Seasonal environmental fluctuations alter the transcriptome dynamics of oocytes and granulosa cells in beef cows

Abstract

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.

Keywords: Beef cows; Granulosa cells; Heat stress; Oocytes; Seasonality; Transcriptome.

Fig. 1

Hormonal protocol used in the present study. Briefly, a 5-day CIDR and FSH synchronization and stimulation protocol was used to induce follicular growth before each OPU session (Winter and Summer). Created with BioRender.com

Fig. 2

Climatic conditions were collected for twenty-one days before each OPU session. A) Average air temperature, B) Relative humidity, C) Temperature humidity index (THI). Data were collected using the Florida Automated Weather Network (FAWN; https://fawn.ifas.ufl.edu/data/reports/)

Fig. 3

Mean ± SEM of rectal temperature (A), plasma cortisol concentration (B), and HSP70 (C) and HSP90 (D) transcript abundance from beef cows during the summer and winter (n = 11). *** Denotates statistical differences when P ≤ 0.05 and * denotates statistical tendencies when P ≥ 0.05 still P < 0.1

Fig. 4

Transcriptomic analyses and differentially expressed genes (DEGs) during summer and winter of oocytes in beef cows. Principal component analysis (PCA; A) and hierarchical heatmap (B) indicating the expression of genes in oocyte’s during summer and winter. Venn diagram (C) showing exclusively and mutually expressed genes between summer and winter and Volcano plot (D) indicating the differentially expressed genes from summer compared to the winter [Individual red and green dots represent the number of up- and downregulated genes, respectively (FC ≥ 2; FDR p-value ≤ 0.05)]. Summer = SUM (n = 4) and Winter = WIN (n = 4)

Fig. 5

Top 20 enriched pathways (A. Upregulated pathways; B. Downregulated pathways) and biological processes (BP; C. Upregulated BP; D. Downregulated BP) in oocytes collected during summer (SUM) and winter (WIN) in beef cows

Fig. 6

Transcriptomic analyses and differentially expressed genes (DEGs) during summer and winter of granulosa cells in beef cows. Principal component analysis (PCA; A) and hierarchical heatmap (B) indicating the expression of genes in granulosa cells during summer and winter. Venn diagram (C) showing exclusively and mutually expressed genes between summer and winter and Volcano plot (D) indicating the differentially expressed genes from summer compared to the winter [Individual red and green dots represent the number of up- and downregulated genes, respectively (FC ≥ 2; FDR p-value ≤ 0.05)]. Summer = SUM (n = 4) and Winter = WIN (n = 4)

Fig. 7

Top 20 enriched pathways (A. Upregulated pathways; B. Downregulated pathways) and biological processes (BP; C. Upregulated BP; D. Downregulated BP) in granulosa cells collected during summer (SUM) and winter (WIN) in beef cows

Fig. 8

Commonly and exclusively differentially expressed genes from summer compared to winter collected oocytes (OO) and granulosa cells (GCs)

Fig. 9

Top pathways and the related commonly DEGs in the oocytes and granulosa cells in summer compared to winter groups

Fig. 10

Top pathways and the related DEGs which exhibited a downregulation in oocytes and granulosa cells in summer compared to winter groups