Influence of seminal plasma, spermatozoa and semen extender on cytokine expression in the porcine endometrium after insemination.
ABSTRACT The effects of semen components or extender alone on the expression of selected cytokines [interleukine (IL)-1β, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10 and transforming growth factor (TGF)-β1] on the porcine endometrium were studied, as well as the presence of polymorphonuclear neutrophilic granulocytes (PMNs). In experiment (Exp) I, groups of gilts were sampled at 5-6h after insemination with fresh semen in extender (Beltsville thawing solution, BTS), spermatozoa in extender (Spz), seminal plasma (SP), or only BTS (control). In Exp II, gilts were sampled 35-40h after insemination with Spz, SP, BTS or only catheter inserted (as control). Immunohistochemical (IHC) labelling of IL-6, IL-10 and TGF-β1 was evident, especially in surface and glandular epithelia of the porcine endometrium. There were no consistent differences in IHC-labelling of the cytokines in relation to different treatments. However, the scores for IL-6 and IL-10 in surface epithelium and sub-epithelial connective tissue compartments were higher at 35-40h than shortly (5-6h) after treatment. Cytoplasmic labelling in the sub-epithelial connective tissue was observed in scattered individual cells but not in PMNs. Shortly (5-6h) after insemination, there were no differences between animals inseminated with BTS (control) and the semen components for any of the cytokine mRNAs. Later however, at 35-40h, lower endometrial expression of TGF-β1 mRNA was observed in the Spz and BTS groups compared with the control (catheter only). The same pattern was found for IL-10 (NS). The mRNA expression of IL-6 in the BTS inseminated group was higher compared to the control group. Insemination with SP resulted in significantly lower PMN cell infiltration in the sub-epithelial connective tissue compared with Spz or BTS groups shortly (5-6h) after insemination. Later (35-40h), a significant difference was found between SP (lower) and the control group (only catheter). To conclude, our results show that insemination and/or inseminated components modulated cytokine expression in the gilt endometrium. The semen extender BTS stimulated immune reactivity, as shown by down-regulation of the suppressive cytokine TGF-β1. Insemination with solely SP clearly decreased PMN cell infiltration of the gilt endometrium. However, no clear relation between the cytokines studied and PMN cell presence was found.
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ABSTRACT: The effects of seminal plasma on the presence of the cytokines transforming growth factor (TGF)-beta1, interleukin (IL)-10 and IL-6 in ovarian follicles and follicular fluid were studied shortly after insemination in gilts.Ovaries from gilts were sampled 5--6 h after insemination with either seminal plasma (SP), fresh semen in extender (Beltsville thawing solution, BTS), spermatozoa in extender (Spz), or only BTS (control). Immunohistochemical (IHC) labeling of TGF-beta1, IL-10 and IL-6 was evident in the ovarian oocytes and granulosa cells independent of stage of follicular development (antral follicles). Theca interna cells were labeled to a high degree in mature follicles. No consistent differences between treatment groups could be observed for any of the cytokines.In follicular fluid, high concentrations of TGF-beta1 were found while the levels of IL-10 and IL-6 were low. There were no differences between treatment groups. Our results show a presence of the cytokines TGF-beta1, IL-6 and IL-10 in oocytes, granulosa and theca cells, as well as in the fluid of mature follicles suggesting a role of these cytokines in intra-ovarian cell communication. However, treatment (SP, fresh semen in BTS, spermatozoa in BTS or BTS) did not influence the IHC-labeling pattern or the levels of these cytokines in follicular fluid shortly after insemination.Acta Veterinaria Scandinavica 09/2013; 55(1):66. · 1.00 Impact Factor
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ABSTRACT: After mating, seminal plasma has an immuno-modulatory effect on the endometrium in some mammals. In jennies, achieving conception via artificial insemination (AI) with frozen-thawed semen is generally much more difficult than in mares. The endometrial inflammatory response is hypothesized to be a contributing factor to the lesser fertility. Following a cross-over experimental design, the uterine inflammatory response of six jennies was evaluated at 6h after AI with frozen-thawed semen (deposited in the uterine body) in the presence or absence of autologous seminal plasma (+SP or -SP). The endometrial cytology and histology of the animals were examined by uterine lavage, uterine swabbing and biopsy. The amount of cyclooxygenase-2 (COX-2) protein in endometrial cells was also evaluated. As a control (C), the same examinations were made before any AI procedure (i.e., when the jennies were in oestrus). Large numbers of polymorphonuclear neutrophils (PMN) were observed in the -SP and +SP cytology and biopsy samples; more than in the C samples. The -SP samples also had intense COX-2 labelling; less labelling was detected in the +SP and C samples (no significant difference between these latter two types). Thus, while the presence of SP does not change the post-AI number of PMNs with regard to that detected in its absence, it does reduce COX-2 protein. Further research into the complex mix of molecules in SP and its effects during AI might help increase the pregnancy rates achieved in jennies.Animal reproduction science 11/2013; · 1.56 Impact Factor
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ABSTRACT: Infusion of seminal plasma in the uterus is known to elicit an instant inflammatory response in the porcine uterus, but whether or not it prepares a uterine immunological response to the presence of conceptuses is not well understood. The presence of a conceptus induced immune changes in leukocyte populations, as measured by flow cytometry and mRNAs for various cytokines by quantitative reverse-transcriptase PCR in porcine endometrium collected on Day 6 and Day 13 from cycling and pregnant animals. Seminal plasma infusion also induced long-term modulatory effects, resulting in more endometrial FoxP3-positive T-regulatory and T-helper cells 6 days after infusion as compared to cycling and pregnant animals. The number of T-cytotoxic and T-null cells did not change between the studied groups. The early molecular effects of seminal plasma were not observed at 13 days post-infusion, although animals on Day 13 of pregnancy did show significantly more T cells of any type investigated. Seminal plasma also showed a delayed effect on cytokine expression, specifically exhibiting a significant increase in interleukin 10 (IL10) and a decrease in granulocyte macrophage colony-stimulating factor (GMCSF) gene expression on Day 13 as compared to Day 6 of cycling or pregnant gilts. The results indicate a delayed regulatory effect of seminal plasma on immune responses in the porcine uterus, which are similar to immune changes generated by the implanting conceptuses. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.Molecular Reproduction and Development 12/2013; · 2.68 Impact Factor