Guan-Chung Wu

Testicular dmrt1 is Involved in the Sexual Fate of the Ovotestis in the Protandrous Black Porgy

ABSTRACT In hermaphroditic fish, the ovotestis can respond to external stimuli so that only one type of gonadal tissue (either ovarian or testicular tissue) will remain reproductively active and the other will recede to a rudimentary stage. However, the molecular mechanism for sexual fate determination is still poorly understood in hermaphroditic fish. In the present study, we examined whether sexual fate determination with respect to testis development is due to differential expression of dmrt1. Expression of dmrt1 was limited to the spermatogonia-surrounding cells (Sertoli cells) throughout testis development. Testicular dmrt1 was differentially expressed in fish (black porgy [Acanthopagrus schlegeli Bleeker]) depending on if fish were destined to be female or male. Expression of dmrt1 in Sertoli cells did not require germ cell factors with busulfan treatment. To examine the role of dmrt1, we used virus-based RNA interference. Deficiency of dmrt1 resulted in a reduced number of germ cells in the testis and stimulated a male-to-female sex change. Higher serum luteinizing hormone levels were detected in 2+- to 3-yr-old male fish as compared to sex-changing female fish. Furthermore, we showed that fish treated in vivo with gonadotropin-releasing hormone (Gnrh) and fish treated in vitro with gonadotropin (Gth) had higher dmrt1 expression in the testis, suggesting that these endocrine factors may affect the male-to-female sex change. Therefore, our data suggest that dmrt1 plays a key role in initial testis differentiation and in later maintenance of male development. We show, to our knowledge for the first time, the functions of dmrt1 in hermaphroditic fish, which indicate that male-phase maintenance may be regulated by the brain-pituitary-gonadal axis via the Gnrh-Gth-Dmrt1 axis.

wnt4 Is Associated with the Development of Ovarian Tissue in the Protandrous Black Porgy, Acanthopagrus schlegeli

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 ≥0-yr-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-yr-old 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.

Differential Dmrt1 transcripts in gonads of the protandrous black porgy, Acanthopagrus schlegeli

Black porgy, Acanthopagrus schlegeli Bleeker, is a marine protandrous hermaphrodite fish. A Dmrt1 cDNA was cloned and characterized and in order to study the process of sex change in this species, mRNA transcripts of Dmrt1 were monitored. Dmrt1 was specifically transcribed in testis and seminal vesicle in 2-year-old black porgy according to RT-PCR and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of Dmrt1 transcripts. Dmrt1 transcripts were at significantly higher levels in bisexual testis than bisexual ovary in 1+ and 2+ year-old fish. Dmrt1 transcripts decreased in the functional and bisexual testis of 3-year-old fish. Much higher levels of Dmrt1 transcripts in the bisexual ovary were detected in 1+ year-old fish than in 2+ and 3-year-old fish. No differences in Dmrt1 transcripts were found in bisexual ovaries of 2+ and 3-year-old fish and female ovaries of 3-year-old fish. The data suggest there is relationship of Dmrt1 to the sex change of protandrous black porgy.

Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

Dual Roles of cyp19a1a in Gonadal Sex Differentiation and Development in the Protandrous Black Porgy, Acanthopagrus schlegeli

Abstract Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with male sex differentiation at the juvenile stage, a bisexual gonad during first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. The present study investigated the role of aromatase (cyp19a1a/Cyp19a1a) in gonadal development in this species, especially in relation to sexual differentiation and sex change. Fish of various ages were treated with estradiol (E2) or aromatase inhibitor (AI) to determine whether manipulation of the hormonal environment has an impact on these processes. We report an integrative immunohistochemical, cellular, and molecular data set describing these interesting phenomena. During male sex differentiation, high levels of cyp19a1a/Cyp19a1a expression were observed in the undifferentiated gonad (4 mo of age), in marked contrast to the low cyp19a1a/Cyp19a1a levels detected in the differentiated testis at the age of 5–6 mo. A low dose of E2 (0.25 mg/kg feed) stimulated testicular growth and function in sexually differentiated fish, whereas a high dose of E2 (6 mg/kg feed) induced female development. Furthermore, administration of AI suppressed male development and promoted female sexual differentiation. An increased number of figla transcripts (an oocyte-specific gene) were observed prior to cyp19a1a expression, concomitant with the development of oogonia and early primary oocytes in the ovaries of both E2- and AI-treated groups. Immunohistochemical Pcna staining showed that the regression of testicular tissue occurred prior to the development of ovarian tissue in both E2- and AI-induced females. The importance of cyp19a1a in female development was further demonstrated by the increase in cyp19a1a transcripts during the naturally occurring sex change. Transcripts of foxl2 increased in the gonads of 2- to 3-yr-old black porgy during the early stages of the natural sex change, followed by a gradual elevation of cyp19a1a levels. The levels of both genes peaked in the resulting ovarian tissue. Thus, cyp19a1a/Cyp19a1a plays dual roles in the gonadal development, namely, in testicular development during the initial period of sexual differentiation and later in ovarian development during the natural sex change.

