Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro.

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development.

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

Global transcriptome analysis of porcine oocytes in correlation with follicle size.

Although our knowledge regarding oocyte quality and development has improved significantly, the molecular mechanisms that regulate and determine oocyte developmental competence are still unclear. Therefore, the objective of this study was to identify and analyze the transcriptome profiles of porcine oocytes derived from large or small follicles using RNA high-throughput sequencing technology. RNA libraries were constructed from oocytes of large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) ovarian follicles and then sequenced in an Illumina HiSeq4000. Transcriptome analysis showed a total of 14,557 genes were commonly detected in both oocyte groups. Genes related to the cell cycle, oocyte meiosis, and quality were among the top highly expressed genes in both groups. Differential expression analysis revealed 60 up- and 262 downregulated genes in the LO compared with the SO group. BRCA2, GPLD1, ZP3, ND3, and ND4L were among the highly abundant and highly significant differentially expressed genes (DEGs). The ontological classification of DEGs indicated that protein processing in endoplasmic reticulum was the top enriched pathway. In addition, biological processes related to cell growth and signaling, gene expression regulations, cytoskeleton, and extracellular matrix organization were among the highly enriched processes. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.

Interrelationships between ovarian follicles grown in culture and possible mediators.

Dominance or cooperation between ovarian follicles can determine the number of ovulations and fecundity, but interrelationships between follicles in mono- and poly-ovulatory species and their mechanisms are poorly understood. The goals of this work were to determine the existence and compare the character of mutual influence of cultured ovarian follicles from a mono-ovulatory species (cow) with established follicular dominance with those from a poly-ovulatory species (pig), in which interrelationship between follicles remain unknown, and to examine the role of ovarian cell proliferation, the insulin-like growth factor I (IGF-I)- oxytocin (OT) system, and steroid hormones in mediating interrelationships among ovarian follicles. Bovine and porcine ovarian follicles were isolated and cultured alone and in pairs, and the percentage of growing follicles was calculated. Porcine follicles were cultured alone and in pairs after addition of exogenous OT and IGF-I (100ngmL-1) or inactivation of endogenous OT and IGF-I by antisera against these hormones (1%). Proliferation of porcine follicular cells was assessed by SDS PAGE-Western immunoblotting, the release of IGF-I, progesterone, androstenedione and estradiol by cultured porcine ovarian follicles was analyzed by RIA/EIA. Overall, our observations suggest (1) competition/dominance (mutual suppression of growth) in bovine ovarian follicles, (2) cooperation (mutual support of growth) in porcine ovarian follicles, (3) that this mutual growth of porcine ovarian follicles was caused by the promotion of cell proliferation, (4) that this mechanism was probably not involved in bovine follicular dominance, (5) that communication between both porcine and bovine follicles affects their secretory activity, and (6) that both follicular dominance in cows and cooperation of follicles in pigs can be mediated by either down- or up-regulation of the IGF-I-OT system, which in turn affects follicular steroidogenesis and promotes follicular cell proliferation and follicular growth.

Caloric restriction and IGF-I administration promote rabbit fecundity: Possible interrelationships and mechanisms of action.

