Transcripts In Oocytes Research Articles

Effect of basic fibroblast growth factor (FGF2) on cumulus cell expansion, in vitro embryo production and gene expression in buffalo (Bubalus bubalis)

The present study was undertaken to evaluate the effect of different concentration of FGF2 viz. 5 ng (T1), 10 ng (T2), and 20 ng/mL (T3) on cumulus cell expansion, oocyte maturation, in vitro embryo production, total cell number (TCN) of the blastocyst, and expression of the FGF2 and FGFR2 transcripts in buffalo oocytes and the embryos. Results showed that the effect of FGF2 on the diameter of buffalo COC was significantly higher (P < 0.05) in the T1 group than the other groups at 24h of maturation. The maturation and cleavage rate of oocytes was significantly higher (P < 0.05) in the T3 group than the control, however, the values did not different (P> 0.05) from other groups. The effect of FGF2 on morula and blastocyst yield did not different (P > 0.05) between treatment groups. However, the TCN of the blastocyst was slightly higher (P > 0.05) in the T3 group than the control and other groups. In subsequent trials, the expression of the FGF2 transcript was higher (P < 0.05) in A-grade of oocytes than the C- and D-grade of oocytes, but the expression was not different (P> 0.05) from the B-grade of oocytes. While the FGFR2 expression was higher (P < 0.05) in cumulus cells than any grades of oocytes. The relative abundance of FGF2 and FGFR2 transcripts was significantly higher (P < 0.05) in the 2-cell stage of the embryo than the other stages of embryos. This study was further extended to characterize the FGF2 ligand-binding site in the D3 domain of the buffalo FGF2 receptor. Bioinformatics analysis showed that the bovine FGF2 ligand-binding site in the D3 domain of buffalo was different from the D3 domain of the cattle.

Processing of Alu small RNAs by DICER/ADAR1 complexes and their RNAi targets.

In addition to adenosine-to-inosine RNA editing activities, ADAR1 has been shown to have various RNA editing-independent activities including modulation of RNAi efficacy. We previously reported that ADAR1 forms a heterodimer complex with DICER and facilitates processing of pre-miRNAs to mature miRNAs. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs). Generation of retrotransposon-derived endo-siRNAs by DICER and their functions in regulation of transcripts in mouse oocytes has been previously reported. However, the synthesis and functions of endo-siRNAs in somatic cells remain largely unknown. Here, we report that ADAR1 together with DICER generates endogenous small RNAs, Alu endo-siRNAs by cleaving long double-stranded regions of inverted Alu repeats. We identified AGO2-loaded Alu endo-siRNAs, which are highly expressed in commonly used cell lines. These Alu endo-siRNAs carrying both sense and antisense Alu sequences seem to target a set of genes containing a single Alu sequence, either antisense or sense, respectively, within their 3′UTR. In silico screening identified potential RNA silencing target genes for these Alu endo-siRNAs. We present results of a proof-of-concept experiment, in which sense Alu endo-siRNAs derived from AluSz and AluJr family elements target CUB Domain Containing Protein 1 mRNAs containing an antisense copy of AluJb in their 3′UTRs and consequently induce apoptosis in HeLa cells. Our results clearly indicate that Alu endo-siRNAs are functional also in somatic cells.

Distinct expression patterns of TLR transcripts in human oocytes and granulosa cells from primordial and primary follicles

Ovulation has long been regarded as a process resembling an inflammatory response. Previously, luteinizing hormone (LH) was shown to induce Toll-like receptor 2 (TLR2) and TLR4 in granulosa cells from preovulatory hormone-dependent follicles. However, whether this could already initiate before the hormone-dependent phase is currently unknown.The aim of this study was to investigate TLR genes in human oocytes and granulosa cells from primordial and primary ovarian follicles during the hormone-independent phase. A class-comparison study of existing oocyte and granulosa cell RNA sequencing transcriptomes from primordial (n = 539 follicles) and primary (n = 261) follicles collected from three patients was examined. This revealed a distinct expression pattern of TLR3, TLR4 and TLR5 transcripts. Interestingly, the TLR3 protein was differentially detected in both the oocyte and the granulosa cells in primordial and primary follicles, suggesting that TLR3 is maternally contributed both as mRNA and protein. Intracellularly, the compartmentalized TLR3 dot-like staining in the intersection between the oocyte and the surrounding primordial granulosa cells. The TLR4 protein was detected in both primordial and primary follicles, with a notable staining in the granulosa cells. We functionally challenged ovaries in vitro, by polyinosinic:polycytidylic acid (poly I:C) and LPS, known to activate TLR3 and TLR4, respectively, and found a tendency for increased IL-6 production, which was particular evident in the LPS-treated group. Based on the expression of TLRs, it is notably that human primordial and primary follicles express genes that would allow them to respond to innate immune proteins and cytokines during follicle activation.

