br Material and methods br Results br
Material and methods
Discussion Both progesterone and estradiol are major ovarian steroids crucial for the development and homeostasis of the female reproductive tract (Patel et al., 2015). In the cervix, estrogen softens the cervical stroma affecting the extracellular matrix (ECM) with changes in collagen and elastin as well as allowing water permeation (Nallasamy et al., 2017) while progesterone promotes cervical closure by changing the structural organization of collagen and elastin to increase the tissue stiffness of the ECM (Neubauer et al., 2011, Nallasamy et al., 2017). Within the cervical mucosa and stroma, ESR1 and PGR were found to have similarly high expression during both estrus and diestrus, suggesting that PGR and ESR1 are working synergistically. This supports previous research, as it is known that progesterone responsiveness is dependent on the presence of an estrogenic drive (Tsai et al., 1998). Moreover, the gene coding for PGR is a well-known ESR1 target in the human female reproductive tract (Patel et al., 2015). Additionally, this work shows similar expression patterns of ESR1 and ESR2, with both present in the nucleus and cytoplasm of epithelium of the endocervix as well as the nucleus of smooth muscle of the cervix in estrus and late pregnancy, similar to PGR. According to Larsen and Hwang, the activity of the progesterone is pleiotropic, acting not only through nuclear activation of canonically steroid regulated genes, but also through non-genomic actions mediated by membrane receptors acting via G protein-coupled signaling cascades (Larsen and Hwang, 2011). We observed a similar pattern of PGR expression in the cervical mucosa and stroma throughout the estrous cycle, as previously reported in mare endometrium (Silva et al., 2014). Both our study and Silva’s showed an increased expression of PGR in the cervix during estrus and diestrus compared to pregnancy. Additionally, we saw PGR protein staining in the nucleus and cytoplasm of epithelium GDC-0941 in cervical mucosa, as well as in the nuclei and cytoplasm of smooth muscle for all reproductive stages evaluated, similar to findings in humans (Patel et al., 2015). Our results did not show statistical differences in cervical ESR1 expression between estrus and diestrus, however, Silva and coworkers observed that estrogen secreted in the pre-ovulatory period increased the endometrial transcripts for ESR1, while exposure to progesterone in diestrus down-regulated ESR1 expression (Silva et al., 2014). This is discrepancy is likely to be due to differences in tissue-specific expression; Loux et al. also observed significant differences in gene expression between the endometrium and cervical mucosa in the mare (Loux et al., 2017). Progesterone receptors are responsible for modulating the expression of genes stimulating collagen synthesis, as well as those inhibiting collagen breakdown (Neubauer et al., 2011). Specifically, PGR antagonizes estrogen-induced collagenase expression (Kyo et al., 2010) and inhibits hyaluronate synthesis in human cervical mucosal fibroblasts (Uchida et al., 2007). Therefore, it was surprising to see no significant differences in expression levels of PGR between the different reproductive stages observed. This lack of change could be due to a variety of reasons, including an increased concentration of progesterone itself, an increase in down-stream elements increasing the functional action of the sex-steroids themselves, or simply because other factors are coming into play to facilitate the changes in collagen composition. When we analyzed the cervical mucosa and stroma in late equine pregnancy, expression levels for ESR1 and PGR were lower than those observed in estrus and diestrus. During this stage of pregnancy, progesterone metabolites such as 5α-dihydroprogesterone (5α-DHP) are approximately three times higher than progesterone in diestrus (Scholtz et al., 2014). As 5α-DHP is known to be bioactive, and to have an affinity to PGR equivalent to progesterone itself (Scholtz et al., 2014), the high levels of progestogens seen in late gestation may preclude the need for high levels of the receptor. Additionally, we know that PGR has non-genomic signaling, including the activation of intracellular signaling pathways, modulation of putative cell surface receptors, ion channels, and cytoplasmic second messenger cascades (Singh et al., 2013), suggesting these mechanisms could be at play in the pregnant cervix as well.