First evidence of bone morphogenetic protein 1 expression and activity in sheep ovarian follicles

Abstract

Bone morphogenetic protein (BMP) 1 is a vertebrate metalloproteinase of the astacin family. BMP1 plays a key role in regulating the formation of the extracellular matrix (ECM), particularly by processing the C-propeptide of fibrillar procollagens. BMP1 also promotes BMP signaling by releasing BMP signaling molecules from complexes with the BMP-antagonist chordin. As a result of BMP1's dual role in both ECM formation and BMP signaling, we hypothesized that BMP1 could play a role in ovarian physiology. Using the sheep ovary as a model system, we showed that BMP1 was expressed in the ovary throughout early fetal stages to adulthood. Furthermore, in adult ovaries, BMP1 was expressed along with chordin, BMP4, and twisted gastrulation, which together form an extracellular regulatory complex for BMP signaling. Within ovine ovaries, immunohistochemical localization demonstrated that BMP1 was present in granulosa cells at all stages of follicular development, from primordial to large antral follicles, and that the levels of BMP1 were not affected by the final follicle selection mechanism. In cultured granulosa cells, BMP1 expression was not affected by gonadotropins, but BMP4 and activin A had opposing effects on the levels of BMP1 mRNA. BMP1 appeared to be secreted into the follicular fluid of antral follicles, where it is able to exert procollagen C-proteinase and chordinase activities. Interestingly, BMP1 activity in follicular fluid decreased with follicular growth.

FIG. 1.

Expression of BMP1, CHRD, TWSG1, and BMP4 mRNAs in adult sheep tissues. Total mRNA was extracted from the tissues indicated and reverse transcribed before PCR analysis. Amplification of the RPL19 gene amplification was used as a cDNA quality and quantity control.

FIG. 2.

BMP1 mRNA expression in developing sheep gonads. Total RNA was extracted from sheep gonads obtained during embryogenesis (between 25 and 141 days postcoitum) or at birth and reverse transcribed before PCR analysis. The GAPDH gene was used as a cDNA quality control.

FIG. 3.

Immunostaining for BMP1 in the sheep ovary. Photomicrographs of antral (a and c), preantral (d), secondary (e and f), primary (f), and primordial (g) follicles are shown. The photomicrographs shown in a and c–g correspond to immunostaining with anti-BMP1 rabbit polyclonal antibody. The photomicrographs shown in b and h correspond to immunostaining with control rabbit IgG. GC, granulosa cell; OO, oocyte; TC, theca cell.

FIG. 4.

In vivo regulation of BMP1 mRNA expression during terminal follicular maturation. Total RNA extracted from granulosa cells isolated from small (S; n = 5), large (L; n = 3), and preovulatory (PO; n = 4) ovine follicles was reverse transcribed and analyzed by real-time PCR. Data (mean ± SEM) represent the percentage of expression of BMP1 mRNA relative to the RPL19 reference gene.

FIG. 5.

In vitro regulation of BMP1 mRNA expression by the main regulators of folliculogenesis. Granulosa cells from small (A) or large (B) ovine follicles were cultured for 24 h in the presence of fetal ovine serum and then grown for 48 h in serum-free medium in the absence (control) or presence of FSH (50 ng/ml), LH (50 ng/ml), IGF1 (10 ng/ml), BMP4 (50 ng/ml), activin A (50 ng/ml), or TGFB1 (5 ng/ml). At the end of the culture period, mRNA was extracted and reverse transcribed before quantitative real-time PCR analysis. Data are presented as the percentage of BMP1 mRNA levels (mean ± SEM, n = 5) relative to the RPL19 reference gene. *P < 0.05 or **P < 0.01 for each treatment vs. control.

FIG. 6.

Evidence of procollagen C-proteinase activity in sheep ovarian follicular fluids. Neat follicular fluid samples were incubated with ¹⁴C-labeled type I procollagen overnight at 37°C, as described in Materials and Methods. The digests were separated by SDS-PAGE, and the radiolabeled collagen chains were detected using a PhosphorImager. Recombinant BMP1 was used as a positive control in which almost all of the procollagen was converted to pN collagen, a normal intermediate in the conversion of procollagen to collagen in which the N-propeptides, but not the C-propeptides, are retained. A) SDS-PAGE gel (7%) of [¹⁴C]procollagen digests. Procollagen C-proteinase (PCP) activity is readily detected by the appearance of the pNalpha2 collagen chain. Follicular fluids from small (S), large (L), and preovulatory (PO) follicles were assayed for PCP activity. B) Graph representing the results of densitometric quantification of the extent of procollagen cleavage (mean ± SD) following overnight incubation with recombinant BMP1 (BMP1), recombinant BMP1 with EDTA (EDTA), or follicular fluids from S, L, or PO follicles. *P < 0.01 by one-way ANOVA and Tukey HSD. C) PCP activity in follicular fluid samples is inhibited by EDTA. Pools of follicular fluid samples from small or large follicles were incubated with ¹⁴C-labeled type I procollagen in the presence or absence of EDTA. Addition of EDTA inhibited the metalloproteinase-dependent conversion of procollagen to pN collagen.

FIG. 7.

Evidence of chordinase activity in sheep ovarian follicular fluids. A) Schematic depicting the domain structure of N-terminal c-Myc-tagged chordin. B) c-Myc-tagged chordin from the cell culture medium of transfected 293EBNA cells was incubated with follicular fluid samples or with recombinant BMP1, in the presence or absence of EDTA, overnight at 37°C. Control incubations were also performed using unconditioned cell culture medium in place of Myc-tagged chordin to identify bands resulting from the nonspecific binding of 9E10 to untagged proteins on the Western blot. Samples were separated by electrophoresis, and the chordin cleavage products were detected by Western blotting using the anti-Myc monoclonal antibody 9E10. Chordinase activity was inhibited in samples treated with EDTA. In follicular fluid samples, chordinase activity could be detected a reduction in the amount of full-length chordin and by the appearance of an approximately 17-kDa chordin cleavage product, corresponding to fragment 1. Both fragment 1 and fragment 2 were produced by recombinant BMP1 (see Discussion). Chordinase activity appears to be slightly higher in follicular fluid samples from small follicles.