In brief: Embryo production technologies can affect early developmental processes critical for pregnancy establishment in cattle. This study reveals that in vitro-derived embryos exhibit impaired elongation and altered lineage patterning compared to those derived from artificial insemination. Abstract: This study aimed to characterize and compare cell lineage specification in the embryonic disc (ED) of elongating bovine conceptuses derived from artificial insemination or from in vitro-produced embryos, including fresh and cryopreserved blastocysts. Conceptuses were recovered on day 16 of gestation, measured, and EDs were dissected for whole-mount immunofluorescence using markers of epiblast, mesoderm, and endoderm lineages. Image analysis was performed to assess disc morphometry, epiblast cell number, and spatial organization of lineage-specific markers. Conceptuses derived from artificial insemination were longer and exhibited larger EDs compared to those derived following transfer of in vitro-produced embryos, particularly vitrified embryos, which showed reduced elongation and altered disc morphology. Epiblast cell numbers were lower in vitrified conceptuses but scaled proportionally with disc size across all groups. The initiation of gastrulation and hypoblast differentiation had occurred in most conceptuses, regardless of origin; however, in vitro-produced conceptuses displayed altered spatial organization of mesodermal and endodermal markers, with less defined patterning compared to artificial insemination-derived conceptuses. These findings indicate that while key developmental processes are preserved, in vitro embryo production, especially when combined with cryopreservation, is associated with impaired elongation and disrupted spatial organization of cell lineages during early development. Such alterations may contribute to reduced developmental competence and increased embryonic loss.

Despite the extensive use of in vitro embryo production (IVP) in cattle, its efficiency remains variable, mainly due to suboptimal conditions during oocyte in vitro maturation (IVM). Oocyte developmental competence is influenced by the local signaling environment, particularly by interactions between the oocyte and cumulus cells. Among the mediators involved in this process, amphiregulin (AREG) was identified as an important regulator of oocyte maturation. This study evaluated the effect of supplementing maturation media with AREG during bovine IVM on oocyte maturation, meiotic spindle organization, and subsequent embryo development. Oocytes were matured under three conditions: control (0 ng/mL AREG), A50 (50 ng/mL AREG), and A100 (100 ng/mL AREG). Supplementation with A100 significantly increased cumulus expansion compared with the control and A50 groups (P < 0.05). The A100 group also showed higher rates of progression to metaphase II (>89%) and a lower proportion of oocytes arrested at the germinal vesicle breakdown stage (P < 0.05). In addition, A100 supplementation reduced severe chromosome misalignment and spindle multipolarity and α-tubulin volume (P < 0.05). Embryo development was improved in the A100 group, as evidenced by higher cleavage rates on D3 (>80%), increased proportions of embryos reaching the ≥ 8-cell stage, and greater blastocyst and hatching rates on D7 compared with the control and A50 groups (P < 0.05). No differences among treatments were detected in blastocyst global 5 methylcytosine levels (P > 0.05). In summary, 100 ng/mL AREG supplementation during bovine IVM improves oocyte quality and embryo developmental competence without altering global DNA methylation.

The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized the study of gene expression in individual cells, providing unprecedented insights into cellular heterogeneity and developmental processes. The application of scRNA-seq to oocyte biology has facilitated the identification of species-specific transcriptional signatures and developmental trajectories, enhancing our understanding of oogenesis. This chapter presents a detailed protocol for scRNA-seq analysis of growing bovine oocytes.

The acquisition of oocyte competence in cattle, encompassing both cytoplasmic and nuclear maturation, is essential for successful fertilization and embryonic development. This competence is progressively achieved during the latter stages of the oocyte growth phase and completed within the dominant follicle (DF). The unique hormonal and immunological environment of the DF during oestrous supports oocyte "capacitation," a process involving organelle reorganization, mRNA storage and meiosis resumption, which fully prepares the oocyte for fertilization. These changes differentiate oocytes from the DF from those of subordinate follicles, explaining why only oocytes from the DF mature and ovulate successfully. Despite advances in assisted reproductive technologies like in vitro maturation (IVM) and in vitro fertilization (IVF), developmental outcomes remain inferior compared to in vivo matured oocytes, largely due to incomplete or altered oocyte maturation in vitro. Blastocyst rates after IVM/IVF are substantially lower (~35%) than those from in vivo matured oocytes (58-78%). The heterogeneity of oocytes retrieved from antral follicles and the lack of exposure to the natural follicular environment during IVM are key factors limiting developmental competence. Here we describe the molecular changes in bovine oocytes from DFs, collected at 24 and 2 h before ovulation without ovarian stimulation, using single-cell RNA sequencing and bisulfite sequencing to assess gene expression and DNA methylation dynamics. Results revealed significant shifts in transcripts related to oxidative phosphorylation, highlighting the crucial role of energy metabolism during oocyte capacitation. DNA methylation changes were subtle but indicated a more dynamic and less stable epigenome in fully-grown oocytes than previously assumed. Overall, understanding the gene expression and epigenetic landscape during oocyte maturation in the DF offers valuable insights into improving oocyte quality and ART outcomes in cattle. Optimizing the maturation environment to better mimic natural follicular conditions could enhance reproductive efficiency in bovine production systems.

