Seminal fluid expands the uterine gamma/delta T cell pool during early pregnancy in mice

Seminal fluid elicits an immune response in the uterine mucosa after mating that impacts embryo implantation and pregnancy, but the underlying molecular and cellular events are unclear. In this study, we report RNA sequencing to analyze the uterine response to seminal fluid after mating. Females exposed to seminal fluid of intact males exhibited gene expression changes on D3.5 post-coitum (pc) just prior to embryo implantation, compared to females mated with males surgically rendered seminal plasma deficient. Functional enrichment analysis revealed genes related to T cell activation amongst those with the largest fold-changes. Using flow cytometry we then showed profound changes in uterine T cell abundance and phenotype regulated by seminal fluid contact. While CD4⁺ and CD8⁺ T cells were elevated by seminal fluid, the most conspicuous change was in CD4^-CD8^- T cells expressing γδ T cell receptors (TCR). Mating with intact males caused a 8.3-fold increase in γδ T cell abundance compared to estrous virgin females, and a 22.4-fold increase in the proportion of γδ T cells expressing proliferation marker Ki67. Vγ6⁺ cells were the most abundant subpopulation in the uterus, followed by Vγ4⁺ and Vγ1⁺ T cells, and all three were similarly expanded after mating. Seminal plasma was critical for γδ T cell accumulation and activation in the endometrium, and similar changes occurred in uterine-draining lymph nodes but not spleen. These findings identify γδ T cells as prominent in the immune response to seminal fluid and imply key roles in uterine immune regulation and reproductive success.