The aim of these in vivo and in vitro studies was to examine the influence of caloric restriction (CR), and the administration of insulin-like growth factor (IGF-I), on rabbit fecundity and to understand the interrelationships between CR and IGF-I, as well as the endocrine and intracellular mechanisms of their effects. Female rabbits were subjected to 50% CR, injections of IGF-I (20 µg/animal/day) and a combination of the two for 10 d before and 2 d after ovulation induced by 25 IU PMSG and 0.25 IU hCG. On the day of ovulation blood samples were collected and analyzed IGF-I, leptin, progesterone (P4) and estradiol (E2) concentrations by RIA. Some animals from each group were killed in their periovulatory period and weighed, as were their ovaries. Granulosa cells isolated from ovaries of does subjected or not to CR were cultured for 2 d with and without IGF-I (100 ng/mL). Accumulation of markers of cell proliferation (PCNA and cyclin B1), apoptosis (bax), MAP/ERK1,2 kinase (MAPK), protein kinase A (PKA) and IGF-I were evaluated by immunocytochemistry. In addition, E2 release by cells isolated from ovaries of animals subjected or not to CR and cultured with and without IGF-I (1, 10, 100, 1000 or 10000 ng/mL) was assessed by RIA. The remaining animals were kept until parturition, when the number of pups was recorded. CR did not affect animal and ovarian weight, but significantly increased the number of pups per litter and plasma levels of IGF-I and decreased plasma leptin and P4, but not E2 concentration. Injections of IGF-I did not influence body and ovarian weights, but increased the number of pups per litter and plasma IGF-I and leptin concentration and reduced plasma E2 but not P4 level. IGF-I administration did not modify the main effects of CR, although it prevented the CR-induced decrease in plasma P4 level. CR reduced accumulation of PCNA, bax, promoted accumulation of cyclin B1 but not of MAPK, PKA or IGF-I within ovarian granulosa cells. Addition of IGF-I to culture medium reduced accumulation of bax, MAPK, and IGF-I and promoted PKA accumulation and E2 release. CR promoted the stimulatory effect of IGF-I on E2 output. Thus, CR can increase rabbit fecundity, probably via changes in IGF-I, leptin and steroid hormones released, which in turn can affect ovarian cell cycle, apoptosis, and response to IGF-I. Furthermore, they demonstrate the stimulatory influence of IGF-I on rabbit fecundity, which was associated with changes in plasma leptin, E2 and ovarian cell apoptosis, PKA, MAPK, IGF-I and E2 release. The promotion of IGF-I output by CR and the ability of IGF-I to mimic/replace but not to modify CR effects on fecundity, plasma IGF-I, and ovarian cell apoptosis suggest that IGF can mediate the action of CR on these reproductive indexes. In contrast, differences in the action of CR and IGF-I on other hormones, ovarian cell proliferation, protein kinases and IGF-I suggest that CR action on these indexes is not mediated by IGF-I. We thus demonstrate that both CR and IGF-I administration can increase rabbit fecundity, and that their effects can be mediated by changes in reproductive hormones, ovarian cell proliferation, apoptosis, and the response of ovarian cells to IGF-I.

Metabolic state defines the response of rabbit ovarian cells to leptin.

Leptin is a hormone that mediates the effect of the metabolic state on several biological functions, including reproduction. Leptin affects reproductive functions via alterations in the release of hormonal regulators. However, the extent to which caloric restriction (CR) can affect the complex processes of reproduction by other mechanisms, such as altering ovarian functions via direct binding/response to leptin, is unknown. Therefore, the aim of the present study was to show basic ovarian cell functions and CR on the response of ovarian cells to leptin. Female rabbits were subjected to 50% CR restriction for 10days before ovulation. On the day of ovulation, both control and CR animals were sacrificed. Isolated granulosa cells were cultured for 2days with and without leptin (100ng/ml), and the accumulation of various markers was evaluated using immunocytochemistry; i.e., cell proliferation (PCNA and cyclin B1), apoptosis (bax), MAP/ERK1,2 kinase (MAPK), protein kinase A (PKA), and IGF-I. In addition, the release of IGF-I and estradiol (E2) by cells cultured with and without leptin (1, 10, 100, 1000, or 10,000ng/ml) was assessed by radioimmunoassay (RIA). In the granulosa cells of control animals, leptin promoted cyclin B1, MAPK, and PKA accumulation, but not that of PCNA, and reduced bax and IGF-I accumulation. These cells responded to leptin by increased IGF-I, but not E2 release. In cells of CR animals, leptin increased cyclin B1 accumulation, but decreased PCNA, MAPK, and IGF-I expression. Bax and PKA were not affected. Leptin resulted in a decrease in IGF-I release. CR modulated the influence of leptin on E2 release dose dependently, i.e., E2 increased at 10 and decreased at 10,000ng/ml. Therefore, CR modified the influence of leptin on PCNA, E2, bax, PKA, MAPK, and IGF-I release, but it did not change the effect of leptin on cyclin B1 and IGF-I accumulation within the cells. Our data showed that leptin directly affected proliferation, apoptosis, and hormone release by ovarian cells, probably via PKA- and MAPK-dependent pathways. Furthermore, it was demonstrated that nutrition could influence reproduction by affecting the response of ovarian cells to leptin.

RNA polymerase II transcriptional silencing in growing and fully grown germinal vesicle oocytes isolated from gonadotropin-stimulated and non-stimulated gilts.

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.

Nuclear and nucleolar reprogramming during the first cell cycle in bovine nuclear transfer embryos.

The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed at 0.5, 1, 2, 3, 4, 8, 12, and 16 h postactivation (hpa). Parthenogenetic (PA) embryos were used as control. The SCNT and PA embryos were processed for lacmoid staining, autoradiography, transmission electron microscopy (TEM), and immunofluorescence localization of: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa. Likewise, starved and nonstarved fibroblasts were processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillogranular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. In conclusion, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development.

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro.

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.