wnt 4 Is Associated with the Development of Ovarian Tissue in the Protandrous Black Porgy , Acanthopagrus schlegeli 1

The protandrous black porgy, Acanthopagrus schlegeli, has a striking life cycle with sex differentiation at the juvenile stage, mono-male development, and male-to-female sex change (with vitellogenic oocytes) at age 3 yr. In the present study, we investigated the possible roles of wnt4 in gonadal development in a nonmammalian model organism (protandrous black porgy), especially in relation to sex differentiation, ovarian growth, and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment had an effect on these processes. Furthermore, a natural sex change ( 2-yr-old fish) and a nonchemical method to induce an early sex change ( 1-yr-old fish) via the removal of testicular tissue were examined in this study. We present herein an integrative immunohistochemical, cellular, and molecular data set describing these phenomena. During gonadal sex differentiation, no increase in wnt4 expression was detected. A profile of increased wnt4 expression and decreased cyp19a1a expression was associated with ovarian growth (proliferation of oogonia and development of ovarian lamellae) in 1-yr-old fish. Both E2 and AI induced an increase in wnt4 transcripts and resulted in ovarian development in 0yr-old and 1-yr-old fish. Increased wnt4 transcripts were found in ovarian tissue undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in 2-yrold fish. Removal of testicular tissue in 1-yr-old fish resulted in successful early sex change (with vitellogenic oocytes) 6 mo after the excision. During the process of the early sex change (3 mo after testis excision), the fish ovary became active and had increased diameter of the primary oocytes; this was in accord with increased ovarian wnt4 expression but not sf1, foxl2, and genes in the steroidogenic pathway, including cyp19a1a. Wnt4 staining further confirmed the profile of wnt4 expression associated with ovarian development. The results of the present study suggest that wnt4 has important roles in late ovarian growth (e.g., oogonia proliferation and structure of ovarian lamellae) and the natural sex change (vitellogenic oocytes) in the protandrous black porgy. aromatase, cyp19a1a, early development, estradiol, gonadal development, oocyte development, sex change, sex differentiation, wnt4 INTRODUCTION Black porgy, Acanthopagrus schlegeli Bleeker, are marine protandrous hermaphroditic fish that have a striking life cycle. The fish are functional males for the first 2 yr of life, but they transform into females during the third year [1, 2]. A differentiated bisexual gonad divided by connective tissue is observed in 4to 5-mo-old fish [3]. The testicular tissue further develops to become a functional testis for the first and second reproductive seasons. The ovarian compartment contains few oogonia and primary oocytes in the germinal epithelium around the ovarian cavity between sex differentiation (4-mo-old fish) and the first reproductive season [3]. During the postspawning (March to May) and nonspawning (June to August) periods after the first reproductive season, the testicular tissue regresses, and the ovarian tissue expands to become the dominant tissue (with ovarian lamellae) in the bisexual gonad, although the germ cells remain at the primary oocyte stage [3]. The ovarian tissue continues to be the predominant tissue in the bisexual gonad until the prespawning season (September to December) in 1-yr-old fish, at which point it regresses concomitantly with the growth of testicular tissue. The testicular tissue resumes dominance and activity for the second reproductive season. A similar pattern of ovarian development is observed during the postspawning and nonspawning seasons in 2-yr-old fish, but the ovarian tissue does not regress in about 40%–50% of the population. The fish will subsequently change sex by developing vitellogenic oocytes in the approaching third reproductive season [3, 4]. Plasma estradiol (E2) levels during the late prespawning and spawning periods are correlated with the natural sex change in 2to 3-yr-old fish [1, 2], and the natural sex change is blocked by long-term aromatase inhibitor (AI) administration [5]. Plasma E2 is maintained at low levels, however, when ovarian tissue develops to become the dominant tissue (with ovarian lamellae) during the postspawning and nonspawning seasons in 1-yr-old and 2-yr-old fish [2, 4, 6]. Furthermore, ovarian tissue is arrested at the primary oocyte stage until the sex change [2, 5, 7]. In our recent investigations, cyp19a1a transcript levels were high before male sex differentiation and dramatically decreased after the testis underwent early spermatogenesis [8, 9]. Testicular tissue development was blocked by long-term AI administration, and ovarian tissue developed after testis regression in juvenile fish [9]. As such, it would be relevant to understand the mechanisms behind ovarian development (with ovarian lamellae) in a low E2 environment. Few species have been studied in relation to Wnt4 (wingless-type MMTV integration site family, member 4) in gonad development. Wnt4 has been reported to be related to ovarian development in mice [10]. It induces the expression of downstream genes such as Fst and has been observed to exhibit sexually dimorphic expression and is essential for the development of mouse ovaries [11]. Wnt4-mutant females Partially supported by the National Science Council (Taiwan) and the Center for Marine Bioscience and Biotechnology (National Taiwan Ocean University, Keelung, Taiwan). Correspondence: FAX: 886 2 2462 1579; e-mail: B0044@mail.ntou. edu.tw Received: 6 March 2009. First decision: 3 April 2009. Accepted: 1 July 2009. 2009 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod. org ISSN: 0006-3363 1073 D ow nladed rom w w w . biolrod.org. D ow naded rom http/academ ic.p.com /biolreprod/artitract/81/6/1073/2557792 by gest on 15 Feruary 2019 show a lack of mullerian ducts, an increase in ectopically steroidogenic cell migration, and the activation of testosterone biosynthesis [10, 12]. Wnt4 is predominantly expressed in the ovary in mammals [10] but has no sexually dimorphic expression during female differentiation of nonmammalian vertebrates, including chickens [13], turtles [14], and frogs [15]. The profile and possible roles of wnt4 expression in relation to gonad development are not yet clear in teleost fish. We propose that Wnt4/wnt4 has an important role in ovarian development, especially in relation to early formation of ovarian lamellae and the late sex change in the protandrous black porgy. In the present study, protandrous black porgy exhibiting unique development of a bisexual gonad and a natural sex change were chosen to study wnt4 profiles in relation to the following: 1) gonadal sex differentiation (i.e., from undifferentiated to differentiated juveniles [ 0-yr-old fish]) given the functional mono-male sex pattern observed in this species during the first and second reproductive seasons, 2) E2 activity during sex differentiation and gonad development using exogenous E2 and AI treatments in 0-yr-old fish and AI treatment in 1-yr-old fish, 3) the natural sex change from male to female in 2to 3-yr-old fish, and 4) the testicular effects on the development of ovarian tissue in the bisexual gonad and the expression of wnt4 by in vivo surgical manipulation of testicular tissue and ovarian tissue. MATERIALS AND METHODS