Effects of vinclozolin exposure on the expression and activity of SIRT1 and SIRT6 in the porcine ovary.

Within the mammalian reproductive system sirtuin 1 and 6 (SIRT1, SIRT6) are considered to contribute to steroid hormone signaling and control of reproductive physiology. Therefore, the specific question is whether and how a commonly used dicarboximide fungicide with antiandrogenic activity, vinclozolin (Vnz) alters SIRT1 and SIRT6 expression and whether both investigated sirtuins positively affect survival of the follicles after vinclozolin exposure. Immunocytochemistry and immunohistochemistry were performed to localize SIRT1 and SIRT6 expression in cultured granulosa cells (GCs; 48 hours) and whole ovarian follicles (24 hours) after treatment with two androgens, testosterone (T; 10-7 M) and dihydrotestosterone (DHT; 10-7 M), and an antiandrogen, Vnz (1.4 x 10-5 M), separately and in combinations. Granulosal and follicular mRNA and protein expression of both sirtuins was also investigated by real-time PCR and Western blot. In addition, their concentration and activity was studied by immunoenzymatic and fluorescence assays. Our observations: (1) demonstrate the presence of both investigated sirtuins in ovarian cells, (2) show their potential involvement in the control of follicular atresia because of increased SIRT1/SIRT6 expression and SIRT1 activity after exposure to Vnz, (3) represent the first data on the interrelationships between sirtuins and androgens in porcine ovarian cells. Based on these findings and our previous results we can conclude, that SIRT1 and SIRT6 do not exert the protective effects in ovarian follicles after vinclozolin exposure. These novel data on the role of SIRT1/SIRT6 in porcine ovarian follicles shows that in the presence of the investigated fungicide, sirtuins are upregulated, which can induce apoptosis of follicular cells. Furthermore the androgen receptor sensitivity to ligands, especially environmental ones (for example: vinclozolin) might be directly linked with the mechanism of action of both investigated sirtuins in the porcine ovary, which requires further investigation.

Identification of perivascular and stromal mesenchymal stem/progenitor cells in porcine endometrium.

Mammalian uterus contains a population of mesenchymal stem/progenitor cells that likely contribute to endometrial regeneration during each reproductive cycle. In human and mouse, they reside in perivascular, epithelial and stromal compartments of the endometrial functionalis and basalis. Here, we aimed to identify tissue resident cells expressing mesenchymal stem cell markers CD29, CD44, CD90, CD105, CD140b and CD146 in the porcine endometrium. We used single immunofluorescence and Western blotting. Each of these markers was detected in small cells surrounding endometrial blood vessels. CD105 and CD146 were also expressed in single stromal cells. A few stromal and perivascular cells showed the presence of pluripotency marker Oct4 in the cytoplasm, but not in the nucleus, which may imply they are not truly pluripotent. Endometrial cell cultures were examined for the expression of CD29, CD44, CD90, CD105 and CD140b proteins and tested in wound-healing assay and culture model of chemotaxis. In conclusion, our results demonstrate perivascular location of prospective mesenchymal stem/progenitor cells in the porcine endometrium and may suggest that stromal CD105+ and CD146+ cells represent more mature precursors originating from their perivascular ancestors.