Differential expression of focimatrix and steroidogenic enzymes before size deviation during waves of follicular development in bovine ovarian follicles.

During the growth of bovine follicles, one emerges from a wave as the largest and dominant follicle. What regulates dominance is not known but candidates include oestradiol, transforming growth factor beta beta1 (TGFB1), and recently CYP11AI (cholesterol side-chain cleavage) and focal intra-epithelial matrix (focimatrix). To examine this, pairs of bovine ovaries with 2 or more follicles of equal size (>5mm) and hence in a wave before deviation, were collected at an abattoir (6.7+/-SEM 0.1mm diameter; n=14 animals, 35 follicles in total). These follicles were dissected and follicular fluid collected to measure progesterone and oestradiol concentrations. A portion of the follicle wall was processed for histological classification of health or atresia and granulosa cells were harvested for quantitative RT-PCR of focimatrix components [COL4A1 (collagen type IV alpha1), LAMB2 (laminin beta2) and HSPG2 (perlecan)], steroidogenic enzymes [CYP11A1 and CYP19A1] and TGFB1. For statistical analyses follicles within each animal were grouped into either the highest (oestradiol, CYP11A1) or lowest (TGFB1) expression (n=14) for comparison with the remaining follicles (n=21). When grouped on oestradiol no other parameters differed significantly, and when grouped on TGFB1 some parameters were different however the levels were also lower, and not higher as expected. When grouped on CYP11A1 other parameters were significantly elevated in the high CYP11A1 group (COL4A1P<0.05; LAMB2P<0.01; HSPG2P<0.01 and CYP19A1P<0.001). This suggests that steroidogenesis and focimatrix might be important in a follicle attaining dominance.

Matti N ，Irving-Rodgers HF ，Hatzirodos N ，Sullivan TR ，Rodgers RJ ... -  《-》

Studies of granulosa cell maturation in dominant and subordinate bovine follicles: novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1.

Irving-Rodgers HF ，Harland ML ，Sullivan TR ，Rodgers RJ ... -  《-》

A novel basal lamina matrix of the stratified epithelium of the ovarian follicle.

Irving-Rodgers HF ，Harland ML ，Rodgers RJ 《MATRIX BIOLOGY》

Spatial differences within the membrana granulosa in the expression of focimatrix and steroidogenic capacity.

In the ovarian follicular membrana granulosa there are morphological and functional differences between cells adjacent to the follicular fluid lumen, or aligning the basal lamina. Amongst the observed functional differences are steroidogenic capacity and expression levels of a novel basal lamina, focimatrix; both of which increase in the later stages of antral follicle growth. A number of different studies have produced apparently inconsistent results as to which cell layers are more steroidogenic. To examine this systematically, individual bovine follicles, confirmed as healthy by post hoc histological examination, were used to isolate populations of apical and basal granulosa cells. Cell counts revealed that the respective groups did not differ in the numbers of cells, thus confirming the separation of these populations. We measured gene expression (quantitative RT-PCR, n=8-10, follicle diameter 14.0±0.5 mm) and protein levels (Western immunoblotting, n=14, follicle diameter 11.9±0.5 mm) and hormone production from granulosa cells (2.5×10(5) viable cells/well in serum-free conditions for 24 h, n=15, diameter 12±0.5 mm). Levels of mRNA of HSD3B1 and CYP19A1 and three focimatrix genes COL4A1, HSPG2 and LAMB2 and LHCGR were significantly lower in apical granulosa cells (P<0.05), whereas, expression of CYP11A1 and HSD17B1 were not different (P>0.05). The protein levels of steroidogenic enzymes P450scc and P450arom were significantly higher in apical cells (P<0.05), whereas those of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase type 1 were not different (P>0.05). Progesterone production was significantly lower and oestradiol production was significantly higher in apical granulosa cells (P<0.05). These results confirm that apical and basal cells are functionally different, and the differences might be explained by the location of cells of different ages and maturity within the membrana granulosa. Discrepancies in the literature on their steroidogenic capacity may reflect differences in the steroidogenic parameters measured.

Nguyen T ，Lee S ，Hatzirodos N ，Hummitzsch K ，Sullivan TR ，Rodgers RJ ，Irving-Rodgers HF ... -  《-》

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle.

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta2 and gamma1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha1, laminins beta2 and gamma1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

Irving-Rodgers HF ，Catanzariti KD ，Aspden WJ ，D'Occhio MJ ，Rodgers RJ ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》