Granulosa Cell Co-culture Research Articles

WNT4 promotes macrophage polarization via granulosa cell M-CSF and reduces granulosa cell apoptosis in endometriosis

BackgroundWNT4 gene polymorphism are common in endometriosis and may functionally link estrogen and estrogen receptor signaling. Previous study confirmed estrogen and estrogen receptor signaling recruit macrophage to promote the pathogenesis of endometriosis. To investigate the effect of WNT4 in endometriosis involved in macrophage polarization and whether WNT4 could reduce the apoptosis of granulosa cells. MethodsAn observational study consisting of 8 cases of women with endometriosis (diagnosed by surgery and histology) and 22 mice of endometriosis animal model was conducted. Granulosa cells were isolated from 16 patients with endometriosis and co-cultured with macrophage under WNT4 treatment using TUNEL assay, quantitative reverse transcription PCR, flow cytometry and ELISA analysis. 22 mice of endometriosis animal model confirmed the WNT4 treatment effects using histology and immunohistochemistry, Western blot and flow cytometry. ResultsWe observed that the apoptotic proportion of granulosa cells was significantly decreased and M2 macrophage was significantly increased after WNT4 treatment during the granulosa cell and macrophage co-culture system. To reveal the underlying mechanism for this, we conducted a series of experiments and found that high expression of granulosa cell M-CSF led to the M2 polarization of macrophages. The animal model also suggested that the anti-apoptotic effect of WNT4 on granulosa cells were conducted by the M2 polarized macrophage. ConclusionsWNT4 could reduce granulosa cell apoptosis and improve ovarian reserve by promoting macrophage polarization in endometriosis. M-CSF secreted by granulosa cell after WNT4 treatment was the main mediator of macrophage polarization.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

Effect of addition of follicular fluid, oviductal or granulosa cell co-culture on In vitro cleavage and embryonic development in sheep oocytes

Effect of addition of follicular fluid, oviductal or granulosa cell co-culture on In vitro cleavage and embryonic development in sheep oocytes

Comparative effects of retinoic acid, granulosa cells conditioned medium or forskolin in combination with granulosa cell co-culturing on mouse germ cell differentiation.

Devising of an appropriate in vitro culture method for germ cells differentiation in the presence of soluble factors has attracted considerable attention, which results will provide new insight into reproductive biology. In this study, we compared the effects of forskolin, retinoic acid (RA) or granulosa cell-conditioned medium in the presence or absence of granulosa cell co-culturing on germ cell differentiation from embryonic stem cells (ESCs). Embryonic stem cells were differentiated using embryoid bodies (EBs) for 5 days, and then EB-derived cells were co-cultured with or without adult mouse granulosa cells using monolayer protocol and treated with 50 µM forskolin, 1 µM RA and 50% granulosa cell-conditioned medium for 4 days. Granulosa cell-conditioned medium significantly increased the levels of Scp3, Rec8, Mvh and Gdf9 expression in the granulosa cell co-culture method compared to untreated cells. A significant elevation of Stra8, Rec8 and Mvh was observed after treatment with RA in the absence of granulosa cells and there was no significant increase in the levels of expression of germ cell-specific genes after treatment with forskolin compared to control. Furthermore, forskolin and RA significantly increased viability and proliferation of germ-like cells, compared with granulosa cell-conditioned medium. Our study revealed that granulosa cell-conditioned medium and RA effectively can induce germ cell differentiation from ESCs, however combined application of granulosa cell-conditioned medium and co-culturing with granulosa cells had synergic effect on germ cell development in vitro as optimized protocol.

