Activin/Inhibin in Fundulus heteroclitus Ovary: Cloning of the Subunits and Effects on Oocyte Maturation . Program No. 238.17. School of Natural and Health Sciences, Barry University, Miami Shores, Florida 33161. Germinal Vesicle Prophase I. GVBD Metaphase II.
Activin/Inhibin in Fundulus heteroclitus Ovary: Cloning of the Subunits and Effects on Oocyte Maturation
Program No. 238.17
School of Natural and Health Sciences, Barry University, Miami Shores, Florida 33161
Germinal Vesicle Prophase I
GVBD Metaphase II
Fig. 4 Effects of Inhibin on DHP-induced Oocyte Maturation
Prematurational ovarian follicles
(1.3 mm in diameter)
Fundulus heteroclitus (7-10 cm)
Fig. 6 Activin/Inhibin ßB subunit Protein Alignment
Fig.5 Partial Nucleotide Sequence and Conceptual Translation
of the F. heteroclitus Activin/Inhibin ßB subunit
MATERIALS AND METHODS
Amino Acids are indicated by brown single letter code above the nucleotide sequence. Position of
Cysteine are highlighted in blue. X: Stop codon. Polyadenylation signal is underlined.
Sequence homology analysis were performed using the Basic Local Alignment Search Tool
(BLAST, NCBI); Global multiple sequence alignments were done using Clustalw (EBI-EBML); Color letters indicate the target amino acid sequences used for primer design (Table 1), Fundulus sequence is highlighted in yellow
Activins and inhibins are gonadal dimeric proteins, structurally related to the transforming growth factor-. Inhibin A and B are composed of an -subunit and one of two -subunits (A or B) respectively. Activins A, B and AB consist of two -subunits. The role of these various peptides as local regulators of ovarian function is not fully understood. To confirm the presence of activin/inhibin-like molecules in the F. heteroclitus ovary, and to define their role on oocyte maturation, the expression of the activin/inhibin subunits was determined by reverse transcription-polymerase chain reaction (PCR) using ovarian mRNA as template. Degenerate primers for the PCR were designed based on sequence homology of known activin/inhibin-subunits. PCR products were cloned and sequenced. BLAST and Clustal analysis indicated that several clones encodes amino acids that show strong homology to the carboxy-terminal of the -subunit. Incubation of isolated ovarian follicles with porcine inhibin caused a significant dose-dependent but reversible inhibition, while human recombinant activin A slightly enhanced the steroid-induced oocyte maturation. Results present evidence for the presence of activin/inhibin-like molecules in the F. heteroclitus ovary and their possible role as modulator of oocyte maturation. Supported by NIH-MBRS SCORE (GM 45455-08), RISE (GM 59244-01A1), and NSF (DBI-0116080) Grants.
Gesulla Toussaint, Teresa Petrino, and Yu-Wai P. Lin
In addition to steroid hormones, the gonads synthesize several peptide regulators, including the activins and inhibins (reviewed by Findlay et al., 1987; Ackland et al.,1992), whose potential roles as local modulators of gonadal function are not yet clearly defined (Fig. 1). These peptides are related to the transforming growth factor –β (TGF- β) super family. Inhibins are heterodimeric glycoproteins composed of an α-subunit and one of the two β-subunits (βA or βB), giving rise to biologically active forms termed inhibin A and inhibin B. Activins consist of two β-subunits, in any combination (reviewed by Ying, 1988).
Evidence that these gonadal peptides can serve as paracrine and/ or autocrine regulators of gonadal functions was demonstrated by their ability to modulate steroidogenesis, proliferation of spermatogonia, follicle development, oocyte competence and fertilization in mammals (Mather et al., 1997;Alak et al., 1996;Stock et al., 1997), as well as in lower vertebrates (Lin et al., 1999;Pang and Ge, 2002).
Using the teleost Fundulus heteroclitus as a model, this study aimed to investigate the role of activin and inhibin, particularly, on the oocyte maturation, which is a complex process regulated by the interplay of multiple factors present in the follicular environment. Oocyte maturation has been extensively studied in teleost. In Fundulus, this process is triggered by the action of gonadotropic hormones on the granulosa cells to produce the steroid 17α,20β-dihydroprogesterone (DHP, the maturation- inducing hormone in this species) that acts directly on the oocyte to reinitiate meiosis (Petrino et al., 1993) (Fig.1 and 2). Resumption of meiosis or oocyte maturation can be readily monitored by the dissolution of the oocyte nucleus, a process known as germinal vesicle breakdown (GVBD) (Fig. 2). In addition, the presence of activin and inhibin in the Fundulus ovarian tissue is also being investigated by reverse transcription and polymerase chain reaction (RT-PCR).
Isolation of the activin/inhibin B subunit: Based on the amino acid sequences conserved in homologous protein of other species, we designed several degenerate oligonucleotide primers (Table 1 and Fig. 6). With Beta B2F (forward primer)and Beta B4R (reverse primer), a partial cDNA fragment was obtained by RT-PCR (Polymerase Chain Reaction). Additional gene specific primers (Fig. 6) were synthesized based on this sequence and used in conjunction with the 5' and 3' RACE primer adapters (Ambion) to amplify the fragments corresponding to the 5' and 3' ends of Fundulus mRNA.
In contrast to the activin effect, inhibin significantly decreased DHP-induced GVBD during the first 24 hr of culture at all concentration of DHP used. This effect of inhibin was dose-dependent and reversible. This inhibitory effect became less evident after 40 hr with the highest doses of DHP, but it persisted with the lowest doses of DHP.
Subcloning of PCR products: PCR products were isolated and purified from agarose gels using the MinElute gel extraction kit (Qiagen), ligated into a pGEM-T plasmid vector (Promega), and transformed into JM109 High Efficiency Competent Cells (Promega). Transformants were isolated based on blue/ white color selection, insert size confirmed by PCR and sequenced (DNA Core Lab, UM, FL).
Statistics: Data are presented as mean ± SEM from three or more experiments performed at different dates. Statistical comparisons were conducted by analysis of variance, and the means were subsequently compared by Tukey’s test or Hall-Sidak method (all pairwise multiple comparisons). Differences were considered significant if P0.05. Same letter indicate significantly different from each other.
Fig. 3 Effects of Activin on DHP-induced Oocyte Maturation
Recombinant human activin A alone did not induce oocyte maturation (GVBD) in F. heteroclitus. However, DHP-induced GVBD was significantly enhanced by activin A in a dose-dependent manner after 48 hr. No activin effect was observed during the first 24 hr of culture.
Sister John Karen Frei, O.P., PhD.
Dr. Flona Redway
NIH-MBRS SCORE Grant: GM 45455-08
RISE Grant: GM 59244-01A1
NSF Grant: DBI-0116080
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