130 EXPRESSION OF STEROIDOGENIC ENZYMES IN THE CAT OVARY DURING FOLLICULAR GROWTH

Abstract

This study was designed to describe the expression of steroidogenic enzymes in the various compartments of the feline ovary, from primordial to preovulatory follicles: P450 side-chain cleavage (P450scc), 17α-hydroxylase (17α-OH), 3β-hydroxysteroid dehydrogenase (3β-HSD). Nine female cats were ovariectomized during behavioural oestrus. After validation of their specificity by Western blot on feline tissue, 3 polyclonal antibodies obtained in the rabbit were used for immunohistochemistry (anti-bovine P450scc; anti-pig 17α-OH; anti-human 3β-HSD). Three successive ovarian cryosections were incubated with antibodies and 2 sections were used as controls (purified immunoglobulin G from a nonimmunized rabbit). Immunostaining was revealed by streptavidin-biotin system (LSAB kit, DAKO S.A., Trappes, France), with aminoethylcarbazole as a chromogen. The staining was evaluated according to its localization, intensity (from 1 to 4) and number of cell layers stained. Histological examination after haematoxylin-eosin-safran staining allowed follicle diameter measurement and evaluation of the atresia. Analysis of variance was used to compare follicles from various size classes. A total of 343 follicles from 140 to 3250 μm in diameter were observed (i.e. all follicles present on the slides), of which 15.2% were atretic. The observations were similar for the 3 enzymes. From the preantral stage onward (diameter >140 μm), more than 85% of the healthy follicles had the theca interna positive for P450scc, 3β-HSD and 17α-OH (respectively, 87.3, 86.0 and 99.0%). Approximately 20 to 25% were also positive in the theca externa. Granulosa were stained in 9.5% (P450scc), 0.8% (3β-HSD) and 1.8% (17α-OH) of the healthy follicles, but always with a low intensity (<2). The follicular size did not affect the proportion of stained follicles, but the staining intensity decreased progressively until 1200 μm and increased thereafter. The number of positive layers decreased continuously until 1500 μm in diameter. Between 90 and 95% of the preovulatory follicles (>2000 μm) were positive in the theca interna; the signal was more intense and the number of stained layers was higher than in smaller follicles. The 3 enzymes were detected in all the atretic follicles, with a similar intensity as in healthy ones; they were also present in the interstitial tissue, but with a higher intensity. The staining pattern in the various ovarian compartments suggests that the major steroidogenic tissues in the cat ovary are the theca interna of healthy follicles and the interstitial tissue. The lack of expression of the 3 key enzymes in the granulosa cells, even in preovulatory follicles, strongly suggests that these cells, shaping the future corpus luteum, are probably unable to produce progesterone.