Influence of seminal plasma, spermatozoa and semen extender on cytokine expression in the porcine endometrium after insemination
Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. Animal reproduction science
(Impact Factor: 1.51).
02/2011; 123(3-4):210-20. DOI: 10.1016/j.anireprosci.2010.11.016
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.
Available from: Elisabeth Persson
- "We have previously studied the effects of SP on the cytokines TGF-β1, IL-6 and IL-10 in different parts of the oviduct  and endometrium  both shortly after insemination (5–6 h) and later (35–40 h). The results showed that at 35–40 h after infusion, not only SP but also semen extender (BTS) and spermatozoa in BTS, up-regulated TGF-β1 mRNA expression compared with the control (catheter insertion) in the oviduct (isthmus and infundibulum). "
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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.
Available from: Andrzej Madej
- "These two prostaglandins are also involved in acute and chronic inflammatory conditions  . Seminal plasma suppresses the migration of polymorphonuclear neutrophil granulocytes (PMNs) into the uterus, decreases PMNs infiltration of the endometrium and enhances the rate of disappearance of uterine inflammation in gilts following breeding  . Rozeboom , et al.  suggested that non-estrogenic factors in seminal plasma may protect spermatozoa from an inflamed uterine environment and consequently a suitable population of viable spermatozoa is able to reach the site of fertilization. "
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ABSTRACT: The objectives of this study were to explore whether heparin-binding proteins, separated by fast protein liquid chromatography from boar seminal plasma influence the release of pros- taglandins F2 α, (PGF2α), E2 (PGE2) and interleu- kin-6 (IL-6) by porcine endometrial and cervical cells and even bovine endometrial cells. In Ex- periment I, we showed that release of PGF2α by endometrial epithelial, endometrial stromal and cervical stromal cells to the medium was inhi- bited (p < 0.05) to 9.0% - 60.6% after 24 h incu- bation with 125 μg of heparin-binding proteins. Tumor necrosis factor α (TNFα) stimulated re- lease of IL-6 by endometrial and cervical stromal cells after 24 h incubation, but in the presence of heparin-binding proteins, this stimulation was attenuated. Release of PGF2α by cryopreserved (Experiment II) and primary (Experiment III) cer- vical stromal cells was significantly inhibited after 3 h incubation with 66 - 95.4 μg of heparin- binding proteins. A significant inhibition of PGE2 release by cryopreserved and primary cervical stromal cells was already achieved after incuba-tion with 16.5 - 23.9 μg of heparin-binding pro-teins. The release of IL-6 by cryopreserved cells was stimulated after 3 h incubation with heparin- binding proteins in a dose dependent manner in contrast to the release of IL-6 by freshly isolated cervical stromal cells. We also found (Experi-ment IV) that porcine heparin-binding seminal plasma proteins inhibited release of PGF2α and stimulated release of IL-6 by bovine endometrial epithelial cells. In conclusion, a group of hepa- rin-binding proteins separated by fast protein liquid chromatography from boar seminal plas- ma inhibit PGF2α, PGE2 and stimulate IL-6 re-lease by porcine endometrial and cervical cells, and even by bovine endometrial cells. Thus, these proteins have a similar effect as the entire seminal plasma.
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ABSTRACT: Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-β1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.
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