Developmental Expression of Key Steroidogenic Enzymes in the Brain of Protandrous Black Porgy Fish, Acanthopagrus schlegeli

In the present study, we tested the hypothesis that the brain of the black porgy fish, Acanthopagrus schlegeli, has the capacity for de novo steroidogenesis and that these neurosteroids may impact sex differentiation. Gonadal histology and Dmrt1 gene expression revealed that the fish were not sex differentiated until 155 dah (days after hatching). We further demonstrated the developmental expressions of the mRNAs encoding for four key neurosteroidogenic enzymes, namely, the cytochrome P450 side chain cleavage (CYP11A1), 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (3βHSD), cytochrome P450c17 (CYP17) and aromatase (CYP19b) in the brain at different post‐hatching developmental ages. The results indicated that steroidogenic genes are expressed in brain from the earliest sampling time, 60 dah. Quantitative real‐time polymerase chain reaction analysis demonstrated significantly higher expression levels of these enzymes at 120 dah compared to 60 dah in all the brain regions. However, the increase for 3βHSD was significant only in hypothalamus and midbrain, whereas it was significant only in forebrain and hypothalamus for CYP19b. A decline in mRNA levels were observed for all the genes at 155 dah except in midbrain for CYP11A1 and in hindbrain for CYP19b. Analysis of aromatase enzyme activity showed a significant increase in aromatase activity in the forebrain at 120 dah. Thus, the present study demonstrated for the first time an age‐ and/or region dependent expression of the mRNAs encoding the steroidogenic enzyme genes in the brain of black porgy. The presence of key steroidogenic enzymes as early as 60 dah, before gonadal sex differentiation, demonstrates that steroid biosynthetic capacity in brain precedes histological gonad differentiation. The mRNA transcripts of these genes showed a synchronous peak at 120 dah, suggesting that oestradiol may be locally formed in most parts of the brain. The study suggests an important role for brain aromatase in male black porgy brain sex differentiation, and considers the possibility of a role for this enzyme in neurogenesis.

Estradiol-17β Induced a Reversible Sex Change in the Fingerlings of Protandrous Black Porgy, Acanthopagrus schlegeli Bleeker: The Possible Roles of Luteinizing Hormone in Sex Change

Abstract The objectives of the present study were to investigate the effects of oral administration of estradiol-17β (E2) on luteinizing hormone (LH) in plasma, aromatase activity in gonad, and sex change in the fingerlings of protandrous black porgy, Acanthopagus schlegeli Bleeker. The expression of estrogen receptor (ER) and androgen receptor (AR) transcripts in gonad was also analyzed. Undifferentiated (2-mo-old) black porgy were divided into two groups, one fed a control diet and the other a diet mixed with E2 (6.0 mg/kg feed) for 5 mo. Fish treated with E2 for 3 mo showed complete suppression of spermatogenesis and spermiation and induced sex change with primary oocytes. Aromatase activity in forebrain and midbrain was increased in the control in December–March (during the spawning season). E2 stimulated aromatase activity in the brain. Higher gonadal aromatase activity in concordance with elevated levels of plasma LH was observed in the E2 group compared with the control. After 2-mo of E2 termination, regressed testicular tissue recovered and controlled females gradually reversed back to functional males in January and March. Plasma LH levels were higher in the E2-terminated group during the period of reversible sex change (from a controlled female to male) compared with the control. The expression of ER and AR transcripts was closely related to the development of testis and ovary. The data showed that E2 induced a reversible sex change with high plasma LH. Increase of gonadal aromatase and decrease of ER/AR were associated with controlled sex change. Plasma LH levels were correlated with the conversion from a controlled female to male in black porgy.

The Expression of amh and amhr2 Is Associated with the Development of Gonadal Tissue and Sex Change in the Protandrous Black Porgy, Acanthopagrus schlegeli