In brief:In vitro maturation is an essential tool in reproductive technologies, though its impact on oocyte quality remains a concern. This study shows that in vitro maturation alters gene expression and DNA methylation in bovine oocytes compared to in vivo matured oocytes, potentially compromising oocyte quality and developmental competence. Abstract:In vitro maturation of oocytes is a routine step in assisted reproduction but is associated with lower embryo development rates compared to oocyte maturation in vivo. We analyzed the genomic profiles of oocytes from the same cow, either matured in vivo or in vitro, using single-cell methylome and transcriptome sequencing, along with transcriptome analysis of corresponding cumulus cells. Both the transcriptome and methylome of the oocytes matured in vitro were altered. Notable changes included alterations in CpG islands associated with imprinted genes, including decreased methylation levels in MEST (PEG1), NNAT (both implicated in large offspring syndrome), and MIMT1. Transcriptomic analysis of their cumulus cells highlighted impaired mitochondrial function, hypoxia responses, and cell adhesion. Our findings highlight the extent to which the maturation environment can influence key epigenetic regulators and mRNA profiles that affect oocyte quality and subsequent developmental outcomes. The data provide a valuable resource for optimizing assisted reproductive technologies.

In brief:Bovine oocytes and follicle cells express the nPR throughout their growth; moreover, nPR expression is colocalised with mitochondria in secondary follicle stage oocytes. Potential nPR target genes which gain expression during oocyte growth populate pathways associated with nuclear and mitochondrial function. Abstract:Previous work from our group demonstrated that blocking nuclear progesterone receptor (nPR) signalling during bovine in vitro oocyte maturation decreases embryo development following subsequent in vitro fertilization and embryo culture, suggesting a critical role for nPR activity in oocyte developmental competence. The objective of this study was to characterize the expression, localization, and function role of the nPR during bovine oocyte growth. Bovine cumulus-oocyte complexes (COCs) were recovered via ovarian slicing. Oocyte diameter was measured and oocytes were allocated to groups according to their size for protein expression and immunofluorescence analysis. In addition, ovarian cortex slides were prepared for immunohistochemistry. To determine a putative functional role of nPR during oocyte growth, an in-silico analysis of genes with increased expression during bovine oocyte growth was conducted to identify those containing the progesterone response element (PRE) core sequence in their promoter regions. The results demonstrated continuous nPR expression during follicle growth in both oocytes and follicular cells. The in-silico analysis revealed that 20% of genes with increased expression during oocyte growth contained a PRE in their promoter regions and were enriched in nuclear and mitochondrial pathways. MitoTracker labelling revealed extensive nPR colocalisation with mitochondria in small bovine oocytes (40-100 μm), which exhibited the highest mitochondrial activity. This study provides new insights into nPR expression in bovine oocytes, COCs, and follicle cells during folliculogenesis and oocyte growth, and suggests an association between nPR and mitochondria in the latter process.

A previous RNA-Seq study revealed that the transcript abundance of specificity protein 1 (SP1) was significantly higher in Day 7 bovine blastocysts compared to conceptuses on Days 10, 13, 16, and 19, suggesting a stage-specific role in early bovine embryo development. The present study aimed to characterize the mRNA expression of SP1 and associated candidate genes (ACSS1, C1QBP, ATF3, MAT2A, and POLD1) during early bovine embryo development from the 2-cell to blastocyst stage. Further, the effects of SP1 inhibition on embryo development were evaluated by culturing embryos with the SP1 inhibitor, mithramycin A (MT) at varying concentrations (0, 25, 50, 100, and 1000 nM). As further validation, we examined expression of SP1 and associated genes by interrogating transcriptomic data from Day 4 (16-cell stage) embryos cultured in vitro or in vivo in the oviducts of lactating or nonlactating dairy cows. The relative abundance of SP1 peaked at the time of embryonic genome activation, being higher (P < 0.05) in 8- and 16-cell embryos compared to the 2-cell stage, and decreasing thereafter (at the morula and blastocyst stages). Similarly, transcript abundance for most of the selected candidate genes involved in the SP1 network were upregulated (P < 0.05) at the 8- and 16-cell stage, but not at other stages investigated. Inhibition of SP1 with MT did not affect embryo development up to the 8-cell stage but reduced (P < 0.05) the proportion of embryos reaching the 16-cell and blastocyst stages in a dose-dependent manner. Moreover, blastocysts produced in the presence of MT contained fewer (P < 0.05) cells than blastocysts developed without MT. Expression of SP1 and associated genes in 16-cell stage (Day 4) embryos produced either in vitro or in vivo was higher (P < 0.05) compared to in vitro-produced 2- to 4- cell stage (Day 2) embryos. These findings suggest an essential role of SP1 during early embryo development, particularly around the time of embryonic genomic activation.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

