Pig Cumulus Cells Research Articles

Role and action mechanisms of miR-149 and miR-31 in regulating function of pig cumulus cells and oocytes

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor β2 (TGFB2), respectively, in the transforming growth factor-β (TGF-β) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-β signaling, suggesting that they might be used as markers for pig oocyte quality.

Effects of CRH and ACTH exposure during in vitro maturation on competence of pig and mouse oocytes

Although it is known that stresses on females damage oocytes with increased production of stress hormones, whether corticotrophin-releasing hormone (CRH) or adrenocorticotropic hormone (ACTH) harm oocytes directly are largely unknown. We demonstrated that CRH exposure during in vitro maturation impaired competence of both pig and mouse cumulus-oocyte-complexes (COCs), and it impaired competence and induced apoptosis in pig cumulus-denuded oocytes (DOs) but not in mouse DOs. CRH receptor 1 was expressed in pig DOs and in cumulus cells (CCs) of both species but not in mouse DOs. In the presence of CRH, whereas mouse CCs underwent apoptosis, pig CCs did not. While pig CCs did, mouse CCs did not express CRH-binding protein. ACTH did not affect competence of either pig or mouse COCs or DOs although they all expressed ACTH receptor. Both pig and mouse CCs expressed steroidogenic acute regulatory protein (StAR), and ACTH enhanced their progesterone production while alleviating their apoptosis. Neither pig nor mouse DOs expressed StAR, but ACTH inhibited maturation-promoting factor and decelerated meiotic progression of DOs suggesting activation of protein kinase A (PKA). In conclusion, CRH impaired pig and mouse oocyte competence by interacting with CRH receptor and inducing CCs apoptosis, respectively. ACTH activated PKA in both DOs and CCs although it showed no effect on oocyte competence.

RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming.

The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes.

Involvement of EGFR and Smad2/3 Activation in FSH-Induced Porcine Oocytes-Cumulus Cell Complex Maturation.

It has been shown that the activation of both EGF receptor and SMAD2 and SMAD3 in mouse cumulus cells is essential for their preovulatory maturation. In the present study we investigated whether these changes are also required for the final differentiation of pig cumulus cells. Porcine oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the absence or in the presence of the following inhibitors: AG1478 (an EGFR tyrosine kinase inhibitor), SB431542 (a specific inhibitor of SMAD2 and SMAD3 activation) and SIS3 (a specific inhibitor of SMAD3 activation). We found that EGFR inhibitor AG1478 completely blocked FSH-induced cumulus expansion by preventing hyaluronic acid production and its incorporation within the extracellular matrix. In addition, in the presence of AG1478, most of FSH-stimulated OCCs were in germinal vesicle stage (GV) and did not mature. Conversely, AG1478 did not affect the stimulation of progesterone synthesis by FSH. In agreement, EGF was unable to stimulate progesterone synthesis by OCCs cultured in vitro. The SB431542, that inhibits the activation of SMAD2 and SMAD3, only partially inhibited cumulus expansion by slightly decreasing both total production and matrix accumulation of hyaluronic acid. Surprisingly, SIS, that inhibits only SMAD3, produced an inhibitory effect higher than SB431542. A divergent effect of the two inhibitors was observed on steroid synthesis: FSH-induced progesterone synthesis increased in the presence of SB431542 and decreased in the presence of SIS. Both inhibitors did not affect the induction of oocyte meiotic resumption promoted by FSH. These results suggest that in pig, like in mice, FSH stimulates cumulus cells to produce EGF that acting in an autocrine way induces the formation of the expanded matrix and the signaling for oocyte meiotic resumption. The results also suggest that SMAD2 and SMAD3 activation is required for optimal stimulation of hyaluronic acid and proteins involved in the organization of this polymer in the pig OCC matrix. Finally, FSH stimulation of progesterone synthesis seems to be independent from EGF, while it appears to be counteracted by SMAD2, but not SMAD3 activation, thereby suggesting that specific SMAD2 signaling pathway is probably indispensable to prevent luteinization of cumulus cells. Supported by grant GACR 523/08/0111 and VEGA 2/0153/08. (poster)

Daidzein does affect progesterone secretion by pig cumulus cells but it does not impair oocytes IVM

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 μM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development.

Covalent transfer of heavy chains of inter-alpha-trypsin inhibitor family proteins to hyaluronan in in vivo and in vitro expanded porcine oocyte-cumulus complexes.

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with IalphaI antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization.

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.