The protandrous black porgy, Acanthopagrus schlegeli, has a striking life cycle, with sex differentiation at the juvenile stage, mono-male development, a bisexual gonad during the first 2 yr of life, and a male-to-female sex change (with vitellogenic oocytes) at 3 yr of age. In the present study, we investigated the possible roles of amh and amhr2 in gonadal development in a nonmammalian model organism (protandrous black porgy), especially in relation to sex differentiation, testicular and ovarian growth, and sex change. Fish of various ages were treated with estradiol or an aromatase inhibitor to induce the fish to become female. Furthermore, a natural sex change (2+-yr-old [>2 yr and <3 yr] fish) and a nonchemical method to surgically remove one of the pair of gonads to examine the possible roles of amh in the natural sex change were conducted. We present integrative in situ hybridization, immunohistochemical, cellular, and molecular data describing these phenomena. During gonadal sex differentiation, an increase in amh and amhr2 expression was detected. Higher levels of amh and amhr2 transcripts were observed in the testicular tissue when compared to the ovarian tissue in the bisexual gonad of 1+-yr-old (>1 yr and <2 yr) fish. Transcripts of amh reached peak levels in November (prespermatogenesis period) and then declined to the lowest levels in January (spawning period). Chemical-induced ovarian tissue had very low amh transcript levels but high levels of amhr2. Active testes had significantly higher amh and amhr2 expression levels as compared to inactive testes. In contrast, no difference in the expression of amh and amhr2 between active and inactive ovarian tissues was found. Transcripts of amh were expressed in the somatic cells of the spermatogonia and vitellogenic oocytes, and amhr2 was expressed in the somatic cells of the spermatogonia. Transcripts of amh decreased in the testicular tissue 5 mo before occurrence of the sex change into a female. In contrast, testicular amh expression remained high if the fish remained male. Human chorionic gonadotropin regulated amh and amhr2 expression in the testicular tissue but not in the ovarian tissue. The present results suggest that amh plays important roles in early testicular and ovarian development, late ovarian growth (e.g., vitellogenic oocytes), and natural sex change in the protandrous black porgy.

The Expression of nr0b1 and nr5a4 During Gonad Development and Sex Change in Protandrous Black Porgy Fish, Acanthopagrus schlegeli

Abstract Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle, with a mono-male sex differentiation at the juvenile stage and male-to-female sex change at 3 yr of age. We report for the first time integrative molecular data on these interesting phenomena. Sex differentiation occurred between 4 and 5 mo of age. Testicular nr5a4 transcripts increased to high levels during sex differentiation (5 mo old), whereas nr0b1 (Dax-1) did not increase until the age of 8 mo. High nr5a4 and nr0b1 expression in testicular tissue, in contrast to low nr5a4 and high nr0b1 expression in ovarian tissue, were found in the male phase of 0+- to 2-yr-old fish (before sex change). Increased nr5a4, decreased nr0b1, and increased cyp19a1a were found in the ovarian tissues undergoing development from primary oocytes to vitellogenic oocytes during the natural sex change in 2+-yr-old fish. Removal of testicular tissue in 1+-yr-old fish resulted in both increased ovarian nr5a4 and genes in the steroidogenic pathway and decreased nr0b1 together with the appearance of vitellogenic oocytes. Ovary developed into the active stage with the increased expression of star and steroidogenic enzymes, including aromatase, in concordance with the decreased expression of nr0b1 in the testis-excised fish. Long-term estradiol (E2) administration resulted in early sex change, but the ovaries were mainly with primary oocytes. Low nr5a4, high nr0b1, and low steroidogenic enzymes, including cyp19a1a expression, were also observed in these E2-fed ovarian tissues. Thus, nr5a4 but not nr0b1 was associated with male sex differentiation. Testicular development required cooperative functions of both nr5a4 and nr0b1. The present study suggests that nr5a4 and nr0b1 have an antagonistic interaction for the oocyte development. Testicular tissue exerted inhibitory effects on ovarian development. It is probable that nr0b1 regulates the timing of vitellogenic development and sex change in black porgy.

Current status of genetic and endocrine factors in the sex change of protandrous black porgy, Acanthopagrus schlegeli (Teleostean).

Abstract: Black porgy, Acanthopagrus schlegeli Bleeker, a marine protandrous hermaphrodite fish, is functionally male for the first 2 years of life, but begins to sexually change to female after the third year. Testicular tissue and ovarian tissue are separated by connective tissue in the bisexual gonad. This sex pattern provides a unique model to study the mechanism of sex change in fish. The annual profiles of plasma estradiol, vitellogenin, and 11‐ketotestosterone concentrations in males were significantly different from those in the 3‐year‐old females. Oral administration of estradiol stimulated high levels of gonadal aromatase activity, plasma luteinizing hormone (LH) levels, and sex change in the 2‐year‐old fish. Oral administration with aromatase inhibitors for 1 year further blocked the natural sex change in 3‐year‐old black porgy and all fish became functional males. Transcripts of estrogen receptor (ER), androgen receptor, and gonadotropin receptors in the ovarian tissue of bisexual gonad were significantly less expressed than those in the bisexual testicular tissue. ER and aromatase transcripts were much higher in the vitellogenic ovary than those in the bisexual ovarian tissue. Plasma LH levels were higher in male fish than sex‐changing fish during postspawning and nonspawning season in 2+‐year‐old black porgy. We are also conducting investigations on the role of the genetic factors (Dmrt 1, Sox 9, Sf‐1, and Dax‐1) in sex development and sex change. An endocrine mechanism of sex change in black porgy is proposed.