Semen Modulates Inflammation and Angiogenesis in the Reproductive Tract of Female Rabbits

Abstract

The maternal environment modulates immune responses to facilitate embryo development and ensure pregnancy. Unraveling this modulation could improve the livestock breeding systems. Here it is hypothesized that the exposure of the female rabbit reproductive tract to semen, as well as to early embryos, modulates inflammation and angiogenesis among different tissue segments. qPCR analysis of the gene expression changes of the anti-inflammatory interleukin-10 (IL10) and transforming growth factor beta family (TGFβ1-3) and the angiogenesis mediator vascular endothelial growth factor (VEGF-A) were examined in response to mating or insemination with sperm-free seminal plasma (SP). Reproductive tract segment (cervix to infundibulum) samples were obtained in Experiment 1, 20 h after gonadotropin-releasing hormone (GnRH) stimulation (control), natural mating (NM) or vaginal infusion with sperm-free SP (SP-AI). Additionally, segmented samples were also obtained at 10, 24, 36, 68 or 72 h after GnRH-stimulation and natural mating (Experiment 2). The results of gene expression, analyzed by quantitative PCR, showed that NM effects were mainly localized in the uterine tissues, depicting clear temporal variation, while SP-AI effects were restricted to the oviduct. Changes in anti-inflammatory and angiogenesis mediators indicate an early response in the uterus and a late modulation in the oviduct either induced by semen or preimplantation embryos. This knowledge could be used in the implementation of physiological strategies in breeding systems to face the new challenges on rabbit productivity and sustainability.

Keywords: angiogenesis; endometrium; gene expression; inflammation; oviduct; rabbit; seminal plasma; spermatozoa.

Figure 1

Representation of the experimental design and tissue sections obtained from does. Sequential tissue segments derived from does were endocervix, distal uterus, proximal uterus, utero-tubal junction, distal isthmus, ampulla and infundibulum. Intramuscular injection of 0.03 mg gonadotropin-releasing hormone (GnRH) was used to induce ovulation in all groups of both experiments.

Figure 2

Results of Experiment 1. Gene expression of (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ3 and (e) VEGF-A in differentially expressed segments (p < 0.05) (DistUt: distal uterus, ProxUt: proximal uterus, UTJ: utero-tubal junction, Amp: ampulla and Inf: infundibulum) of the does internal reproductive tract after 20 h of the induction of the ovulation with 0.03 mg gonadotropin-releasing hormone (GnRH) intramuscularly (20 h_C, n = 3), 20 h post-GnRH-stimulation and seminal plasma vaginal infusion (20h_SP-AI, n = 3) and 20 h post-GnRH-stimulation and natural mating (20 h_NM, n = 3). Fold changes relative to the control of the ovulation group are shown. Different letters (a,b) represent statistical differences between groups (p < 0.05). Median (minimum, maximum).

Figure 3

Results of Experiment 2. Gene expression of (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ3 and (e) VEGF-A was statistically significant in rabbit endocervix (Cvx), distal uterus (DistUt), proximal uterus (ProxUt), utero-tubal junction (UTJ), distal isthmus (Isth), ampulla (Amp) and infundibulum (Inf) after 10, 24, 36, 68 or 72 h post-induction of the ovulation (intramuscular injection of 0.03 mg gonadotropin-releasing hormone) and natural mating (n = 3/collection time). Fold changes relative to 10 h post-mating group are shown. Different letters (a–c) represent statistical differences between groups (p < 0.05). Median (minimum, maximum).

Figure 4

Heatmap composition of the gene expression changes among tissues in the different groups included in the study. Changes in IL10, TGFβ1, TGFβ2, TFGβ3 and VEGF-A expression among tissues (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; distal isthmus, Isth; ampulla, Amp; and infundibulum, Inf) (a) at 20 h after the induction of ovulation with 0.03 mg of gonadotropin-releasing hormone (GnRH) intramuscularly, as the control of ovulation (20 h_C); 20 h post-GnRH stimulation and sperm-free seminal plasma vaginal infusion (20 h_SP-AI); and 20 h post-GnRH stimulation and natural mating (20 h_NM); (b) at 10, 24, 36, 68 and 72 h post-GnRH-stimulation and natural mating. Row Z-scores of the mean fold change relative to the reference group (UTJ) are shown. Red indicates upregulation, and green indicates downregulation.