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Ejaculate fraction-driven changes in porcine uterine gene expression and histological profiles

ABSTRACT: Seminal plasma contains bioactive components capable of modulating the uterine environment. Although the boar ejaculate is fractionated, with each portion differing in composition, the cumulative effect of these fractions on the female reproductive tract remains poorly understood. This study evaluated how stepwise inclusion of ejaculate fractions affects uterine gene expression, cell proliferation, and immune response after artificial insemination (AI). Semen doses were prepared using three combinations: FR1 (sperm-rich fraction), FR2 (FR1 + intermediate fraction), and FR3 (FR2 + post-sperm fraction). Each dose was used for intrauterine AI in sows (n=5 per group), with a non-inseminated control group (n=5). Uterine tissues (U) were collected six days post-estrus onset for transcriptomic and histological analysis. RNA-sequencing analysis identified the highest number of differentially expressed genes (DEGs; FDR<0.05 and |FC|≥1) in U-FR2 group (n=602), followed by U-FR3 (n=465) and U-FR1 (n=414), relative to the control. Most DEGs were down-regulated in inseminated sows, including immune-related genes such as VNN2. Gene ontology analysis revealed conserved biological processes across groups, including metabolism, immune modulation, and epithelial remodeling. Histological assessment showed reduced Ki-67 positivity and decreased CD3+ T-cell infiltration in all the inseminated groups (p<0.05) compared to the control. Notably, uteri from U-FR2- and U-FR3-treated sows showed a faster transition toward a secretory phenotype (% of glandular tissue; p<0.05). These findings demonstrate that the inclusion of different ejaculate fractions triggers distinct uterine transcriptomic and histological responses, highlighting the role of SP composition on the uterine environment and suggesting that fraction-specific SP components may influence early pregnancy outcomes.

ORGANISM(S): Sus scrofa

PROVIDER: GSE298329 | GEO | 2026/05/23

REPOSITORIES: GEO

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Project description:The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction (SRF); F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows (p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we have found a specific protein cargo in uterine and oviductal EVs depending on the type of semen fraction the sow was inseminated with, and these insemination with different seminal fractions results in the oviductal and uterine secretion of specific EVs proteins are closely associated with reproductive processes.

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Ejaculate fraction-driven changes in porcine uterine gene expression and histological profiles

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