Deletion of BAF250a affects oocyte epigenetic modifications and embryonic development.

BRG1-associated factor 250a (BAF250a) is a component of the SWI/SNF adenosine triphosphate-dependent chromatin remodeling complex, which has been shown to control chromatin structure and transcription. BAF250a was reported to be a key component of the gene regulatory machinery in embryonic stem cells controlling self-renewal, differentiation, and cell lineage decisions. Here we constructed Baf250aF/F ;Gdf9-cre (Baf250aCKO ) mice to specifically delete BAF250a in oocytes to investigate the role of maternal BAF250a in female germ cells and embryo development. Our results showed that BAF250a deletion did not affect folliculogenesis, ovulation, and fertilization, but it caused late embryonic death. RNA sequencing analysis showed that the expression of genes involved in cell proliferation and differentiation, tissue morphogenesis, histone modification, and nucleosome remodeling were perturbed in Baf250aCKO MII oocytes. We showed that covalent histone modifications such as H3K27me3 and H3K27ac were also significantly affected in oocytes, which may reduce oocyte quality and lead to birth defects. In addition, the DNA methylation level of Igf2r, Snrpn, and Peg3 differentially methylated regions was decreased in Baf250aCKO oocytes. Quantitative real-time polymerase chain reaction analysis showed that the relative messenger RNA (mRNA) expression levels of Igf2r and Snrpn were significantly increased. The mRNA expression level of Dnmt1, Dnmt3a, Dnmt3l, and Uhrf1 was decreased, and the protein expression in these genes was also reduced, which might be the cause for impaired imprinting establishment. In conclusion, our results demonstrate that BAF250a plays an important role in oocyte transcription regulation, epigenetic modifications, and embryo development.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well-known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the ‘before IVM’ group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT-qPCR. The current study particularly focused on genes belonging to ‘positive regulation of transcription, DNA-dependent’, ‘positive regulation of gene expression’, ‘positive regulation of macromolecule metabolic process’ and ‘positive regulation of transcription from RNA polymerase II promoter’ ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

The most abundant maternal lncRNA Sirena1 acts post-transcriptionally and impacts mitochondrial distribution.

Tens of thousands of rapidly evolving long non-coding RNA (lncRNA) genes have been identified, but functions were assigned to relatively few of them. The lncRNA contribution to the mouse oocyte physiology remains unknown. We report the evolutionary history and functional analysis of Sirena1, the most expressed lncRNA and the 10th most abundant poly(A) transcript in mouse oocytes. Sirena1 appeared in the common ancestor of mouse and rat and became engaged in two different post-transcriptional regulations. First, antisense oriented Elob pseudogene insertion into Sirena1 exon 1 is a source of small RNAs targeting Elob mRNA via RNA interference. Second, Sirena1 evolved functional cytoplasmic polyadenylation elements, an unexpected feature borrowed from translation control of specific maternal mRNAs. Sirena1 knock-out does not affect fertility, but causes minor dysregulation of the maternal transcriptome. This includes increased levels of Elob and mitochondrial mRNAs. Mitochondria in Sirena1−/− oocytes disperse from the perinuclear compartment, but do not change in number or ultrastructure. Taken together, Sirena1 contributes to RNA interference and mitochondrial aggregation in mouse oocytes. Sirena1 exemplifies how lncRNAs stochastically engage or even repurpose molecular mechanisms during evolution. Simultaneously, Sirena1 expression levels and unique functional features contrast with the lack of functional importance assessed under laboratory conditions.