Vitrification and rapid-freezing of cumulus cells from rabbits and pigs

To use adult somatic cloning technology in animal breeding, this technology should be complemented with nuclear donor cell cryopreservation. Two different conventional nonequilibrium methods (vitrification, V: 3.58M EG and 2.82M DMSO in PBS plus 20% FCS and rapid-freezing, RF: 0.25M sucrose, 2.25M EG and 2.25M DMSO in PBS plus 20% FCS) were assayed here on different cumuli types from rabbits and pigs. In rabbits, the cell proliferation capability of fully disaggregated cumuli was not affected by cryopreservation procedures (V: 100% and RF: 82%). Vitrified samples from partially or non-disaggregated cumuli showed the lowest proliferation frequencies (4% and 0%, respectively). In pigs, differences in cell proliferation capability were only observed between vitrified non-disagreggated cumuli and vitrified or rapid-frozen, fully disaggregated cumuli (72% vs 100% or 100%, respectively; P < 0.05). In both species, in vitro cultured sub-confluent samples were able to survive to a second cryopreservation treatment, maintaining the cell proliferation capability in nearly 50% of thawed samples. In conclusion, before cryopreservation, disaggregation of cumulus cells from both species into small clusters of cells improved their viability after thawing. These results allow us to efficiently, easily and rapidly store rabbit and pig cumulus cells, from selected high-merit females.

Short time priming of pig cumulus-oocyte complexes with fsh and forskolin in the presence of hypoxanthine stimulates cumulus cells to secrete a meiosis-activating substance

This study evaluated the effect of forskolin and FSH on pig oocyte maturation when cultured in a maturation inhibiting system. Ovaries from prepubertal gilts were collected at a local slaughterhouse. Oocytes were cultured in a hypoxanthine (HX 4 mM) containing M 199 for 24 or 40 h with or without forskolin and FSH treatment. After the culture, we examined germinal vesicle breakdown (GVBD) and polar body (PB) formation. Two experiments were designed. (1) Cumulus enclosed oocytes (CEO) were cultured for 24 or 40 h with or without different doses of forskolin and FSH. (2) CEO were primed by forskolin and FSH for different times and then transferred into an HX-medium for a further culture. The total culture period was 24 h. The results revealed that 4 mM HX markedly prevented pig CEO from undergoing GVBD. After 24 and 40 h culture, FSH (50–200 U/L) stimulated oocytes to resume meiosis by overcoming the inhibition of HX. Both GVBD and PB formation were increased (P < 0.002 and 0.01 respectively) after 40 h exposed to FSH. Forskolin showed a biphasic effect on CEO maturation. Within 24 h forskolin, in combination with HX, inhibited oocytes maturation. The GVBD percentage was significantly decreased compared to HX alone group (2% to 20%, P < 0.01), whereas no inhibition was observed after 40 h of culture. The second experiment showed that forskolin (3 u M) and FSH (100 U/L) priming CEO could timedependently induce oocyte maturation by overriding the inhibition of HX. After 30 and 60 min priming by FSH or forskolin, the GVBD and PB percentage was significantly increased (P < 0.002 and 0.01 respectively). No difference of GVBD percentage was observed between FSH short time priming group and FSH long time presentation group. In conclusion, we found that forskolin and FSH in vitro can stimulate pig cumulus cells to secrete a meiosisactivating substance which induces the oocyte to overcome the inhibition of hypoxanthine and undergo GVBD.

Oocytectomy does not influence synthesis of hyaluronic acid by pig cumulus cells: retention of hyaluronic acid after insulin-like growth factor-I treatment in serum-free medium.

Mouse oocytes secrete a factor that enables cumulus cells to undergo expansion in response to FSH (1 microg/ml), whereas expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. The aim of this study was to assess FSH-induced synthesis of hyaluronic acid (HA) by porcine cumulus cells before and after oocytectomy. In addition, we studied the effect of insulin-like growth factor-I (IGF-I) on the ability of cumulus cells to synthesize and retain HA in response to FSH in serum-free medium. Porcine oocyte-cumulus complexes and complexes from which the oocytes had been removed by oocytectomy were cultured for 24 h in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride, fetal calf serum (FCS, 5%), and FSH. After 24 h, incorporation of [(3)H]glucosamine into HA was measured either in complexes alone (retained HA) or in medium plus complexes (total HA). Specificity of incorporation of radioactivity into HA was confirmed by the sensitivity to highly specific Streptomyces hyaluronidase. Our results suggest that 1) the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis; 2) oocytes do not influence retention of HA within the complex; 3) FSH-induced synthesis of HA by cumulus cells is decreased in medium with polyvinylpyrrolidone (PVP)-supplemented (total and retained HA) compared to FCS-supplemented medium; 4) IGF-I enabled cumulus cells to synthesize HA in response to FSH in PVP-supplemented medium in a manner similar to that observed when serum is present in the medium.