FTO protects human granulosa cells from chemotherapy-induced cytotoxicity

BackgroundPremature ovarian failure (POF) is a serious problem for young women who receive chemotherapy, and its pathophysiological basis is the dysfunction of granulosa cells. According to previous reports, menstrual-derived stem cells (MenSCs) can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF. Fat mass- and obesity-associated (FTO) was reported to be associated with oocyte development and maturation. FTO was decreased in POF and may be a biomarker for the occurrence of POF. Knockdown of FTO in granulosa cells promoted cell apoptosis and inhibited proliferation. But the relationship between FTO and ovarian repair was still unclear. This study was aimed at investigating the FTO expression level and the role of FTO in the MenSCs recovering the function of injured granulosa cells.MethodFirst, cisplatin was used to establish a granulosa cell injury model. Then, the MenSCs and injured granulosa cell coculture model and POF mouse model were established in this study to explore the role of FTO. Furthermore, gain- and loss-of-function studies, small interfering RNA transfection, and meclofenamic acid (MA), a highly selective inhibitor of FTO, studies were also conducted to clarify the regulatory mechanism of FTO in granulosa cells.ResultsMenSCs coculture could improve the function of injured granulosa cells by increasing the expression of FTO. MenSCs transplantation restored the expression of FTO in the ovaries of POF mice. Overexpression of FTO restored the injured cell proliferation and decreased apoptosis by regulating the expression of BNIP3. Down-regulation of FTO got the opposite results.ConclusionsIn the treatment of MenSCs, FTO has a protective effect, which could improve the viability of granulosa cells after cisplatin treatment by decreasing the expression of BNIP3. Meanwhile, FTO may provide new insight into therapeutic targets for the chemotherapy-induced POF.

In vitro buffalo embryo production under heat stress: Role of granulosa cells co-culture as a monolayer

Effect ofheat shock and co-culture of monolayer granulosa cells (GCs) on in vitroembryo developmental competence in Egyptian buffalo was the aim of the present study.Cumulus oocytes complexes (COCs) were collected from ovaries (n= 448) of cyclicslaughtered buffalo cows. Good quality immature oocytes (n= 1512) were subjected to in vitro maturation and fertilization. Post 18-22 h of fertilization, presumptive embryos were randomly assigned into four groups: (G1) No heat shock (38.5OC), (G2) Heat shock (40.5°C) and (G3) Co-culture with GCs monolayer and heat shock or (G4) Co-culture with GCs monolayer and no heat shock. Heat-shockedembryos were exposed to temperature of40.5°Cfor the first two hours of culture before exposing to 38.5°C afterward up to the end of culture perio d (8 days). The data were analyzed by General LinearUnivariate model using SPSS. Oocyte expansionrate was 90.8±1.1%. This percentage is higher than nuclear maturation rate (oocytes at Telophase and Metaphase II) as detected byaceto-orcein staining by about 17%. Cleavage rate as recorded at day 3 post fertilization (day 0) was higher (p≤0.05) for G1 and G4 (71.1±10.5% and 70.5±7.9 with GCs, respectively).On the other hand, cleavage rate was dramatically decreased (P ≤0.05) for embryos of G2 (43.7±7.0%) compared with that of G3 (80.2±7.0%). In addition, embryos of G3showed approximately the same rate ofdeveloped embryos(Morula and blastocyst stages at day 8 of culture) as of G1 (50.9±5.3 and51.7±7.9%, respectively). In conclusion, using co-culture of GCs as a monolayer enhances cleavage rate and subsequent embryo development of buffalo embryos under heat stress condition.

The Attenuating Effect of the Intraovarian Bone Morphogenetic Protein 4 on Age-Related Endoplasmic Reticulum Stress in Chicken Follicular Cells.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca2+-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

A paracrine interaction between granulosa cells and leukocytes in the preovulatory follicle causes the increase in follicular G-CSF levels

