A1260008124BcvtH

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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
INTRODUCTION
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 ß
(TGF- ß) super family. Inhibins are
heterodimeric glycoproteins composed of an
a-subunit and one of the two ß-subunits (ßA or
ßB), giving rise to biologically active forms
termed inhibin A and inhibin B. Activins consist
of two ß-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
17a,20ß-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).
Fig. 2
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 Tukeys test or
Hall-Sidak method (all pairwise multiple
comparisons). Differences were considered
significant if P?0.05. Same letter indicate
significantly different from each other.
Fig. 1
RESULTS

• Animals and in vitro culture of ovarian
  follicles Killifish (Fundulus heteroclitus) were
  collected from salt marshes in St. Augustine, FL.
  Fish were maintained in a 25 gal. aquarium on a
  14/10 hr light/dark cycle, at 25ºC, and were fed
  three times a day with flake food (TetraMin).
• Ovaries were removed from females and placed in
  75 Leibovitz L-15 medium with L-glutamine
  (Sigma) containing 100?g gentamicin/ml, pH 7.5.
• Fully grown follicles (1.2-1.4 mm in diameter)
  were manually isolated from several ovaries with
  the aid of fine forceps under a stereomicroscope,
  pooled, and randomly distributed into 24-well
  tissue culture trays (Costar), containing 20
  follicles/1ml L-15 media/ well. Incubations were
  carried out at room temperature and GVBD was
  scored at 24 and 48 hr.
• 17a,20ß-dihydroprogesterone (DHP) was obtained
  from Steraloids Inc.(Newport, RI) and dissolved
  in ethanol. Porcine inhibin was obtained from
  Sigma (St. Louis, MO) and dissolved in culture
  media. Recombinant human activin A was supplied
  by Dr. Parlow (NIDDKs National Hormone and
  Pituitary Program and NICHD) and prepared in
  culture media.
• RNA isolation mRNA was isolated from Fundulus
  heteroclitus ovaries (1-2 g) after homogenization
  of the tissue in a Lysis/binding Solution using
  the Poly(A)Pure mRNA isolation kit (Ambion).
  This RNA preparation was used for the reverse
  transcription reactions (RT) and rapid
  amplification of 3' and 5' cDNA ends (RACE).
•  

Fig. 3 Effects of Activin on DHP-induced
Oocyte Maturation
CONCLUSIONS

• This study provides evidence for the presence of
  the activin/inhibin ßB subunit in the ovary of F.
  heteroclitus.
• A multiple sequence alignment comparison of the
  Fundulus Activin/Inhibin Beta B protein sequence
  with human (gi9257225 ), chicken (gi1429377),
  Xenopus (gi386028), goldfish (gi2209351) and
  zebrafish (gi516357) sequences (Fig. 6) shows
   high amino acid sequence homology at the carboxy
  terminal. Fundulus ßB subunit has well conserved
  Cysteine positions when aligned with other
  species.  
• Results indicate that these gonadal peptides play
  a paracrine role (acting on neighboring cells) in
  modulating the oocyte maturation induced by
  steroid in this species.
• The effects of activin and inhibin are distinct
  and opposed to each other. These effects are
  reversible, and dependent not only on the
  concentration of the peptides but also on the
  dose of the inducing steroid. While inhibin
  appears to delay the onset of GVBD triggered by
  the steroid, activin enhances the steroid-induced
  GVBD by increasing the number of oocytes
  responding to a low dose of steroid.

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.
REFERENCES
ACKNOWLEDGEMENTS
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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