Specific locations and amounts of denatured collagen and collagen-specific chaperone HSP47 in the oviducts and uteri of old cows as compared with those of heifers

Abstract

Collagen, the most abundant extra-cellular matrix in oviducts and uteri, performs critical roles in pregnancies. We hypothesised that the locations and amounts of both denatured collagen and the collagen-specific molecular chaperone 47-kDa heat shock protein (HSP47) in the oviducts and uteri of old cows are different compared with those of young heifers because of repeated pregnancies. Since detecting damaged collagen in tissues is challenging, we developed a new method that uses a denatured collagen detection reagent. Then, we compared damaged collagen in the oviducts and uteri between post-pubertal growing nulliparous heifers (22.1 ± 1.0 months old) and old multiparous cows (143.1 ± 15.6 months old). Further, we evaluated the relationship between denatured collagen and HSP47 by combining this method with fluorescence immunohistochemistry. Picro-sirius red staining showed collagen in almost all parts of the oviducts and uteri. Expectedly, damaged collagen was increased in the oviducts and uteri of old cows. However, damaged collagen and HSP47 were not located in the same area in old cows. The number of fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. These organs of old cows showed higher HSP47 protein amounts than those of heifers. However, the uteri, but not oviducts, of old cows had lower HSP47 mRNA amounts than those of heifers. These findings revealed the specific location and amounts of denatured collagen and HSP47 in the oviducts and uteri of old cows compared with those of heifers.

Anti‐Müllerian hormone stimulates expression of the collagen‐specific chaperone 47‐kDa heat shock protein in bovine uterine epithelial cells

“…Immunohistochemistry performed in the present study showed co‐localization of AMHR2, HSP47, and collagen IV in the epithelial layers, thus showing epithelial cells express these proteins. These observations were supported by our recent studies of AMHR2 (Ferdousy et al, 2020a), AMH (Ferdousy et al, 2020b), and HSP47 (Ferdousy & Kadokawa, 2022) expression in the epithelial layers of the uteri of young heifers and aged cows. In the present study, we found that AMH induces HSP47 expression in uterine epithelial cells.…”

“…For immunohistochemistry and immunocytochemistry, we used the anti‐HSP47 rabbit polyclonal antibody (AP7366B; Abcepta Inc., San Diego, CA, USA) that were used in bovine CAR and ICAR endometria (Ferdousy & Kadokawa, 2022) experiments. The rabbit anti‐human HSP47 antibody recognizes the mature C‐terminal of human HSP47 (corresponding to amino acids 390–418; FLVRDTQSGSLLFIGRLVRPKGDKMRDEL).…”

“…Therefore, we performed immunocytochemistry to confirm the purity of the cultured cells, using anti‐bovine pan cytokeratin mouse monoclonal antibody and anti‐bovine vimentin mouse monoclonal antibody (C2931 and V2258, respectively, both from Sigma‐Aldrich [St. Louis, MO, USA] and diluted as 1:1,000). These antibodies have previously been used for immunocytochemistry in cultured bovine oviduct epithelial cells (García et al, 2017), as well as in bovine CAR and ICAR endometria (Ferdousy et al, 2020b; Ferdousy & Kadokawa, 2022).…”

“…Collagen denaturation is increased in various diseases, including cancer, osteoporosis, and arthritis (Fields, 2013; Ito & Nagata, 2019). We recently showed that denatured collagen levels increase in the uteri of aged cows due to repeated pregnancies, using a new method of visualizing denatured collagen (Ferdousy & Kadokawa, 2022). Subsequently, we discovered increased levels of the 47‐kDa heat shock protein (HSP47), the sole procollagen‐specific molecular chaperone, in the uteri of aged cows compared to that of young ones (Ferdousy & Kadokawa, 2022).…”

“…We recently showed that denatured collagen levels increase in the uteri of aged cows due to repeated pregnancies, using a new method of visualizing denatured collagen (Ferdousy & Kadokawa, 2022). Subsequently, we discovered increased levels of the 47‐kDa heat shock protein (HSP47), the sole procollagen‐specific molecular chaperone, in the uteri of aged cows compared to that of young ones (Ferdousy & Kadokawa, 2022). HSP47 (encoded by SERPINH1 ) is essential for correct folding of the unique triple helical structure of collagen (Ito & Nagata, 2019).…”