ObjectiveFollicular granulocyte colony-stimulating factor (G-CSF) is a new biomarker of oocyte quality and embryo implantation in in vitro fertilization (IVF) cycles. Its role in reproduction is poorly understood. Our study aimed to investigate the mechanisms and cells responsible for G-CSF production in the preovulatory follicle.DesignLaboratory research study.SettingSingle-center study.InterventionsGranulosa cells and leukocytes were isolated from the follicular fluids (FF) or the blood of women undergoing IVF and from the blood of a control group of women with spontaneous ovulatory cycles to perform cocultures.Main outcome measureG-CSF-secreted protein was quantified in the conditioned media of cocultures.ResultsG-CSF secretion was considerably increased in cocultures of granulosa cells and leukocytes. This effect was maximal when leukocytes were isolated from the blood of women in the late follicular phase of the menstrual cycle or from the FF of women undergoing IVF. The leukocyte population isolated from the FF samples of women undergoing IVF had a higher proportion of granulocytes than that isolated from the corresponding blood samples. Leukocytes induced the synthesis and secretion of G-CSF by granulosa cells. Among a range of other FF cytokines/chemokines, only growth-regulated oncogene alpha (GROα) was also increased.ConclusionThe notable rise in G-CSF at the time of ovulation coincides with the accumulation of follicular granulocytes, which stimulate G-CSF production by granulosa cells via paracrine interactions. High follicular G-CSF concentrations may occur in follicles with optimal granulosa–leukocyte interactions, which could explain the increased implantation rate of embryos arising from these follicles.

MiR-644-5p carried by bone mesenchymal stem cell-derived exosomes targets regulation of p53 to inhibit ovarian granulosa cell apoptosis

BackgroundThis article aims to reveal the therapeutic effects and potential mechanisms of bone mesenchymal stem cell (BMSC)-derived exosomes on premature ovarian failure (POF).MethodsExosomes were collected from BMSCs and were used to treat cisplatin-induced POF mouse models. Pathological changes of ovarian tissue were detected by using HE staining and by Western blot that detected the expression of apoptosis-related proteins. In cisplatin-induced primary granulosa cell injury, exosomes were co-cultured with the granulosa cells. The apoptosis or viability of granulosa cells was analyzed by flow cytometry or MTT, respectively. In Target scan and microT-CDS databases, an intersection of miRNAs targeting to p53 was found. The expressions of miRNAs in BMSC-derived exosomes were detected by qRT-PCR. Besides, miR-664-5p targeted to p53 of cells was verified by dual-luciferase reporter assay.ResultsBMSC-derived exosomes improved the follicular morphology of POF mice and inhibited the expression of apoptosis-related protein. By co-culture of exosomes and primary granulosa cells, BMSC-derived exosomes repressed cisplatin-induced granulosa cells apoptosis and increased cells viability, while these effects were abrogated after the exosome-containing RNA was degraded by RNase. By Target scan, microT-CDS and qRT-PCR, miR-664-5p was regarded as the dominated RNA in BMSC-derived exosomes. By dual-luciferase reporter assay, miR-664-5p negatively regulated p53 luciferase activity. After shRNA interfering miR-664-5p of BMSC, BMSC-derived exosomes exerted no protective effect on cisplatin-induced granulosa cell apoptosis.ConclusionOur results indicated that miR-644-5p carried by BMSC-derived exosomes inhibited the apoptosis of ovarian granulosa cell by targeting p53 of cells, suggesting that miR-644-5p had the potential to treat POF and restore ovarian function.

The Cell Type-Specific Expression of Lhcgr in Mouse Ovarian Cells: Evidence for a DNA-Demethylation-Dependent Mechanism.

The luteinizing hormone receptor (LHCGR) is expressed at low levels in mural granulosa cells and cumulus cells of antral follicles and is induced dramatically in granulosa cells but not in cumulus cells by follicle-stimulating hormone (FSH). Therefore, we hypothesized that FSH not only activates transcription factors controlling Lhcgr expression but also alters other events to permit and enhance Lhcgr expression in granulosa cells but not in cumulus cells. In granulosa cells, the level of DNA methylation in the Lhcgr promoter region was significantly decreased by equine chorionic gonadotropin (eCG) in vivo. However, in cumulus cells, hypermethylation of the Lhcgr promoter remained after eCG stimulation. eCG induced estrogen production from testosterone (T) and retinoic acid (RA) synthesis in granulosa cells. When either T or RA in the presence or absence of FSH was added to granulosa cell cultures, the combined treatment with FSH and RA induced demethylation of Lhcgr-promoter region and Lhcgr expression. FSH-dependent RA synthesis was negatively regulated by coculture of granulosa cells with denuded oocytes, suggesting that oocyte-secreted factors downregulate RA production in cumulus cells where Lhcgr expression was not induced. Strikingly, treatment of cultured cumulus-oocyte complexes with a SMAD inhibitor, SB431542, significantly induced RA production, demethylation of Lhcgr-promoter region, and Lhcgr expression in cumulus cells. These results indicate the demethylation of the Lhcgr-promoter region is mediated, at least in part, by RA synthesis and is a key mechanism regulating the cell type-specific differentiation during follicular development.