Progesterone Increases Mitochondria Membrane Potential in Non-human Primate Oocytes and Embryos

Mitochondrial activity is critical and correlates with embryo development. The identification of a novel human mitochondrial progesterone receptor (PR-M) that increases cellular respiration brings into question a role for progesterone in oocyte and preimplantation embryo development. Oocytes and embryos were generated from three Rhesus non-human primates (Macaca mulatta) undergoing in vitro fertilization. Immunohistochemical (IHC) staining for the progesterone receptor and mitochondria, RT-PCR with product sequencing for a mitochondrial progesterone receptor, and mitochondrial membrane determination with JC-1 staining were performed. IHC staining with selective antibodies to the progesterone receptor showed non-nuclear staining. Staining was absent in mouse control embryos. RT-PCR with product sequencing demonstrated PR-M transcript in Rhesus oocytes and embryos, which was absent in mouse embryos. Treatment of Rhesus oocytes and embryos with progesterone showed increased mitochondrial membrane potential, which was absent in mouse embryos. Our results support that progesterone increases mitochondrial membrane potential in oocytes and developing embryos. This is likely an in vivo mechanism to support preimplantation embryo development, and brings up the possibility of in vitro manipulation of culture media for optimization of growth.

Acute low-dose bisphenol S exposure affects mouse oocyte quality

Bisphenol S (BPS) is widely used to replace the known endocrine disruptor BPA in various products. We evaluated the effect of acute in vivo BPS exposure on oocyte quality, simulating the oral route of exposure via oral gavage. Eight-week-old ICR female mice (N = 15 per experimental group) were exposed to vehicle or BPS1–BPS4 (0.001, 0.1, 10, and 100 ng BPS x g bw−1 day−1, respectively) for seven days. Oocytes were isolated and matured in vitro. We observed that BPS exposure increased aberrant spindle formation in mature oocytes and induced DNA damage. Moreover, BPS3 significantly increased the chromatin repressive marks 5-methyl cytosine (5meC) and H3K27me2 in immature oocytes. In the BPS2 group, the increase in 5meC occurred during oocyte maturation. Transcriptome analysis revealed differential expression of early embryonic development transcripts in BPS2-exposed oocytes. These findings indicate that the biological effect of BPS is non-monotonic, affecting oocyte quality even at concentrations that are orders of magnitude below those measured in humans.

183 Effects of epidermal growth factor and progesterone on invitro oocyte growth, meiotic resumption, and expression of maturation-related transcripts in oocytes from bovine small antral follicles

Oocytes from small antral follicles are not able to undergo nuclear maturation because they still need to accumulate mRNA and proteins. We hypothesised that invitro growth of cumulus-oocyte complexes (COCs) of these follicles in the presence of epidermal growth factor (EGF) and progesterone (P4) increase the levels of transcripts and have a positive impact on maturation. This study aimed to evaluate the effects of EGF and P4 on growth, maturation, and expression of mRNAs for growth and differentiation factor 9 (GDF9), cyclin B1, oocyte-specific linker histone (H1FOO), quinase cMOS, poly(A) ribonuclease (PARN), and initiation factor 4E (eIF4E) after growth and prematuration of COCs. Bovine COCs (n=400) were aspirated from antral follicles (1-3mm) and cultured for 48h in control medium (TCM-199 plus 5.0mgmL−1 LH, 0.5mgmL−1 FSH, and 5% fetal bovine serum) alone or with EGF (10ngmL−1), P4 (100 µM), or both EGF and P4. After growth, the COCs were pre-matured for 20h in TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, and 10μM cilostamide. Then, COCs were matured invitro in the same medium used for prematuration, but without cilostamide. Oocyte diameter and meiotic progression were evaluated after growth, prematuration, and maturation periods. After prematuration, oocytes were stored until RNA extraction. Total RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, quantification of mRNAs for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH was performed by real-time PCR. The delta-delta-cycle threshold (ΔΔCT) method was used to normalise the data. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test. Differences in expression of mRNAs were analysed by Kruskal-Wallis test (P&amp;lt;0.05). The results showed that, compared with time 0, an increase in oocyte diameter was observed after 48h of culture for COCs in all groups (P&amp;lt;0.05), except for those cultured in control medium alone or with only P4. However, when compared with control medium, no effects of EGF, P4, or both were seen after the growth and prematuration periods. After prematuration, a higher percentages of oocytes at the germinal vesicle stage was observed for COCs cultured with P4 compared with those cultured in TCM-199 (P&amp;lt;0.05). After IVM, the rate of meiosis resumption was significantly reduced in oocytes cultured with P4 (P&amp;lt;0.05). The levels of mRNA for cMOS, H1FOO, and cyclin B1 in oocytes cultured with P4 were higher than those cultured in the control medium (P&amp;lt;0.05). The mRNA levels for eIF4E in oocytes cultured with both EGF and P4 were increased (P&amp;lt;0.05) compared with those cultured with EGF. The levels of transcripts for PARN in oocytes cultured with both EGF and P4 were higher than those in COCs cultured with only P4. In contrast, mRNA levels of GDF9 did not show differences between treatments. In conclusion, P4 inhibited oocyte meiotic resumption and increased mRNA levels for cMOS, H1FOO, and cyclin B1 in COCs after culture and prematuration invitro.

