184 FOLLICULOGENESIS AND DONOR AGE INFLUENCE MATRIX METALLOPROTEINASE-2 EXPRESSION IN THE DOMESTIC CAT

Abstract

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are known to play key roles in the remodelling of extracellular matrix during ovarian folliculogenesis, especially during the final stage of follicle development. To date, little is known about the significance of MMPs and TIMPs during preantral follicle development. This study determined the expression of MMPs and TIMP-1 during various stages of cat folliculogenesis, largely for the purpose of securing information useful to improving in vitro follicle culture. Primordial (~10 follicles/cat), primary (~5 follicles/cat), secondary (~9 follicles/cat), early antral (~9 follicles/cat), and antral (~4 follicles/cat) follicles were physically isolated from ovaries recovered from 15 cats (5 months to 3 years old during follicular stage) undergoing ovariohysterectomy and assessed for expression of MMP-1, -2, -3, -7, -9, and -13 as well as TIMP-1 using real-time quantitative polymerase chain reaction (q-PCR; 2–4 replicates/follicle stage). Additional ovaries were obtained from three prepubertal (6 months old) and three adult (1 year old) cats and ovarian pieces were fixed in Bouin's solution and assessed for MMP-2 and -13 localization using immunohistochemistry. MMP expression data were analysed using the Kruskull-Wallis one-way ANOVA. Follicles from all stages of development expressed MMPs and TIMP-1. Specifically, expression of MMP-2 increased (P < 0.05) as folliculogenesis progressed (10-fold increases from primordial to early-antral and antral stage). There were no differences (P > 0.05) in the expression of other MMPs among follicular classes. For TIMP-1, there was a tendency (P = 0.07) for increased expression after antrum formation (early antral and antral stages). Immunohistochemistry analysis revealed that MMP-2 was expressed in both the oocyte and somatic cells of all follicular stages in prepubertal cats. However, MMP-2 expression was limited to granulosa and theca cells of antral follicles in adult females. MMP-13 was expressed in the granulosa and theca cells of primary, secondary, and antral stage follicles, and there were no differences (P > 0.05) in localization patterns for this protein between prepubertal and adult females. In summary, the study is the first to report the expression of MMPs as well as TIMP-1 in isolated cat follicles. The difference in MMP-2 expression between prepubertal and adult cats suggests that there may be age-specific requirements for in vitro follicle growth. We are keenly interested in this information for underpinning the development of new in vitro microenvironments for growing immature cat follicles. We suspect that such information will be crucial for understanding how to promote the remodelling of the extracellular matrix by creating degradable biomaterials containing MMP-sensitive peptides to allow optimal follicle expansion.