Effects of chlorogenic acid on the maturation and fertilisation of bovine oocytes and their embryonic development with a comparative bovine granulosa cell co-culture

Effects of chlorogenic acid on the maturation and fertilisation of bovine oocytes and their embryonic development with a comparative bovine granulosa cell co-culture

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 103 b-ATMSCs/mL (p = 0.051), group 104 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

Intracellular lipid dysregulation interferes with leukocyte function in the ovaries of meat-type hens under unrestricted feed intake

Meat-type Red-feather country hens fed ad libitum (AD-hens) exhibit obesity-associated morbidities and a number of ovarian irregularities. Leukocyte participations in ovarian activities are unstudied in AD-hens. In contrast to feed-restricted hens (R-hens), ovulatory process of the F1 follicle appeared delayed in AD-hens in association with reduced F1 follicle progesterone content, gelatinase A (MMP-2) and collagenase-3 (MMP-13) activities coincident with elevated IL-1β and no production (P<0.05), and increased leukocyte infiltration of inflamed necrotic follicle walls. Extracts of AD-hen F1 follicle walls induced greater leukocyte migration than extracts from F1 follicle wall extracts of R-hens (P<0.05). Co-cultures of granulosa cells with increasing numbers of leukocytes from either AD-hens or R-hens exhibited dose dependent reductions in progesterone production and increases in cell death. AD-hen leukocytes were less proapoptotic than their R counterparts (P<0.05). Granulosa MMP-13 and MMP-2 activities were also suppressed in the co-cultures with heterophils or monocytes in a dose-dependent manner (P<0.05). AD heterophils and R monocytes had a greater inhibitory effect on MMP activities in the co-cultures than their respective counterparts (P<0.05). Both basal and LPS-induced IL-1β secretion and MMP-22 or MMP-2 activities in freshly isolated AD-hen leukocytes were reduced (P<0.05). Exposure of AD or R leukocytes to 0.5mM palmitate impaired IL-1β secretion and MMP-22 or MMP-2 activity. Inhibition of ceramide synthesis with FB1 and ROS production with n-MPG scavenging rescued MMP activity and IL-1β production in palmitate treated heterophils, but exacerbated monocyte suppression. These latter findings suggest that intracellular lipid dysregulation in leukocytes contributes to ovarian dysfunction in AD-hens.

Porcine embryo production following in vitro fertilization and intracytoplasmic sperm injection from vitrified immature oocytes matured with a granulosa cell co-culture system

This study was designed to evaluate the capacity of vitrified–warmed porcine immature oocytes to mature and to be fertilized using in vitro fertilization or intracytoplasmic sperm injection, and to determine the subsequent embryo development. Immature oocytes were vitrified using ethylene glycol and dimethylsulphoxide as cryoprotectants and the Cryolock method. After warming oocytes were cultured 44h for maturation. Oocytes were randomly distributed in three treatment groups and subjected to in vitro fertilization (Experiment 1) or intracytoplasmic sperm injection (Experiment 2) procedures. The results indicate that the embryo development was higher in denuded oocytes co-cultured with granulosa cells (NkO-CC group) fertilized by in vitro fertilization or intracytoplasmic sperm injection compared to cumulus-cell oocyte complexes (COCs group), showing no significant differences with control. Vitrified denuded oocytes matured with a co-culture system NkO-CC group, displayed higher cleavage rate and blastocyst production than vitrified COCs group. Blastocysts were successfully obtained after IVF and ICSI procedures; however, the development to the blastocyst stage was better after IVF. These results show that the vitrification–warming media, the employment of a granulosa cell co-culture system and the Cryolock method during vitrification, increased the nuclear and cytoplasmic maturation of vitrified porcine immature oocytes. Further experiments are required to enhance porcine embryo production after vitrification.

