Search

Background: Examining the mechanistic cellular responses to heat stress could aid in addressing the increasing prevalence of decreased fertility due to elevated ambient temperatures. Here, we aimed to study the differential responses of oocytes and granulosa cells to thermal fluctuations due to seasonal differences. Dry beef cows (n = 10) were housed together, synchronized and subjected to a stimulation protocol to induce follicular growth before ovum pick-up (OPU). Two OPU's were conducted (summer and winter) to collect cumulus-oocyte-complexes (COCs) and granulosa cells. In addition, rectal temperatures and circulating blood samples were collected during OPU. Oocytes were separated from the adherent cumulus cells, and granulosa cells were isolated from the collected OPU fluid. RNA was extracted from pools of oocytes and granulosa cells, followed by library preparation and RNA-sequencing. Blood samples were further processed for the isolation of plasma and leukocytes. The transcript abundance of HSP70 and HSP90 in leukocytes was evaluated using RT-qPCR, and plasma cortisol levels were evaluated by immunoassay. Environmental data were collected daily for three weeks before each OPU session. Data were analyzed using MIXED, Glimmix or GENMOD procedures of SAS, according to each variable distribution., Results: Air temperatures (27.5 °C vs. 11.5 °C), average max air temperatures (33.7 °C vs. 16.9 °C), and temperature-humidity indexes, THI (79.16 vs. 53.39) were shown to contrast significantly comparing both the summer and winter seasons, respectively. Rectal temperatures (Summer: 39.2 ± 0.2 °C; Winter: 38.8 ± 0.2 °C) and leukocyte HSP70 transcript abundance (Summer: 4.18 ± 0.47 arbitrary units; Winter: 2.69 ± 0.66 arbitrary units) were shown to increase in the summer compared to the winter. No visual differences persisted in HSP90 transcript abundance in leukocytes and plasma cortisol concentrations during seasonal changes. Additionally, during the summer, 446 and 940 transcripts were up and downregulated in oocytes, while 1083 and 1126 transcripts were up and downregulated in the corresponding granulosa cells, respectively (Fold Change ≤ -2 or ≥ 2 and FDR ≤ 0.05). Downregulated transcripts in the oocytes were found to be involved in ECM-receptor interaction and focal adhesion pathways, while the upregulated transcripts were involved in protein digestion and absorption, ABC transporters, and oocyte meiosis pathways. Downregulated transcripts in the granulosa cells were shown to be involved in cell adhesion molecules, chemokine signaling, and cytokine-cytokine receptor interaction pathways, while those upregulated transcripts were involved in protein processing and metabolic pathways., Conclusion: In conclusion, seasonal changes dramatically alter the gene expression profiles of oocytes and granulosa cells in beef cows, which may in part explain the seasonal discrepancies in pregnancy success rates during diverging climatic weather conditions., (© 2024. The Author(s).)

The bidirectional communication between the oocyte and the companion somatic cells in the follicular environment is known to be mediated by either a direct communication via gap junction or transzonal projections or indirectly through endocrine, paracrine and autocrine signaling factors. Extracellular vesicles (EVs), which are found in various biological fluids, including follicular fluid (FF) are known to play important roles in mediating the communication between the oocyte and the surrounding somatic cells through shuttling bioactive molecules to facilitate follicular growth and oocyte maturation. As vesicles in the extracellular space are known to reflect the physiological status of the donor or the releasing cells, molecules carried by the EVs in the follicular environment could be markers of the internal and external stressors. EVs exhibit greater degree of heterogeneity in their size, biogenesis and the bioactive molecule they carry. The process of biogenesis of EVs is known to be regulated by several proteins associated with the endosomal sorting complex required for transport (ESCRT) proteins. The type of EVs and surface proteins markers vary according to the type of protein involved in their biogenesis. EVs are recently reported to play indispensable role in promoting cell-to-cell communication during follicular growth. Recent advancements in EV research opened the possibilities to load EVs with specific molecules like miRNA, siRNA, CRISPR-cas9 complex and protein, which showed a new horizon for their application in therapeutics. The present review explores the biogenesis, the role and the future prospects of EVs with a special emphasis given to follicular growth and oocyte maturation., (Copyright © 2020.)

Dietary intake in early lactating cows is outmatched by milk production. These cows experience a negative energy balance, resulting in a distinct blood metabolism and poor reproductive function due to impaired ovulation and increased embryo loss. We hypothesize that oocytes from lactating cows undergoing transient metabolic stress exhibit a different epigenetic profile crucial for developmental competence. To investigate this, we collected oocytes from metabolically-profiled cows at early- and mid-postpartum stages and characterized their epigenetic landscape compared with control heifers using whole-genome bisulfite sequencing. Early-postpartum cows were metabolically deficient with a significantly lower energy balance and significantly higher concentrations of non-esterified fatty acids and beta-hydroxybutyrate than mid-postpartum animals and control heifers. Accordingly, 32,990 early-postpartum-specific differentially methylated regions (DMRs) were found in genes involved in metabolic pathways, carbon metabolism, and fatty acid metabolism, likely descriptive of the epigenetic regulation of metabolism in early-postpartum oocytes. DMRs found overlapping CpG islands and exons of imprinted genes such as MEST and GNAS in early-postpartum oocytes suggest that early lactation metabolic stress may affect imprint acquisition, which could explain the embryo loss. This whole-genome approach introduces potential candidate genes governing the link between metabolic stress and the reproductive outcome of oocytes.

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation., (© 2018 Society for Reproduction and Fertility.)

The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells) and cumulus-oocyte complexes (COCs) to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.