189 Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and expression of maturation-related transcripts in bovine oocytes from medium-size antral follicles

The aims of this study were to evaluate the effects of epidermal growth factor (EGF) and progesterone (P4) on maturation and expression transcripts for GDF9, CCNB1, H1FOO, cMOS, PARN, and eIF4E after prematuration of cumulus-oocyte complexes (COCs) from antral follicles. Bovine COCs (3-6mm) were aspirated and pre-matured for 20h in control medium [TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, cilostamide (10μM) and follicular hemisections] alone or supplemented with EGF (10ngmL−1), P4 (100 µM), or both EGF (10ngmL−1) and P4 (100 µM). After that, COCs were matured for 24h in the same medium, without EGF, P4, cilostamide, and follicular hemisections. Oocyte diameters were evaluated with the software Nis Elements (Nikon Instruments Inc.). To evaluate meiotic progression, the oocytes were fixed in 4% paraformaldehyde and transferred to 0.5% Triton X-100. The chromatin configuration during meiosis was assessed by 10μgmL−1 bisbenzimide (Hoechst 33342) and analysed under an epi-fluorescent inverted microscope (DMI4000B; Leica). Oocytes were classified according to the nuclear maturation stage as germinal vesicle, metaphase I, anaphase I, telophase I, and metaphase II. To evaluate mRNA expression, oocytes were stored in micr-centrifuge tubes at −80°C until RNA extraction. RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, mRNA for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH (housekeeping gene) was quantified by real-time PCR and analysed by Kruskal-Wallis test. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test (P&amp;lt;0.05). The results showed that prematuration of COCs in the presence of P4 and both EGF and P4 promoted an increase in oocyte diameter compared with the control or EGF treatment alone. The presence of cilostamide inhibited early meiotic resumption, benefiting oocyte capacitation, but the presence of EGF, P4, or EGF and P4 together in the prematuration medium did not influence meiosis resumption rates. The presence of EGF or P4 in prematuration medium increased the mRNA levels for cMOS in oocytes (P&amp;lt;0.05). The H1FOO mRNA levels in oocytes cultured with EGF and P4 increased significantly compared with oocytes cultured in EGF alone (P&amp;lt;0.05). In contrast, mRNA levels for cyclin B1 in oocytes cultured with P4 were higher than those cultured in the presence of EGF alone (P&amp;lt;0.05). In addition, levels of mRNA for eIF4E showed a significant reduction in oocytes cultured with P4 compared with those pre-matured with EGF or both EGF and P4. The EGF treatment reduced the levels of mRNA for GDF9 compared with control medium. The mRNA levels of PARN did not differ significantly between treatments. In conclusion, EGF, P4, and EGF and P4 combined did not influence oocyte growth and meiotic resumption. However, EGF or P4 increased the mRNA expression of cMOS, whereas EGF reduced the levels of transcripts for GDF9.

