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Transcriptomic Profiles of Bovine Mammary Epithelial Cells Under Heat Stress and During Subsequent Recovery

ABSTRACT: Heat stress (HS) poses a significant challenge to the dairy industry as it negatively affects milk production. However, the molecular mechanism behind mammary gland responses to HS and recovery remains poorly understood. This study aimed to determine the transcriptomic profile and uncover the underlying mechanism using RNA-sequencing and functional analysis in bovine mammary epithelial (MAC-T) cells. MAC-T cells were cultured in 6 groups: 1) basal (undifferentiated, unDiff) medium or 2) lactogenic (differentiated, Diff) medium for four days. Differentiated cells were subject to either 3) non-HS (37℃) or 4) HS (42℃) for 1hr. Two HS groups were further allowed to recover at 37℃ for 2 hr (D2R) and 6 hr (D6R). Among six group comparisons, 1,668 differentially expressed genes (DEGs) were identified. HS vs. D6R groups yielded the highest number of DEGs, followed by HS vs. non-HS, and Diff vs. unDiff groups. Functional analysis suggested that differentiated cells exhibited upregulated genes associated with milk lipid synthesis, indicating their lactogenic state. Moreover, HS suppressed cellular differentiation and activated several heat shock protein (HSP) genes. Additionally, several transcription factors were identified as potential positive or negative regulators of HS response. During recovery, chaperon-mediated protein folding remained elevated and apoptosis regulation was induced after 2 hr. After 6 hrs, response to oxidative stress and cellular homeostasis regulation were enriched. Overall, differentiated MAC-T cells enriched genes in lipid synthesis activities. HS triggered heat shock response and protein misfolding while impaired cellular differentiation in differentiated MAC-T cells. Furthermore, MAC-T cells demonstrated dynamic recovery responses, including regulations on apoptosis and hypoxia

ORGANISM(S): Bos taurus

PROVIDER: GSE249950 | GEO | 2025/12/11

REPOSITORIES: GEO

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	GSE249950_MACT_count.txt.gz	Txt

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Project description:Heat stress (HS) could damage animal reproduction, while the mechanisms are still required to be better understood. Here, we showed that incubation of porcine immature Sertoli cells (iSCs) at 43 °C for 30 minutes followed 36h recovery under normal culture could inhibit the proliferation, promote apoptosis and increase the lactate production. Then, we profiled mRNA expression by RNA sequencing on Control (before HS), HS0.5 (0h after HS) and HS0.5-R36 (36h recovery after HS) groups of iSCs. We identified 126 (92 up- and 64 down-regulated), 3372 (2076 up- and 1296 down-regulated) and 3096 (1772 up- and 1324 down-regulated) differentially expressed (DE) mRNAs respectively in HS0 vs. Control, HS0-R36 vs. HS0 and HS0-R36 vs. Control comparisons. Gene ontology (GO ) enrichment found multiple significantly enriched biological pathways involved in different comparisons, including cellular response to heat, heat shock protein binding, regulation of cellular response to stress, RNA polyadenylation, sterol biosynthetic process, positive regulation of stress-activated MAPK cascade, chaperone-mediated protein folding, ATPase activity, cell cycle process and DNA replication etc. KEGG analysis showed the changed pathways, including cell cycle, MAPK, P53, PI3K-AKT, GnRH, HIF1, Wnt, cAMP signal pathways and Glycolysis/Gluconeogenesis etc. RT-qPCR validation of 9 DEGs (DNAJB1, TRAF6, INSIG1, GADD45G, HDAC6, FKBP4, SERPINE1, PFKP and GALM) showed the expression change trend of most DEGs (except TRAF6) consistent with the RNA-seq. Moreover, 6 of HSPs (HSPA6, HSPB1, HSPD1, HSP90AA1, HSP90AB1 and HSPH1) were validated by RT-qPCR and 5 of 6 (except HSP90AB1) showed the similar expression trend to RNA-seq results. Further validation of HSP90AA1 on protein level via immunostaining and westernbloting showed similar expression trend to RT-qPCR and RNA-seq. Taken together, HS could extensively change the global transcriptome, especially HSPs, to affect proliferation, apoptosis and metabolite production of pig iSCs.

Project description:Eimeria (E.) maxima parasite infects the midgut disrupting the jejunal and ileal mucosal lining causing high morbidity and mortality in chickens. Heat stress (HS) is a seasonal stressor that affects biological functions leading to poor performance. Our objective was to elucidate how HS, E. maxima infection, and their combination affect the ileum transcriptome. Two-hundred and forty 2-week-old males Ross708 chickens were randomly allocated into four treatment groups: thermoneutral control (TNc), thermoneutral infected (TNi), heat stress control (HSc), and heat stress infected (HSi), with 6 replicates each of 10 birds. Infected groups received 200x103 sporulated E. maxima oocysts/bird, and heat treatment groups were raised at 35oC. At 6-day post-treatment, five chickens per group were randomly sampled, and ileum tissues were collected for RNA extraction and sequencing using NGS Illumina sequencing platforms. A total of 413, 3377, 1908, and 2304 DEGs were identified when applying the comparisons: TNc vs HSc, TNc vs TNi, HSi vs HSc, and TNi vs HSi, respectively, at cutoff ≥1.2-fold change (FDR: q<0.05). HSc vs TNc showed upregulation of lipid metabolic pathways and degradation and metabolism of multiple amino acids, and downregulation of most immune-related and protein synthesis pathways. TNc vs TNi displayed upregulation of most of the immune-associated pathways and eukaryotic mRNA maturation pathways, and downregulation of fatty acid metabolism and multiple amino acid metabolism pathways including tryptophan. Comparing HSi versus HSc and TNi revealed that combining the two stressors restored some normal cellular functions, e.g., oxidative phosphorylation and protein synthesis, and reduced the intensity of immune response associated with E. maxima infection. Tryptophan metabolism was upregulated, including genes that contribute to catabolizing tryptophan through serotonin and indole pathways; which might result in reducing the cytoplasmic pool of nutrients and calcium available for the parasite to scavenge and consequently might affect the parasite’s reproductive ability.

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Transcriptomic Profiles of Bovine Mammary Epithelial Cells Under Heat Stress and During Subsequent Recovery

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