintenance of follicular integrity, oocyte quality and maturation capacity. Therefore, we aimed to study effects of melatonin and its interaction with growth factors in sheep preantral follicles. Methods: The influence of different concentrations of Melatonin (5-500 pM) on in vitro culture of preantral follicles (PFs’) isolated from sheep ovaries was studied. Experiments I and II were conducted to standardize the optimum concentration of Melatonin that supports better development of preantral follicles. Experiment III was conducted with the optimum level of Melatonin derived in the Experiments I and II to evaluate the effect of melatonin at 100pM in combination with various growth factors. Result: Overall follicular development was found to be the best in the PFs’ cultured in medium supplemented with 100pM of Melatonin. Melatonin supplementation showed positive effects on the preantral follicular development in combination with different growth factors.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

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