Evolution of imprinting via lineage-specific insertion of retroviral promoters

Imprinted genes are expressed from a single parental allele, with the other allele often silenced by DNA methylation (DNAme) established in the germline. While species-specific imprinted orthologues have been documented, the molecular mechanisms underlying the evolutionary switch from biallelic to imprinted expression are unknown. During mouse oogenesis, gametic differentially methylated regions (gDMRs) acquire DNAme in a transcription-guided manner. Here we show that oocyte transcription initiating in lineage-specific endogenous retroviruses (ERVs) is likely responsible for DNAme establishment at 4/6 mouse-specific and 17/110 human-specific imprinted gDMRs. The latter are divided into Catarrhini- or Hominoidea-specific gDMRs embedded within transcripts initiating in ERVs specific to these primate lineages. Strikingly, imprinting of the maternally methylated genes Impact and Slc38a4 was lost in the offspring of female mice harboring deletions of the relevant murine-specific ERVs upstream of these genes. Our work reveals an evolutionary mechanism whereby maternally silenced genes arise from biallelically expressed progenitors.

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.

Lipopolysaccharide and tumor necrosis factor-alpha alter gene expression of oocytes and cumulus cells during bovine in vitro maturation.

Communication between the oocyte and cumulus facilitates oocyte growth, cell cycle regulation, and metabolism. This communication is mediated by direct contact between oocytes and cumulus cells, and soluble secreted molecules. Secreted molecules involved in this process are known inflammatory mediators. Lipopolysaccharide (LPS) is detected in follicular fluid and is associated with reduced fertility, whereas accumulation of inflammatory mediators in follicular fluid, including tumor necrosis factor-α (TNF-α), is associated with female infertility. Maturation of oocytes in the presence of LPS or TNF-α reduces meiotic maturation and the capacity to develop to the blastocyst. Here we evaluated the abundance of 92 candidate genes involved immune function, epigenetic modifications, embryo development, oocyte secreted factors, apoptosis, cell cycle, and cell signaling in bovine cumulus cells or zona-free oocytes after exposure to LPS or TNF-α during in vitro maturation. We hypothesize that LPS or TNF-α will alter the abundance of transcripts in oocytes and cumulus cell in a cell type dependent manner. Exposure to LPS altered abundance of 31 transcripts in oocytes (including ACVR1V, BMP15, DNMT3A) and 12 transcripts in cumulus cells (including AREG, FGF4, PIK3IP1). Exposure to TNF-α altered 1 transcript in oocytes (IGF2) and 4 transcripts in cumulus cells (GJA1, PLD2, PTGER4, STAT1). Cumulus expansion was reduced after exposure to LPS or TNF-α. Exposing COCs to LPS had a marked effect on expression of targeted transcripts in oocytes. We propose that altered oocyte transcript abundance is associated with reduced meiotic maturation and embryo development observed in oocytes cultured in LPS or TNF-α.

In vitro culture of secondary follicles and prematuration of cumulus-oocyte complexes from antral follicles increase the levels of maturation-related transcripts in bovine oocytes.

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.

Transcription of pericentromeric tandemly repeated DNA transcripts in human preovulatory oocytes

Transcription of pericentromeric tandemly repeated DNA transcripts in human preovulatory oocytes

Expression dynamics of Integrin Subunit Beta 5 in bovine gametes and embryos imply functions in male fertility and early embryonic development.

Integrins have been shown to act as signalling receptors, and they primarily recognise extracellular matrix ligands on the oocyte surface. However, their possible roles in oocyte activation and embryo development are not clearly understood. The objectives of this study were to evaluate expression of Integrin Subunit Beta 5 (ITGβ5) in bovine sperm, oocytes, and early embryos and to ascertain the evolutionary conservation of ITGβ5. To accomplish these objectives, we used western blotting to study expression levels of ITGβ5 protein in sperm and RT-qPCR to determine expression levels of ITGβ5 transcripts in oocytes and embryos. We have also used bioinformatic analysis to determine the evolutionary conservation of the ITGβ5 protein among various species. Western blotting showed that ITGβ5 protein was detectable in bull sperm. Moreover, results of RT-qPCR showed that levels of ITGβ5 were significantly higher in the two-cell embryos, followed by the 8-16-cell embryos. However, no significant difference in expression levels were noted for the morula and blastocyst stages as compared to MII oocytes. Bioinformatic analysis revealed that ITGβ5 is conserved among various species. We conclude that expression of ITGβ5 in bovine gametes and embryos implies an important role in fertilisation and embryogenesis.

