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Quantitative proteomic analysis and verification identify global protein profiling dynamics in pig saliva during the estrus cycle

ABSTRACT: The current estrus detection method is generally time-consuming and has low accuracy. As such, a deeper understanding of the physiological processes during the estrus cycle accelerates the development of estrus detection efficiency and accuracy. This study used label-free acquisition mass spectrometry to explore salivary proteome profiles during the estrus cycle (day −3, day 0, day 3, and day 8) in pigs. A total of 1155 proteins were identified in the label-free analysis, of which 115 were identified as differentially expressed proteins (DEPs) among different groups (P ≤ 0.05). Functional annotation revealed that the DEPs were clustered in calcium ion binding, actin cytoskeleton, and lyase activity. Parallel reaction monitoring (PRM) was applied to verify the relative profiles of protein expression, in which PHB domain-containing protein, growth factor receptor-bound protein 2, elongation factor Tu, carboxypeptidase D, carbonic anhydrase, and trefoil factor 3 were confirmed to be consistent in both label-free and PRM approaches. Comparative proteomic assays on saliva would increase our knowledge of the estrus cycle in sows and provide potential methods for estrus detection.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Saliva

SUBMITTER: Haiyun Xin

LAB HEAD: Jianhao Li

PROVIDER: PXD043141 | Pride | 2023-09-15

REPOSITORIES: Pride

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Project description:The potential for xenoestrogens and other environmental chemicals to alter female reproductive function calls for the study of their affects on endogenous hormone-regulated gene expression pathways during uterine tissue remodeling which occurs as part of the menstrual/estrus cycle. However, our knowledge of these pathways is limited. Here, we characterize changes in the CD-1 mouse uterine transcriptome during proestrus and estrus, which are regulated by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 wk of age and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis identified 2,429 genes differentially expressed in estrus compared to proestrus, indicating that the mouse uterus undergoes remarkable remodeling during the estrus cycle, affecting ~10% of all protein-coding genes. Changes in gene expression associated with structural alteration of the uterus include remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis, MHC class II presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and several transcription factors with poorly understood roles in reproductive tissues, include several genes and gene networks that have been implicated in pathological states. The information presented here builds a background for understanding of mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases. Beginning on postnatal day 42, female CD-1 mice were monitored for reproductive stage cycle for 2 consecutive cycles and then total RNA was isolated from uterine horns at estrus or proestrus. Samples were pooled for microarray as follows. Six litters were used to generate 2 tissue pools for estrus and 2 tissue pools for proestrus, with estrus pool 1 and proestrus pool 1 comprised of mice from the same 3 litters, and estrus pool 2 and proestrus pool 2 comprised of mice from the other 3 litters. Each pool was comprised of uterine tissue RNA from n= 4-6 individual mice. Fluorescent labeling of RNA and hybridization of the Alexa 555-labeled (green) and Alexa 647-labeled (red) RNA samples to Agilent-026655 Whole Mouse Genome Microarray 4x44K v2 (Agilent Technology, Palo Alto, CA; catalog # G4846A) were carried out, with dye swapping for each estrus cycle stage comparison.

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Quantitative proteomic analysis and verification identify global protein profiling dynamics in pig saliva during the estrus cycle

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