Regulation of hyaluronan system in sheep ovaries

Supervisor: Dr Ali Fouladi (afouladi@rvc.ac.uk )

Hyaluronan (HA) is a non-sulphated glycosaminoglycan component of extracellular matrix in mammalian tissues. It is produced in different sizes by three HA synthases with diverse biological functions mediated mainly through its cell membrane receptors CD44 and RHAMM.  HA is catabolised by hyaluronidases which cleave long HA polymers to smaller fragments and finally to its basic units for constant turnover. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. Research in my laboratory has shown that HA synthesis is regulated by steroid hormones (Raheem et al, 2013) and different hormones have differential effects on HAS and Hyal expression in sheep granulosa cells.  The overall aim of this project is to clarify regulation of the HA system in the developing corpus luteum. Sheep ovaries were previously collected from oestrus synchronised ewes prior to and after LH surge and during mid-luteal stage. One ovary from each animal collected on days 5 and 8 after ovulation will be used for analyses of HAS-1, HAS-2, HAS-3, HYAL-1, HYAL-2, VGEF, CD44 and RHAMM protein by IHC. Corpus lutea from the other ovaries will be used for analyses of mRNA expression by quantitative PCR.

In addition, cells from the corpus luteum will be isolated by enzymatic digestion and cultured to confluency by day 5 before treatment with LH (10-100ng/ml) and P4 (40-400ng/ml) and LH+P4 in the presence or absence of RU486 (progesterone receptor antagonist).  Cultured cells will be assessed for cell proliferation (cell titre assay) and development of blood vessels (staining with Anti-CD31, Anti-von Willebrand Factor) at the end of culture. Freshly isolated and cells recovered 1h, 6h, 12h, 24h, 48h, 72h, 96h and 120h after the start of the treatment will be used for analysis of HAS, HYAL-2, and angiogenic factors (VEGF and Angiopoietins) by PCR and their related proteins by Western blot techniques.

Objectives:  

1.    To determine whether LH stimulated progesterone affects the profile of genes and proteins of the HA system in the ovarian follicles and the developing corpus luteum

2.    To determine whether small HA sizes produced by Hyal-2 have angiogenic activity in the corpus luteum

Key references

1. Chavoshinejad R, Marei WF, Hartshorne GM, Fouladi-Nashta AA. (2014) Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells. Reprod Fertil Dev. 2014 Nov 27. doi: 10.1071/RD14294.

2. Raheem, K.A., Marei, W.F., Mifsud, K., Khalid, M., Wathes, D.C., and Fouladi-Nashta, A.A. (2013) Regulation of the hyaluronan system in ovine endometrium by ovarian steroids. Reproduction 145(5), 491-504

3. Irving-Rodgers, H.F., et al., Extracellular Matrix of the Corpus Luteum. Semin Reprod Med, 2006. 24(04): p. 242,250.

4. Young, J.M. and A.S. McNeilly, Theca: the forgotten cell of the ovarian follicle. Reproduction, 2010. 140(4): p. 489-504.

If you would like to apply for this studentship please contact the supervisors in the first instance and then apply via UKPASS. See also the How to Apply box

The deadline for applications is 3rd April 2016