Actin depolymerization disrupts karyosphere capsule integrity but not residual transcription in late oocytes of the grass frog Rana temporaria.

Late diplotene oocytes are characterized by an essential decrease in transcriptional activity. At this time, chromosomes condense and form a compact structure named a karyosphere. The karyosphere of grass frogs Rana temporaria is surrounded by a fibrillar karyosphere capsule (KC). One of the main protein constituents of R. temporaria KC is actin. In this study, we used antibodies against different actin epitopes to trace different forms of actin in the KC. We also investigated the effect of F-actin depolymerization on the oocyte nuclear structures and transcription of chromatin DNA and rDNA in the amplified nucleoli. It was determined that disruption of actin filaments leads to chromosome shrinkage, nucleoli fusion, and distortion of the KC structure, but does not inhibit residual transcription in both the karyosphere and the nucleoli.

Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro†.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 μM were added as control, SB, SB+GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

BOARD INVITED REVIEW: Post-transfer consequences of in vitro-produced embryos in cattle.

In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.

107 Insights from roe deer oocyte transcriptome across embryonic diapause

Embryonic developmental arrest, known as diapause, has been reported in more than 130 species. However, its mechanisms are still not completely understood. In the roe deer, the only known ungulate that exhibits this phenomenon, diapause lasts for approximately 5 months, starting after the rut period in mid-July to early August and ending with embryo elongation and implantation in December/January. Little is known regarding oocyte characteristics during this period. Here, we analysed the roe deer oocyte transcriptome as a model to understand diapause effects on oocyte features during embryonic developmental arrest and reactivation. During regular hunting, immature oocytes were obtained by ovary slicing from diapause and nondiapause stages, and classified according to morphological characteristics. Only oocytes with &amp;gt;2 layers of compact cumulus cells and cytoplasm from 30 hunted females were used for analyses. Immature oocytes were denuded and snap frozen. Additional oocytes were cultured in maturation medium for 20-24h. Matured oocytes with a present polar body were snap frozen. Two pools of 10 immature and mature oocytes for both diapause and nondiapause stages were included (at least 4 donors/pool). Oocyte pools were processed using the Smart-seq 2 single-cell protocol (Illumina Inc., San Diego, CA, USA) for full-length cDNA and library preparation. We performed RNA-seq on an Illumina sequencer. The obtained Fastq files were clipped and analysed with a locally installed version of the Galaxy platform. Sequences were mapped against the roe deer transcriptome (unpublished data) and annotated against human and bovine transcripts. Differentially expressed genes (DEG; false discovery rate &amp;lt;1%) were identified using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) in immature (IMM) and mature (M) oocytes from diapause (D) and nondiapause (ND) stages. Additionally, to evaluate the effects of maturation on oocyte transcript abundancy, DEG between IMM and M oocytes for D and ND were identified. Multidimensional scaling resulted in clustering according to oocyte types. Gene ontology terms for biological processes were assigned using ToppCluster tools. A total of 23066, 23022, 22438, and 22532 transcripts were detected for IMM D, M D, IMM ND and M ND oocytes, respectively. Using false discovery rate filtering, we found 333 and 288 DEG in immature and mature oocytes, respectively. Furthermore, maturation changed the oocyte expression profiles during D (2233 DEG) and ND periods (2589 DEG). Gene ontology classification revealed that most of the DEG in immature oocytes were involved in macromolecule catabolic process and oxidation-reduction process. For mature oocytes, DEG were mostly related to regulation of chromosome condensation and ER and Golgi vesicle-mediated transport. Additionally, the top 20 DEG from IMM D v. M D oocytes were related to mRNA splicing, mRNA metabolic process, and mRNA processing. These processes were not identified in the top 20 DEG from IMM ND v. M ND oocytes. These preliminary results suggest that oocyte transcriptome analysis could disclose new pathways implicated in oocyte competence and embryonic developmental arrest and activation stages.