The Expression of Prostaglandin Endoperoxide Synthase 2 Messenger RNA and the Proportion of Smooth Muscle and Collagen in the Sheep Cervix During the Estrous Cycle

Abstract

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E2 synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.

Progesterone exposure of seasonally anoestrous ewes alters the expression of angiogenic growth factors in preovulatory follicles

“…Immunohistochemistical assays for the proteins of interest were performed on consecutive sections of a follicle using the avidin-biotin peroxidase method as previously described [27][28][29][30], but with secondary antibody concentration of 1:25 biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) in 2% v/v normal goat serum in PBS and color detection being performed using ImmPACT DAB (Vector Laboratories).…”

“…During synthesis, riboprobes were labeled with digoxogenin-II-uridine triphosphate (Roche Applied Bioscineces, Welwyn Garden City, England, UK) for nonradioactive detection following the manufacturer's recommendations. In situ hybridization was performed according to the technique described by Kershaw, et al [28] and Chowdhury, et al [27]. ANG-1, ANG-2, and TIE-2 hybridization was performed for 19 hours in a humidified chamber at 60 C and color development took place for 20 hours before mounting.…”

“…The pattern and intensity of protein and mRNA staining for VEGF, VEGFR-2, ANG-1, ANG-2, and TIE-2 in follicular tissue were determined semi-quantitatively using a histochemical score (HSCORE) method that has been widely reported in a number of studies in our own laboratory [27][28][29][30] and by others [31,32]. Moreover, the HSCORE method of analysis used in this study has been previously validated as a suitable method for semiquantitative estimation of immunohistochemistry and in situ hybridization results [33].…”

“…Moreover, the HSCORE method of analysis used in this study has been previously validated as a suitable method for semiquantitative estimation of immunohistochemistry and in situ hybridization results [33]. In this study, all assessments were carried out in a blinded manner by one experienced assessor using HSCORE, incorporating both the distribution and the intensity of specific staining as described previously [27,28,30,33,34]. Briefly, the percentage of positively stained cells and the intensity of the staining of those cells were recorded at an original magnification of Â400.…”

Traversing the ovine cervix – a challenge for cryopreserved semen and creative science

“…An accompanying feature at this time is the abundant expression of the hyaluronan receptor, CD44, in the epithelial layer (Perry et al, 2010b). In view of the central role that peri-oestrous endocrine changes play in the collagen and elastin remodelling processes that promote cervical relaxation, the research emphasis in ewes in recent years has been on improving understanding of their mechanistic pathways (Mitchell et al, 2002 andKershaw et al, 2007;Leethongdee et al, 2007 andKershaw-Young et al, 2009 and. An overview of the findings from these studies is illustrated in Figure 3.…”