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.

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium).

Background: Oocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles.

Methods: The growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors.

Porcine oocytes matured in a chemically defined medium are transcriptionally active.

The statement that fully-grown porcine oocytes (oocytes from follicles with diameter from 3 to 6 mm) are transcriptionally quiescent is not as strongly supported as it was before. Currently, we know that there is a difference between the transcription profile of germinal vesicle (GV) and metaphase II (MII) oocytes. The goal of our study was to compare the transcription profile of GV, germinal vesicle breakdown (GVBD), metaphase I (MI), and MII oocytes matured in the chemically defined medium FLI.

Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality.

Background: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality.

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.

Improving the Quality of Oocytes with the Help of Nucleolotransfer Therapy.

The nucleolus is an important nucleus sub-organelle found in almost all eukaryotic cells. On the one hand, it is known as a differentiated active site of ribosome biogenesis in somatic cells, but on the other hand, in fully grown oocytes, zygotes, and early embryos (up to the major embryonic genome activation), it is in the form of a particular homogenous and compact structure called a fibrillar sphere. Nowadays, thanks to recent studies, we know many important functions of this, no doubt, interesting membraneless nucleus sub-organelle involved in oocyte maturation, embryonic genome activation, rRNA synthesis, etc.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential .

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured .

Inhibition of miR-152 during In Vitro Maturation Enhances the Developmental Potential of Porcine Embryos.

Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes.

Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle.

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles.

Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells.

Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells.

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.

Oocytes, embryos and pluripotent stem cells from a biomedical perspective.

The veterinary and animal science professions are rapidly developing and their inherent and historical connection to agriculture is challenged by more biomedical and medical directions of research. While some consider this development as a risk of losing identity, it may also be seen as an opportunity for developing further and more sophisticated competences that may ultimately feed back to veterinary and animal science in a synergistic way. The present review describes how agriculture-related studies on bovine embryo production through studies of putative bovine and porcine embryonic stem cells led the way to more sophisticated studies of human induced pluripotent stem cells (iPSCs) using e.

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.

Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos.

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes.

Enucleolation and nucleolus transfer in mammalian oocytes and zygotes.

The oocyte GV/GVs (germinal vesicle/germinal vesicles) and zygot PN/PNs (pronucleus/pronuclei) of some mammals contain clearly visible nucleoli which exhibit an atypical morphological structure. These nucleoli (NCLs) can be relatively easily manipulated, i.e.

microRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles.

Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte-specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing.

Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos.

SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)).

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.

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.

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.

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.

Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle.

Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin).

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.

Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos.

The goal of the present study was to investigate whether key nucleolar proteins involved in ribosomal RNA (rRNA) transcription and processing are transcribed de novo or from maternally inherited messenger RNAs (mRNA) in bovine embryos, and to which extent de novo transcription of these proteins mRNA is required for the development of functional nucleoli during the major activation of the embryonic genome. Immunofluorescence for localization of key nucleolar proteins, autoradiography for detection of transcriptional activity, and transmission electron microscopy were applied to in vitro produced bovine embryos cultured from the 2-cell stage with or without (control groups) alpha-amanitin, which blocks the RNA polymerases II and III transcription and, thus the synthesis of mRNA. In the control groups, weak autoradiographic labeling was initially observed in the periphery of few nuclei at the 4-cell and the early 8-cell stage, and the entire nucleoplasm as well as nucleolus precursor bodies (NBBs) were prominently labelled in all late 8-cell stages.