scholarly journals Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

2021 ◽  
Author(s):  
◽  
Shruti Patel
Keyword(s):  
Growth Factors ◽  
Gap Junctions ◽  
Gap Junction ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Dye Transfer ◽  
Antral Follicles ◽  
Fluorescence Dye

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

scholarly journals Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

2021 ◽  
Author(s):  
◽  
Shruti Patel
Keyword(s):  
Growth Factors ◽  
Gap Junctions ◽  
Gap Junction ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Dye Transfer ◽  
Antral Follicles ◽  
Fluorescence Dye

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

scholarly journals The Role of Steroids, Growth Factors and CAMP Stimulators on the Gap Junction Activity in Cumulus Oocyte Complexes in the Rat

2021 ◽  
Author(s):  
◽  
Manvi Yadav
Keyword(s):  
Growth Factors ◽  
Gap Junction ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Relative Increase ◽  
Primary Objective ◽  
Dye Transfer ◽  
Oocyte Growth ◽  
Phosphodiesterase Type

<p>Bidirectional communication between mammalian oocytes and their surrounding somatic cells is essential for oocyte maturation. Gap junctions promote the transfer of essential metabolites, nucleotides, amino acids and ions from cumulus cells to the oocyte that are crucial for oocyte growth and development. However, the range of factors present in the microenvironment of the developing antral follicle, which modulate gap junction activity of the cumulus-oocyte complexes (COCs), is largely unknown. The primary objective of this study was to determine the effects of various steroids, growth factors and cAMP stimulators on the gap junction activity in rat COCs. The gap junction activity was measured in presence or absence of different treatments using a fluorescence dye and in the presence of milrinone, a phosphodiesterase type 3 inhibitor. The major findings of this study were that cAMP stimulators increased the rate of dye transfer from cumulus cells to the oocyte. Under in vitro conditions it was established that neither steroids nor IGF1 by themselves were able to modulate gap junction activity in rat COCs. Furthermore, forskolin, a potent cAMP stimulator; caused a relative increase in Cx37 gene expression levels following a four hours incubation period. The outcomes from the present study may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes.</p>

scholarly journals The Role of Steroids, Growth Factors and CAMP Stimulators on the Gap Junction Activity in Cumulus Oocyte Complexes in the Rat

2021 ◽  
Author(s):  
◽  
Manvi Yadav
Keyword(s):  
Growth Factors ◽  
Gap Junction ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Relative Increase ◽  
Primary Objective ◽  
Dye Transfer ◽  
Oocyte Growth ◽  
Phosphodiesterase Type

<p>Bidirectional communication between mammalian oocytes and their surrounding somatic cells is essential for oocyte maturation. Gap junctions promote the transfer of essential metabolites, nucleotides, amino acids and ions from cumulus cells to the oocyte that are crucial for oocyte growth and development. However, the range of factors present in the microenvironment of the developing antral follicle, which modulate gap junction activity of the cumulus-oocyte complexes (COCs), is largely unknown. The primary objective of this study was to determine the effects of various steroids, growth factors and cAMP stimulators on the gap junction activity in rat COCs. The gap junction activity was measured in presence or absence of different treatments using a fluorescence dye and in the presence of milrinone, a phosphodiesterase type 3 inhibitor. The major findings of this study were that cAMP stimulators increased the rate of dye transfer from cumulus cells to the oocyte. Under in vitro conditions it was established that neither steroids nor IGF1 by themselves were able to modulate gap junction activity in rat COCs. Furthermore, forskolin, a potent cAMP stimulator; caused a relative increase in Cx37 gene expression levels following a four hours incubation period. The outcomes from the present study may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes.</p>

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 The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1761 ◽  
Author(s):  
Cheng-Jie Zhou ◽  
Sha-Na Wu ◽  
Jiang-Peng Shen ◽  
Dong-Hui Wang ◽  
Xiang-Wei Kong ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Beneficial Effects

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affectsin vivoversusin vitromaturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination usingin vivo- orin vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes),in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), andin vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for bothin vivoandin vitrooocyte maturation. In addition, for oocytes maturedin vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes maturedin vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

scholarly journals The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology

Reproduction ◽  
2004 ◽  
Vol 128 (4) ◽  
pp. 379-386 ◽  
Author(s):  
K P McNatty ◽  
L G Moore ◽  
N L Hudson ◽  
L D Quirke ◽  
S B Lawrence ◽  
...  
Keyword(s):  
Growth Factors ◽  
Point Mutations ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Ovulation Rate ◽  
Growth Stages ◽  
New Paradigm ◽  
Protein Conjugates ◽  
Preovulatory Follicles

Ovulation rate in mammals is determined by a complex exchange of hormonal signals between the pituitary gland and the ovary and by a localised exchange of hormones within ovarian follicles between the oocyte and its adjacent somatic cells. From examination of inherited patterns of ovulation rate in sheep, point mutations have been identified in two oocyte-expressed genes, BMP15 (GDF9B) and GDF9. Animals heterozygous for any of these mutations have higher ovulation rates (that is, + 0.8–3) than wild-type contemporaries, whereas those homozygous for each of these mutations are sterile with ovarian follicular development disrupted during the preantral growth stages. Both GDF9 and BMP15 proteins are present in follicular fluid, indicating that they are secreted products. In vitro studies show that granulosa and/or cumulus cells are an important target for both growth factors. Multiple immunisations of sheep with BMP15 or GDF9 peptide protein conjugates show that both growth factors are essential for normal follicular growth and the maturation of preovulatory follicles. Short-term (that is, primary and booster) immunisation with a GDF9 or BMP15 peptide-protein conjugate has been shown to enhance ovulation rate and lamb production. In summary, recent studies of genetic mutations in sheep highlight the importance of oocyte-secreted factors in regulating ovulation rate, and these discoveries may help to explain why some mammals have a predisposition to produce two or more offspring rather than one.

