Specific locations and amounts of denatured collagen and collagen-specific chaperone HSP47 in the uterine cervices of old cows compared with those of heifers.

Collagen, the most abundant extra-cellular matrix in the reproductive tract, performs a critical role in pregnancy. Although detecting damaged collagen in tissues is challenging, we recently developed a new in situ detection method using a denatured collagen detection reagent in bovine oviducts and uteri. Utilizing this method, we evaluated the hypothesis that the locations and amounts of denatured collagen in the uterine cervices of old cows are different from those in young heifers as a result of repeated pregnancies and deliveries. We compared damaged collagen in the uterine cervix at the mid-luteal phase between post-pubertal growing nulliparous heifers (22.1 ± 1.0 months old; n = 5) and old multiparous cows (143.1 ± 15.6 months old; 9 ± 1 parities; sacrificed at least 3 months after the last parturition by vaginal delivery; n = 5). Picrosirius red staining showed collagen in almost all parts of the cervices. Expectedly, the amount of damaged collagen was increased in the cervices of old cows. Additionally, we combined in situ detection and fluorescence immunohistochemistry of the collagen-specific molecular chaperone, the 47 kDa heat shock protein (HSP47). Increased HSP47 amounts were observed in the cervices from the old cows, but damaged collagen and HSP47 were not located in the same areas. The age differences were confirmed by western blotting using the anti-HSP47 antibody. These findings revealed the specific location and amounts of denatured collagen in the uterine cervices 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.

Uterine collagen is the most abundant component of the uterine extracellular matrix and plays a critical role in pregnancy. The 47-kDa heat shock protein (HSP47) is the sole collagen-specific molecular chaperone. We investigated the mechanisms regulating the expression of HSP47 in the uterus by assessing the effect of anti-Müllerian hormone (AMH) stimulation on HSP47 expression in cultured bovine uterine epithelial cells. AMH receptor type 2 (AMHR2), AMH, and HSP47 expression was assessed by fluorescence immunocytochemistry in uterine epithelial layers of the uteri of Japanese Black cows. The effect of AMH on HSP47 expression was assessed in cultured epithelial cells. The effect of MEK/ERK inhibitor on AMH-induced HSP47 expression was also assessed. We confirmed the expression of AMHR2, AMH, and HSP47 in the uterine epithelial layers. We confirmed the expression of AMHR2, AMH, HSP47, and type IV collagen in cultured uterine epithelial cells. AMH treatment at 10 or 100 ng/ml promoted significant HSP47 expression (p < 0.05). MEK/ERK inhibitor U0126 pretreatment suppressed such AMH stimulation on HSP47. These findings indicate that AMH induced HSP47 protein expression through the ERK pathway in bovine uterine epithelial cells.

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.

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±1.0months old) and old multiparous cows (143.1±15.6months 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.

Spike protein of SARS-CoV-2 suppresses gonadotrophin secretion from bovine anterior pituitaries.

Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells. The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane. Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein having both S1 and S2 regions. The spike protein (0.07-7 pM) suppressed both basal and GnRH-induced LH secretion (P < 0.05). Spike protein (0.7-7 pM) suppressed GnRH-induced (P < 0.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in turn suppresses LH and FSH secretion from AP cells.

Anti-Müllerian hormone receptor type 2 (AMHR2) expression in bovine oviducts and endometria: comparison of AMHR2 mRNA and protein abundance between old Holstein and young and old Wagyu females.

Anti-Müllerian hormone (AMH) is a glycoprotein produced by granulosa cells of preantral and small antral follicles that has multiple important roles in the ovaries. Recent studies have revealed extragonadal AMH regulation of gonadotrophin secretion from bovine gonadotrophs. In this study we investigated whether the primary receptor for AMH, AMH receptor type 2 (AMHR2), is expressed in bovine oviducts and endometria. Reverse transcription-polymerase chain reaction detected expression of AMHR2 mRNA in oviductal and endometrial specimens. Western blotting and immunohistochemistry were performed to analyse AMHR2 protein expression using anti-bovine AMHR2 antibody. Immunohistochemistry revealed robust AMHR2 expression in the tunica mucosa of the ampulla and isthmus, as well as in the glandular and luminal epithelium of the endometrium. AMHR2 mRNA (measured by real-time polymerase chain reaction) and AMHR2 protein expression in these layers did not significantly differ among oestrous phases in adult Wagyu cows (P>0.1). In addition, AMHR2 mRNA and protein expression in these layers did not differ among old Holsteins (mean (±s.e.m.) age 91.9±6.4 months) and young (26.6±0.8 months) and old (98.8±10.2 months) Wagyu cows. Therefore, AMHR2 is expressed in bovine oviducts and endometria.