Ovarian furin (proprotein convertase subtilisin/kexin type3): expression, localization, and potential role in ovulation in the rat

Abstract

The process of ovulation involves weakening of the follicular wall by proteolytic enzymes. The function of FURIN (also known as PCSK3) is to activate various proteolytic enzymes. In the present study, the expression, localization, and function of FURIN were investigated in the periovulatory rat ovary. Immature female rats were injected with equine chorionic gonadotropin followed by human chorionic gonadotropin (hCG) 48 h later to stimulate ovulation. Ovaries were collected at 0, 4, 8, 12, and 24 h after hCG injection. Administration of hCG increased Furin mRNA expression in both intact ovaries and cultured ovarian follicles to maximal levels at 8 and 12 h before decreasing at 24 h. In cultured granulosa cells, Furin mRNA levels were significantly induced at 12 h after hCG. In situ hybridization of Furin mRNA demonstrated expression in the granulosa cells, with predominant expression in the theca layer. Regulation studies demonstrated that Furin mRNA was induced in residual tissue by forskolin or amphiregulin. To examine the role of FURIN in protease activation and ovulation, rats were treated with a FURIN inhibitor and oocyte release was determined. There was a 38% decrease in the number of oocytes released in ovaries treated with the FURIN inhibitor. Likewise, the FURIN inhibitor decreased the activation of MMP2. The induction of Furin mRNA after treatment with hCG, along with the decrease in MMP2 activation and oocyte release after FURIN inhibition, supports the hypothesis that FURIN is upregulated during the preovulatory period, which results in activation of proteinases associated with the breakdown of the follicular wall during ovulation.

FIG. 1.

Stimulation of Furin mRNA expression by hCG in the rat ovary. Real-time PCR analysis shows the expression of Furin mRNA in whole ovary (A), granulosa cells (B), and ovarian follicles (C) after hCG administration. In A, rats were injected with eCG for 48 h and treated with hCG, and ovaries were collected at 0, 4, 8, 12, or 24 h after treatment. For B and C, rats were injected with eCG for 48 h, and granulosa cells (B) or follicles (C) were isolated and cultured in medium alone (Control) or with hCG (1 IU/ml) for 0, 4, 8, 12, or 24 h. Relative levels of mRNA for Furin were normalized to Rpl32 in each sample (mean ± SEM; n = 3 independent culture experiments). Bars with superscripts or asterisks are significantly different (P < 0.05) within each panel.

FIG. 2.

Cellular localization of Furin mRNA in periovulatory rat ovaries. Sections of rat ovaries obtained at 0 h (48 h after eCG) (A, B, I, J), 8 h (C, D, K, L), 12 h (E, F), or 24 h (G, H) after hCG injection were hybridized to the appropriate antisense Furin mRNA probe. Representative bright-field and corresponding dark-field photomicrographs are depicted. A–H illustrate low magnification localization of Furin mRNA throughout the ovary at different times after hCG. Higher magnification of an ovary obtained at 0 h (I, J) demonstrates the expression of Furin mRNA in the oocyte. Higher magnification of an ovary obtained at 8 h after hCG (K, L) demonstrates the expression of Furin mRNA in the theca layer (arrows) and the granulosa cell layer (arrowheads). Sections from an ovary collected at 8 h after hCG hybridized to a sense Furin mRNA probe exhibit only background deposition of silver grains (M, N). F, follicle. White bar = 250 μm (A–H), 20 μm (I and J), 200 μm (K and L), and 100 μm (M and N).

FIG. 3.

Regulation of Furin mRNA by activators or inhibitors of LH-induced intracellular pathways in cultured ovarian residual tissue. A) Real-time PCR analysis shows the expression of Furin mRNA in ovarian residual tissue after culture in medium alone (Control) or with Phorbol 12-myristate 13-acetate (PMA; 20 nM) + forskolin (FSK; 10 μM) for 0, 8, 12, or 24 h. B) Residual tissues were cultured for 12 h with PMA, FSK, or amphiregulin (Areg; 250 ng/ml). C) Residual tissues were also cultured for 12 h with FSK in the presence of the EGFR antagonist AG1478 (1 μM) or the protein kinase A pathway inhibitor H89 (10 μM). Relative levels of mRNA for Furin were normalized to Rpl32 in each sample (mean ± SEM; n = 3 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

FIG. 4.

Inhibition of oocyte release by administration of Furin Inhibitor I in vivo. Rats were injected with eCG, and 48 h later an osmotic minipump (Alzet) was inserted into the peritoneal cavity in proximity with the right ovary. Animals received a minipump with either 50 μg of Furin Inhibitor I or an equivalent concentration of DMSO vehicle control (DMSO). Rats were injected with 10 IU of hCG 1 h after surgery. Twenty four hours after surgery, oocytes were counted in the right oviduct from the treated ovary (mean ± SEM; DMSO vehicle n = 10, FURIN inhibitor n = 9). A group of animals without surgical manipulation served as the control group (no treatment, no minipump; n = 6). Bars with asterisks are significantly different (P < 0.05).

FIG. 5.

Inhibition of MMP2 activation by Furin Inhibitor I. Rats were injected with eCG, and 48 h later residual tissue was collected. Residual tissues were cultured for 12 h with FSK (10 μM) to induce Furin expression, and then DMSO vehicle control or Furin Inhibitor I (50 μM) was added. Residual tissue was removed from culture at 24 h (i.e., 12 h after treatment with the Furin Inhibitor I), and cell membranes were isolated. Cell membrane proteins were subjected to gelatin zymography (A), and band densities were determined via MetaMorph Image Analysis software. Y-axis shows the ratio of active to inactive MMP2 (mean ± SEM; n = 3 independent culture experiments) (B). Bars with asterisks are significantly different (P < 0.05).