Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (> 18 mm) follicles. RNeasy Micro Kit (Qiagen(A (R))) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Expression et effets des WNTs sur l’expansion du cumulus et la maturation de l’ovocyte chez la vache

Les WNTs sont une famille de glycoprotéines qui, secrétées dans le milieu extracellulaire, jouent un rôle important dans l’embryogenèse. Chez l’adulte, leur dérégulation va entrainer diverses affections incluant des troubles du développement accompagnés ou non de malformations mais aussi des cancers. Au niveau de l’ovaire, le rôle des WNTs demeure peu défini même si des études chez l’humain et la souris prouvent l’implication de certains membres de cette famille dans le développement ovarien ainsi que dans les processus de maturation folliculaire et d’ovulation. Dans ce contexte, nous avons voulu évaluer l’expression de quelques membres de la famille des WNTs (-2, -2b, -4, -5a et -5b) durant l’expansion des cellules du cumulus et évaluer l’effet de certains d’entre eux sur le COC ainsi que la maturation de l’ovocyte chez la vache. Les COCs bovins étaient placés dans une solution de maturation in vitro pendant 0, 6, 12 et 22h et les niveaux d’ARNm mesurés par PCR en temps réel. L’abondance de l’ARNm pour WNT-2b était significativement plus élevée après 6h de maturation comparée aux COCs immatures (à 0h), alors que l’ARNm codant pour WNT-2, -4, -5a et -5b n’augmentait qu’en fin de culture. L’addition d’EGF provoquait l’expansion du COC et la progression de l’ovocyte vers la métaphase II (MII) comme nous l’espérions mais, à notre grande surprise, l’ajout de WNT-2b au milieu de maturation provoquait également l’expansion du COC (82% et 69% pour EGF et WNT-2b respectivement) et la progression de l’ovocyte vers le stade MII (62% et 56% EGF et WNT-2b respectivement). La combinaison d’EGF et WNT-2b n’a pas produit de meilleurs résultats. Notre étude met en lumière l’implication des WNTs dans la maturation du COC chez la vache. Leurs voies d’activation restent toutefois à déterminer.

Vitrification with DAP 213 and Cryotop of Ex Situ and In Situ Feline Cumulus-Oocyte Complexes

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulusoocyte complexes (COCs) vitrified with DAP 213 (2 M DMSO, 1 M acetamide, 3 M propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm3) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29-40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Efeitos e regulação da expressão do kit ligante (KL) durante a maturação in vitro do complexo cumulus-oócito bovino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do estádio do ciclo estral e adição de hormônios ao meio de cultivo in vitro sobre a morfologia do complexo cumulus-oócito canino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulação da expressão de fatores secretados pelo oócito (FSOs) e seus receptores durante a maturação in vitro (MIV) bovina e ações no controle da expressão de cumulus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeitos do fator de crescimento dos fibrobalstos 8 (FGF8) durante a maturação in vitro do complexo cumulus-oócito bovino

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Paracrine and autocrine factors in the differentiation of the cumulus-oocyte complex

A better understanding of the paracrine and autocrine regulatory loops within the cumulus-oocyte complex (COC) is fundamental for the improvement of in vitro maturation (IVM) outcomes in humans and domestic species. This review presents the most important local regulators identified in the COC to date with special attention to those secreted by the oocyte and acting on cumulus cells, as well as their roles in different processes crucial for the successful maturation of the COC. An autocrine regulatory loop mediated by epidermal growth factor-like (EGF-like) peptides in cumulus cells triggers COC maturation. During COC differentiation, oocyte secreted factors (OSFs), particularly members of the transforming growth factor-beta (TGF beta) and fibroblast growth factor (FGF) families, regulate meiotic resumption, cumulus expansion, cumulus metabolism, apoptosis and steroidogenesis.

Expressão gênica de células do cumulus e oócitos bovinos sbmetidos à maturação un vitro com diferentes macromoléculas, EGF e Butirolactina I

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sistema NPPC-NPR2 na espécie bovina: identificação em folículos antrais e modulação na concentração de nucleotídeos cíclicos e na expressão gênica de PDE3 em complexos cumulus-oócito

Pós-graduação em Biociências - FCLAS

Fibroblast growth factor 17 and bone morphogenetic protein 15 enhance cumulus expansion and improve quality of in vitro-produced embryos in cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)