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina.

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as “starting material” for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.

In vitro maturation of nude caprine oocytes recovered during oocyte collection

The present study was undertaken to investigate the effect of granulosa cell co-culture, granulosa monolayer and hormone on in vitro maturation of nude caprine oocytes. Nude oocytes (644) having evenly granulated cytoplasm were recovered by puncture technique. The oocytes were divided into 6 groups and matured in TCM-199 medium supplemented with 10% fetal bovine serum (FBS) and granulosa cell co-culture/ granulosa monolayer/ hormones. After maturation (27h), the oocytes were fixed for 15 min in 2.5% (w/v) glutaraldehyde in phosphate buffer saline,washed twice with PBS, stained with 0.1mg/ml DAPI in PBS and mounted on slides. The evaluation of nuclear status was done by epiflourescence microscope by observing either a polar body release or presence of two shiny chromatin spots. The maturation rate of oocytes in group 1, 2, 3, 4, 5 and 6 were 23.47, 32.40, 31.77, 26.00, 38.09 and 51.37%, respectively. The results of the study indicated that TCM-199 supplemented with hormones and coculture granulosa cells significantly increases the maturation rate of in vitro matured nude caprine oocytes.

Photoinduction of granulosa cell and oocyte co-culture to influence in vitro maturation and fertilisation

Infertility issues can be attended to by in vitro maturation (IVM) of an imma- ture oocyte into a developmentally compe- tent oocyte followed by in vitro fertilisation (IVF). The hypothesis presented here is a consolidated logical outcome of indepen- dent research publications concluding by suggesting photoirradiation of granulosa cu- mulus cells and oocytes as means to influ - ence IVM and IVF in humans and animals with possible outcomes discussed. INTRODUCTION Fertility issues are mostly associated with the quality of the oocyte 1 which in turn is determined by its matu- ration signals. One other factor influenc - ing fertility is the fusion of sperm and egg which is determined by cell surface-as- sociated proteins like CD81 and CD9 2,3 . Attending to these factors during in vitro maturation (IVM) or during in vitro fertiliza- tion (IVF) would increase the success rate of embryo formation. IVM has been sug- gested as an effective technique to solve infertility issues and also as a tool in wild- life or endangered species conservation 4 health risks; (ii) Normal ovulation follow- ed by egg retrieval which is used for IVF achieved by ICSI or incubation with sperm-egg overnight followed by embryo transfer; (iii) Instead of mature egg retriev- al, sometimes the cumulus cells (trans- formed granulosa cells) with the oocyte, called COC or cumulus oocyte complex, is retrieved and cultured in vitro and the mature egg is used for IVF by ICSI or incubation with sperm-egg overnight fol- lowed by embryo transfer. The oocyte and a single layer of squamous granu- losa cells, collectively called the primor- dial follicle, transits from primary (unilami- nar) to secondary (multilaminar) to mature (tertiary) follicle, culminating with antrum formation and the release of the oocyte along with few granulosa cells called the corona radiata. The remnant of the granu- losa cells get transformed into corpus lut- eum which is important for maintaining pregnancy through the release of proges-

Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth

s from the 4th Mammalian Embryo Genomics meeting Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth M.M.H. Sohel, S. Seifi Noferesti, D. Salilew-Wondim, M. Hoelker, F. Rings, E. Tholen, J. Udin, C. Looft, K. Schellander, D. Tesfaye ∗ Animal Breeding and Husbandry Group, Institute of Animal Science, University of Bonn, Germany Here we aimed to investigate the expression pattern of the circulating extra-cellular miRNAs in exosome and non-exosomal fraction of follicular fluid consisted of fully grown or growing oocytes and to validate exosome mediated cell–cell communication between follicular cells. For this, follicles of 5–8mm diameter (n=120) were isolated and individual COCs were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown, n=60) and BCB− (growing, n=60) groups. The corresponding follicular fluid, granulosa cells and theca cells were used for further molecular analysis. miRNAs isolated from exosomal and nonexosomal portion of follicular fluid from the two categories was used for cDNA synthesis and subsequent analysis using a humanmiRNA PCR array (with 745miRNA). Results revealed that 25miRNAs (16 up and 9 down regulated) in exosomes and 30miRNAs (21 up and 9 down regulated) in non-exosomal portion of follicular fluid were differentially expressed (fold change≥2 and p<0.05) between growing vs. fully grown oocyte group. Among these miRNAs, miR-654-5p and miR-640 were found to be enriched in exosomal portion of follicular fluid containing growing oocytes, while miR-526b* and miR-373 were highly abundant in exosomal portion of follicular fluid This paper is part of a special issue entitled: 4th Mammalian Embryo Genomicsmeeting, Guest Edited byMarc-Andre Sirard, ClaudeRobert and Julie Nieminen. containing fully grown oocytes. In-silico analysis of miRNAs enriched in follicular fluid containing growing oocytes revealed that genes are involved indifferent signalingpathways like ubiquitin mediated proteolysis, focal adhesion, oocyte meiosis, MAPK signaling pathways to be potential targets, which are crucial for follicular development. Co-culture of granulosa cells with PKH67 dye labeled exosomes showedsuccessfuluptakeof exosomesby thosecells and subsequently elevated the endogenous miRNAs level in 2.5–5.5 fold. In conclusion, the present study highlighted the oocyte growth status dependent differences of circulatingmiRNAprofiles in follicularfluidandexosomemediated uptake of thosemolecules by surrounding cells in follicular microenvironment. http://dx.doi.org/10.1016/j.anireprosci.2014.06.011 Global transcriptome analysis of bovine blastocysts developed under alternative vivo/vitro culture conditions during specifics stages of development A. Gad1,4,∗, U. Besenfelder2, V. Havlicek2, M. Holker1, F. Rings1, I. Dufort3, M.A. Sirard3, K. Schellander1, D. Tesfaye1 1 Institute of Animal Science, University of Bonn, Bonn, Germany 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria 3 Centre de recherche en biologie de la reproduction, Universite Laval, Quebec, Canada 4 Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt During the period frommaturation until blastocyst formation, several critical events occur in embryos which are regulated by a harmonized expression of genes. However, the exact influence of in vitro culture conditions 0378-4320/$ – see front matter

Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells

Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4–BMP7–BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.

Action of Halowax 1051 on Enzymes of Phase I (CYP1A1) and Phase II (SULT1A and COMT) Metabolism in the Pig Ovary

Polychlorinated naphthalenes (PCNs) are a group of organochlorinated compounds exhibiting dioxin-like properties. Previously published data showed the direct action of PCN-rich Halowax 1051 on ovarian follicular steroidogenesis. Taking into consideration that the observed biological effects of PCNs may be frequently side effects of metabolites generated by their detoxification, the aim of this study was to determine the activity and expression of enzymes involved in phase I (cytochrome P450, family 1 (CYP1A1)) and phase II (sulfotransferase (SULT1A) and catechol-O-methyltransferase (COMT)) detoxification metabolism. Cocultures of granulosa and theca interna cells collected from sexually mature pigs were exposed to 1 pg/mL to 10 ng/mL of Halowax 1051 for 1 to 48 hours, after which levels and activities of CYP1A1, SULT1A, and COMT were measured. Dose-dependent increases of CYP1A1 activity and expression were observed. High doses of Halowax 1051 were inhibitory to COMT and SULT1A activity and reduced their protein levels. In conclusion, fast activation of phase I enzymes with simultaneous inhibition of phase II enzymes indicates that the previously observed effect of Halowax 1051 on follicular steroidogenesis may partially result from metabolite action occurring locally in ovarian follicles.