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Bone morphogenetic protein-15 (BMP-15) is a member of the TGFbeta family known to regulate ovarian functions in mammals. The structure and function of BMP-15 in lower vertebrates are less known. In this study, we cloned the zebrafish BMP-15 (zfBMP-15) cDNA and depicted its genomic organization. The zfBMP-15 cDNA encodes a protein of 384 amino acids. The mature protein has 46-51% sequence identities to fugu, chicken, and mammalian BMP-15. It also shares 38-46% homology with growth and differentiation factor-9 in fishes, chicken, and mammals. Phylogenetic analysis further confirms that the zfBMP-15 is most closely related to BMP-15 from other species, whereas the growth and differentiation factor-9 peptides from fish to mammals form a distinct branch. Comparison of zfBMP-15 cDNA with zebrafish genome database revealed that zfBMP-15 is encoded by a gene with two exons and one intron, located on chromosome 6. BMP-15 mRNA is expressed in the ovary and testis and, to a lesser extent, brain, liver, gut, heart, and muscle. Real-time PCR revealed that BMP-15 is expressed in follicles at all stages of development with no significant changes over the course of folliculogenesis. Using in situ hybridization and immunocytochemistry, we detected BMP-15 in both oocytes and follicular cells. Incubation of follicles with antiserum against zfBMP15 increased oocyte maturation, whereas incubation with recombinant human BMP-15 suppressed human chorionic gonadotropin-induced oocyte maturation. These findings suggest that BMP-15 plays a role in regulating gonadal functions in fish, in particular oocyte maturation.

Bone morphogenetic protein-15 (BMP-15) is a member of the TGF-beta superfamily known to regulate ovarian functions in mammals. Recently, we cloned zebrafish BMP-15 (zfBMP-15) cDNA and demonstrated that it may play a role in oocyte maturation. In this study, we further investigated the role of BMP-15 in zebrafish follicular development and oocyte maturation using an antiserum developed for zfBMP-15 and by microinjection of follicles with antisense zfBMP-15 N-morpholino oligonucleotides or an expression construct containing zfBMP-15 cDNA. Injection with antiserum caused a significant decrease in maturation-incompetent [insensitive to maturation-inducing hormone (MIH)] early growth phase follicles and a concomitant increase in mature follicles in vivo. In vitro maturation assays showed that incubation with antiserum resulted in a significant increase in oocyte maturation as compared with follicles incubated in preimmune serum or media control. Next, early growth phase follicles were collected and preincubated with either antiserum, preimmune serum, or medium control before treatment with MIH or human chorionic gonadotropin (hCG). Antiserum significantly increased oocyte maturation in response to MIH, but not to hCG, and enhanced basal maturation rate in longer-term incubations. Knockdown of BMP-15 in early growth stage follicles with a BMP-15 antisense oligonucleotide resulted in increased oocyte maturation, whereas microinjection of BMP-15 cDNA into oocytes significantly reduced MIH- and hCG-induced oocyte maturation in normally competent, mid-growth-phase follicles. Collectively, these findings suggest that BMP-15 modulates follicular growth and prevents premature oocyte maturation in zebrafish, in part, by suppressing the sensitivity of follicles to MIH.

No abstract

TGF-beta is a multifunctional factor involved in regulating a variety of cellular activities. In mammals, TGF-beta is known to regulate reproduction, including ovarian functions. The role of TGF-beta in lower vertebrates, such as fish, is poorly understood. To examine the role of TGF-beta in fish reproduction, cDNAs encoding TGF-beta 1 and the type II TGF-beta receptor (T beta RII) were cloned from the zebrafish ovary using PCR- based strategies. The mature peptide region of the zebrafish TGF-beta 1 shows 70-85% identity with TGF-beta 1 from other species. The zebrafish T beta RII cDNA sequence is the first to be reported from a fish species, and it shows a high level of conservation at the kinase domain. Using RT-PCR, we have detected mRNA expression of TGF-beta 1, T beta RII, as well as its downstream signaling molecules Smad2, 3, and 4 in ovarian follicles at different stages of development. In addition, we have examined the effect of TGF-beta 1 on oocyte maturation. TGF-beta 1 significantly inhibited both gonadotropin- and 17 alpha, 20 beta-dihydroxyprogesterone-induced oocyte maturation in a dose- and time-dependent manner. These findings demonstrate, for the first time, that TGF-beta 1 plays a role in regulating oocyte maturation in fish and suggest that a TGF-beta/Smad signaling pathway is present in the zebrafish ovary.