BackgroundWithin the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent).MethodsBovine antral follicles were dissected, categorized as small (2-4 mm) or large (5-8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM).ResultsWe identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect.ConclusionThis study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

Abstract Study question How does maternal age impact the molecular composition of oocytes during their final meiotic progression? Summary answer Maternal age significantly correlates with proteomic changes during oocyte maturation, particularly in proteostasis and meiosis related proteins, with minimal impact on transcriptome and DNA-methylation patterns. What is known already Oocyte quality declines with maternal age, resulting in diminished developmental competence and higher aneuploidy rates. However, the molecular mechanisms behind this decline in oocyte quality remain elusive. Transcriptomic studies have revealed few differentially expressed genes in advanced maternal age (AMA) oocytes, suggesting that age-related changes in oocyte quality result from a complex interplay of molecular factors, rather than a single cause. However, comprehensive methylation and proteomic data are still lacking. We employed advanced -omics to assess the effect of maternal age on the molecular signature of human oocytes, focusing on DNA-methylation, transcriptome and proteome changes during the final meiotic progression. Study design, size, duration This study included a total of 112 oocytes obtained from young (<35 years, n = 35) and AMA women (>37 years, n = 55), who were recruited in the study from October 2021 to October 2023. Both germinal vesicle (GV, n = 68) and metaphase II (MII, n = 44) oocytes were analysed. Additionally, 19 immature oocytes (GV and metaphase I) from 9 young women were used for validation. Participants/materials, setting, methods Parallel single-cell bisulfite and RNA sequencing was applied to 44 oocytes (26 GV-Young, 6 MII-Young, 8 GV-AMA, 4 MII-AMA) and single-cell proteomics to 68 oocytes (18 GV-Young, 18 MII-Young, 18 GV-AMA, 14 MII-AMA). Additionally, 10 GV and 9 MI oocytes were treated with the proteasome inhibitor MG-312 (0 µM, 10 µM) for 6 hours, followed by rescue in vitro maturation (rIVM) for 36 hours. Chromosomal distribution was assessed by immunocytochemistry. Main results and the role of chance Our analysis revealed no significant changes in DNA methylation patterns in either GV nor MII oocytes associated with AMA. Also, very limited changes were detected in the transcriptome, with transcript levels of only 5 genes in GV oocytes and 7 genes in MII oocytes detected as being significantly changed. In contrast, proteomic analysis, particularly in GV oocytes, revealed significant age-related changes, notably in proteins participating in the proteostasis network (signalosome complex, UCHL1) including chaperones (TRiC-complex, HSP7C, STIP1), and in the cell cycle, including signal transduction factors (1433E, integrins) and cytoskeleton regulators (DYL2, CAPZB, ARHGG) (Rs ≤ |0.5|, p ≤ 0.05). The proteasome complex, which plays a crucial role both in meiosis and the proteostasis network, was found to decline with age; changes were evident in several subunits of the complex (e.g., PRS8, PRS6A, and PRS10; Rs ≤ -0.56, p ≤ 0.05). Compared to controls, treatment of immature oocytes with the proteasome inhibitor MG-132 resulted in either maturation failure or chromosome mislocalization in metaphase plate, further validating the essential role of the proteasome during oocyte maturation. In MII oocytes, 7 proteins showed alterations with age, including the oocyte-specific marker DDX4, which significantly declined in abundance (Rs =-0.6, p ≤ 0.05). Limitations, reasons for caution Unlike GVs that were collected fresh, MII oocytes underwent vitrification and warming before being included in the study due to clinical protocols. These procedures may have unknown effects on the transcriptome and proteome. Wider implications of the findings Our findings suggest that age primarily affects oocyte quality at the post-transcriptional level, potentially through meiosis dysregulation and proteostasis disruption. We also demonstrate the proteasome's vital role in oocyte maturation, suggesting that targeting the proteasome complex may improve oocyte quality in AMA women. Trial registration number Not applicable

