Hyaluronan (HA) content, the ratio of HA fragments and the expression of CD44 in the ovine cervix vary with the stage of the oestrous cycle

Abstract

The complex anatomy of the ovine cervix limits the success of trans-cervical artificial insemination in sheep. However, there is a degree of natural relaxation of cervix at oestrus that is accompanied by an increase in the water content. As hyaluronan (HA) has a high affinity for water molecules, in this study, we tested the hypothesis that the HA content of the cervix, the proportion of different size fragments of HA and expression of its receptor CD44 vary with the stage of the oestrous cycle. Oestrous was synchronized in 25 Welsh mountain ewes, and their cervices were collected either during luteal phase (nZ8) or pre-LH (nZ8) or post-LH (nZ9) surge stage of the oestrous cycle. The pre-LH surge group had the highest HA content (2.96 ng/mg of cervical tissue), which was significantly (P%0.05) higher than that observed for the post-LH surge (2.04 ng/mg) group. The luteal phase group had a mean HA content intermediate between the pre-and post-LH surge groups, and was significantly different from either. The frequency of cervical samples containing both sizes of HA fragments (small and large) was significantly higher (P%0.05) in the pre-LH surge group compared with the luteal and the post-LH surge groups, whereas that in post-LH surge group was significantly (P%0.05) higher than that in the luteal group. The number of cervical samples that contained only small HA fragments was significantly (P%0.05) higher in the luteal group compared with both the pre-and post-LH surge groups, whereas the number of samples containing only large HA fragments was significantly (P%0.05) higher in the post-LH surge group compared with the luteal or pre-LH surge groups. Overall mean expression of CD44 in the vaginal and mid regions was significantly (P%0.001) higher than that in the uterine region, with no difference between the vaginal and mid regions of the cervix. Pattern of CD44 expression depended on the stage of the oestrous cycle. At the luteal stage, CD44 expression did not vary among epithelial, sub-epithelial, circular and longitudinal smooth muscle layers, whereas at the pre-and post-LH surge stages, the expression in the epithelial layer was significantly (P%0.001) higher than that in the other three layers. In general, CD44 expression in the transverse smooth muscle layer was significantly (P%0.05) lower than the expression in all the other layers at all the stages of the oestrous cycle. The results indicated that the HA varied with the steroid status. Higher HA values at a time when cervical relaxation is naturally higher may indicate its involvement in remodelling of the cervix at oestrus.

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

“…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.…”

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells

“…For example, HAS was detected using immunohistochemistry in pig ovary (Miyake et al 2009), ovine cervix (Perry et al 2012) and uterus (Raheem et al 2013) and human endometrium (Nykopp et al 2010). In addition, the HA receptor (CD44) was immunolocalised in ovine cervix (Perry et al 2010) and uterus (Raheem et al 2013), pig ovary (Miyake et al 2006) and oviduct (Tienthai et al 2003), bovine oocytes, embryos and oviduct (Ulbrich et al 2004;Marei et al 2012;Marei et al 2013), cumulus and mural granulosa (Ohta et al 1999;Marei et al 2012) and cervical cells (El Maradny et al 1997). …”

Regulation and roles of the hyaluronan system in mammalian reproduction

“…CD44 has been detected in various segments of the reproductive tract in mouse (Kennel et al 1993), cow (Bergqvist et al 2005a, sheep (Perry et al 2010a), mare (Rodriguez et al 2011) andhuman (López et al 2013) under normal physiological conditions. It has also been detected in cow (Furnus et al 2003), mouse (Matsumoto et al 2004) and human (Campbell et al 1995) embryos.…”

“…The cervical connective tissue is mainly composed of collagen, HA and proteoglycan (Leppert 1992). The HA content of the cervix varies with the stage of oestrus cycle, with the highest and lowest values during pre-LH surge and post-LH surge periods respectively, whereas the value in the luteal stage is intermediate (Perry et al 2010a).…”