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

Changes of follicular fluid composition during estrous cycle: The effects on oocyte maturation and embryo development in vitro

2019 ◽  
Author(s):  
A.A. Mohammed ◽  
T. Al-Shaheen ◽  
S. Al-Suwaiegh
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Extracellular Fluid ◽  
Cumulus Cells ◽  
Fluid Composition ◽  
Lh Surge ◽  
Antral Follicles ◽  
Positive Effects

Oocytes are bathed in extracellular fluid of the antral follicles, which is termed follicular fluid (FF). Follicular fluid is synthesized from secretions of theca, granulosa, and cumulus cells and from a transudate of blood plasma. Oocytes persist in meiotic arrest in antral follicles until luteinizing hormone (LH) surge or removal the oocytes from the ovarian follicles. This suggests that FF before LH surge might contain meiosis inhibiting factor(s). The microvasculatory bed of the follicular wall and the composition of FF undergo changes during follicular growth and development, which is important for oocyte maturation and subsequent embryo development. Therefore, it is expected that FF composition and components might change according to timing of FF aspiration from follicles. Hence, negative or positive effects could be expected when FF supplemented during oocyte maturation in vitro. Nutrition effects on microvasculatory bed of follicles and their sizes. Thus, the nutritional status of animals is a factor affected on oocyte maturation and embryo development. The present article reviews and discusses these effects.

245 CONNEXIN 43 mRNA EXPRESSION AT DIFFERENT TIME POINT IN IN VITRO MATURATION OF BUFFALO OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 220
Author(s):  
S. C. Gupta ◽  
A. Pandey ◽  
N. Gupta
Keyword(s):  
Gap Junctions ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Cumulus Cells ◽  
Embryo Production ◽  
Cx43 Expression ◽  
Additional Advantage ◽  
Significant Difference ◽  
Cx43 Mrna

In advanced technologies of ART, the basic requirement is the production of in vitro-matured oocytes, and embryo production efficiency depends on healthy, matured oocytes. Oocyte growth and development depends on the ability of oocytes and their surrounding cumulus granulosa cells (Eppig et al. 1979 J. Exp. Zool. 208, 111–120). Cumulus cells provide carbohydrate precursors, amino acids, and nucleotides to the oocytes (Brower and Schultz 1982 Dev. Biol. 90, 144–153). Oocytes and cumulus cell gap junctions are required for the coordination of cytoplasmic and nuclear maturation (Carabatsos et al. 2002 Dev. Biol. 226, 167–179). In bovine COC, functional gap junctions are required for the progression of oocyte maturation. Gap junctions allow for metabolic coupling between adjacent granulosa cells. Disruption in the integrity of the gap junction inhibits oocyte maturation (Anderson and Albertini 1976 J. Cell Biol. 71, 680–686). The aim of this study was to analyze the trend of Cx43 mRNA transcript in in vitro-matured oocytes at different times of maturation in the Indian water buffalo to estimate the correlation with expression level. Oocytes collected from slaughterhouse ovaries were matured in TCM-199 medium supplemented with 2.5 mm pyruvate, gentamycin sulfate (10 mg mL–1), β-estradiol (1000 ng mL–1), FSH (500 ng mL–1), LH (500 ng mL–1), and 10% FBS at 38.5°C in 5% CO2 in air. Cumulus–oocyte complexes were used after 0, 6, 12, 18, and 24 h of maturation for the cDNA preparation with cells of a cDNA II Kit. Expression of the Cx43 gene was quantified at different time intervals for maturation with real-time PCR. Statistical analysis was performed with one-way ANOVA, followed by Duncan’s multiple pair-wise comparison. Our results showed that Cx43 mRNA abundance was affected by time of maturation. The expression of Cx43 was significantly higher at 6 h than at 18 and 24 h, whereas the 12-h value was intermediate. Our results are in agreement with decreased Cx43 protein contents in the outer cumulus layers of COC at maturation time points (Calder et al. 2003 Reprod. Biol. Endocrinol. 1, 14) and the expression of Cx43 in oocyte development regulation (Granot et al. 2002 Biol. Reprod. 66, 568–573). When Cx43 expression was compared among immature oocytes, denuded oocytes, cumulus cells, and COC at 6 h, there was no significant difference. However, 6-h-matured COC showed significantly higher expression than other groups. Further, our study supported the role of cumulus cells in COC in Cx43-mediated communication (Vozzi et al. 2001 Reproduction 122, 619–628). Differential expression of Cx43 mRNA among varying COC classes indicates that this gene may be a useful marker for oocyte quality to improve in vitro production or somatic cell nuclear transfer rates. Marker genes that predict developmental competence could be used in the optimization of maturation and culture conditions. Understanding the molecular mechanism involved in in vitro oocyte maturation would be an additional advantage in analyzing this complex biological phenomenon to improve embryo production.

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