Abstract Background Mammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. Results Single-cell RNA-seq libraries were generated from oocytes of known diameters (< 60 to > 120 μm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 μm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100–109 versus 110–119 μm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. Conclusions The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

The ovulation of a mature oocyte at metaphase II of meiosis, with optimal potential to undergo fertilisation by a sperm cell, complete meiosis and sustain the switch to mitotic division, and support early embryo development, involves a protracted and disrupted/delayed series of processes. Many of these are targeted for exploitation in vivo, or recapitulation in vitro, by the livestock industry. Reproductive technologies, including AI, multiple ovulation embryo transfer, ovum pick-up, in vitro embryo production, and oestrus and ovulation synchronisation, offer practitioners and producers the opportunity to produce offspring from genetically valuable dams in much greater numbers than they would normally have in their lifetime, while in vitro oocyte and follicle culture are important platforms for researchers to interrogate the physiological mechanisms driving fertility. The majority of these technologies target the ovarian follicle and the oocyte within; thus, the quality and capability of the recovered oocyte determine the success of the reproductive intervention. Molecular and microscopical technologies have grown exponentially, providing powerful platforms to interrogate the molecular mechanisms which are integral to or affected by ART. The development of the bovine oocyte from its differentiation in the ovary to ovulation is described in the light of its relevance to key aspects of individual interventions, while highlighting the historical timeline.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Follicular fluid (FF), a product of vascular transudate and granulosa and thecal cell secretions, is the milieu that has evolved to support oocyte growth and maturation which plays a central role in oocyte quality determination. Therefore, a suboptimal FF composition may be reflected in compromised oocyte progression through maturation, fertilization, or embryo development. To date, the composition of bovine FF remains understudied. To address this, we comprehensively characterized the metabolomic constituency of bovine FF in the period during which the oocyte undergoes meiotic maturation. More specifically, FF from pre (−24 h) and peri (−2 h)-ovulatory follicles was profiled by high-throughput untargeted ultra-HPLC tandem mass spectroscopy. A total of 634 metabolites were identified, comprising lipids (37.1%), amino acids (30.0%), xenobiotics (11.5%), nucleotides (6.8%), carbohydrates (4.4%), cofactors and vitamins (4.4%), peptides (3.6%), and energy substrates (2.1%). The concentrations of 67 metabolites were significantly affected by the stage of follicle development, 33.3% (n = 21) were reduced (P ≤ 0.05) by a mean of 9.0-fold, whereas 46 were elevated (P ≤ 0.05) by a mean of 1.7-fold in peri- vs pre-ovulatory FF. The most pronounced individual metabolite concentration decreases were observed in hypoxanthine (98.9-fold), xanthine (65.7-fold), 17β-oestradiol (12.4-fold), and inosine (4.6-fold). In contrast, the greatest increases were in retinal (4.9-fold), 1-methyl-5-imidazoleacetate (2.7-fold), and isovalerylcarnitine (2.7-fold). This global metabolomic analysis of bovine FF temporal dynamics provides new information for understanding the environment supporting oocyte maturation and facilitating ovulation that has the potential for improving oocyte quality both invivo and in vitro. Lay Summary The ovaries are part of the female reproductive system, and they produce and store eggs in structures known as ‘follicles’. Depending on the species, one or more follicles release an egg from the ovary during ovulation. FF, which is formed from the secretions of follicle cells and substances delivered from the bloodstream, bathes the eggs as they develop within their follicles. For pregnancy to happen, the egg must be capable of being fertilised by a sperm cell, developing into an embryo and implanting it in the womb. FF has evolved to support the egg to achieve this. Using the cow as a model, this study looks at the composition of FF during the final hours before ovulation, when the egg becomes mature and ready for fertilisation. More than 600 different substances were identified, providing new information, that has the potential to improve egg quality.

The oocyte is the basis of life, supporting development from a fertilized cell to an independent multicellular organism. The oocyte's competence to drive the first cell cycles postfertilization are critical to embryonic survival and subsequent successful pregnancy. Coupled with the complex processes of follicle assembly, activation, differentiation, growth, and terminal maturation, oocyte developmental competence is gradually acquired during oocyte growth and meiotic maturation. Most reproduction management technologies and interventions are centered around these highly coordinated processes, targeting the ovarian follicle and the oocyte within. Thus, our objective was to highlight key aspects of oocyte and follicle development in cattle, and to discuss recent advances in oocyte and follicle-centered reproductive biotechnologies.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife