Development of the Genital Duct System in the Protandrous Black Porgy Acanthopagrus schlegeli.код для вставкиСкачать
THE ANATOMICAL RECORD 294:494–505 (2011) Development of the Genital Duct System in the Protandrous Black Porgy, Acanthopagrus schlegeli MONG-FONG LEE,1 JING-DUAN HUANG,2 AND CHING-FONG CHANG3* Department of Aquaculture, National Penghu University, Penghu 88046, Taiwan 2 Institute of Marine Biology, National Taiwan Ocean University, Keelung 20224, Taiwan 3 Department of Aquaculture, National Taiwan Ocean University, Keelung 20224, Taiwan 1 ABSTRACT Protandrous black porgies, Acanthopagrus schlegeli, have a striking life cycle, which includes early sex differentiation, bisexual gonads, and a male-to-female sex change at three years of age. We found novel features of and insights into the development of the genital duct system in relation to the gonadal stage during early gonadal development and natural sex change. We found that the genital ducts developed at 16–20 weeks of age during sex differentiation. The gonad developed ‘‘ovarian cavity cracks’’ and became ‘‘four-stranded’’ during the ﬁrst prespawning period and then proceeded to the development of genital ducts before 1 year of age. Two ovarian cavities of the paired gonads combined, developed, and extended caudally to form the oviduct, making up the inner duct of the genital ducts. The testicular main cavities also extended and fused together to form the outer duct of the genital duct system, that is, the sperm duct. The coexistence of an outer sperm duct and an inner oviduct constituted a unique ‘‘double cannula genital duct’’ structure. Gradually the inferior walls of the oviduct intermingled with those of the sperm duct, and the circular lumen of the sperm duct changed into an ‘‘M-shaped canal.’’ Finally, the sperm duct and oviduct separated completely at the distal part of genital duct system. During natural sex change, the male reproductive passage regressed and degenerated and was replaced by connective tissue. The oviduct arrested as a blunt end during male phase and, ﬁnally, extended and connected to the genital pore during the C 2011 Wiley-Liss, Inc. female phase. Anat Rec, 294:494–505, 2011. V Key words: gonadal development; genital pore; oviduct; protandrous ﬁsh; sex change; sex differentiation; sperm duct In male mammals, Sertoli and Leydig cells of the fetal testes secrete anti-Müllerian hormone and testosterone, which promote the degeneration of the Müllerian ducts and the maintenance of the Wolfﬁan ducts, respectively. In the male, the Wolfﬁan ducts are induced to form the efferent ductules, epididymis, vas deferens, and seminal vesicles (Drews, 2000), whereas in the female, the Wolfﬁan ducts regress due to the absence of anti-Müllerian hormone and testosterone. Consequently, in the female, the Müllerian ducts develop into the uterine tube, uterus, and part of the vagina (Huhtaniemi, 1994). The C 2011 WILEY-LISS, INC. V Grant sponsor: National Science Council, Taiwan. *Correspondence to: Ching-Fong Chang, Department of Aquaculture, National Taiwan Ocean University, Keelung 20224, Taiwan. Fax: þ886-2-2462-1579. E-mail: B0044@mail. ntou.edu.tw Received 22 March 2010; Accepted 8 July 2010 DOI 10.1002/ar.21339 Published online 2 February 2011 in Wiley Online Library (wileyonlinelibrary.com). GENITAL DUCTS IN THE PROTANDROUS BLACK PORGY uterine tube of mammalian vertebrates is divided into three regions, the infundibulium, ampulla, and isthmus, and the male reproductive passages include the efferent ductules and epididymal duct (Constantinescu, 2007). In contrast, studies on the genital duct systems of teleost ﬁshes are very limited and scattered, and the characterizations of male genital ducts are also ambiguous. The sperm ducts of the teleosts are considered unique in having evolved as an extension from the testis canal, which is not of nephric origin (Liem et al., 2001). Two potential paths to the development ovaries and oviducts in teleosts have been suggested: the merging of two edges of the genital ridge to enclose an isolated hollow and the lateral growth of one edge of the genital fold fusing with the peritoneum. Both of these enclose part of the coelom and then extend caudally as the oviduct (Barton, 2007). The male reproductive passages of four salmonid species (Lahnsteiner et al., 1993) and three cyprinid species (Lahnsteiner et al., 1994) have been described and include seminiferous tubules, testicular efferent ducts, and testicular main ducts according to terminology by Grier et al. (1980). Suzuki and Shibata (2004) investigated the development of genital ducts in the medaka, Oryzias latipes. They interpreted the ducts in the intragonad of females and males to be ovarian cavities and efferent ducts, and the ducts in the extragonad of those as oviducts and sperm ducts, respectively (Suzuki and Shibata, 2004). Recent studies on the development of genital ducts during sex change in the protogynous honeycomb grouper, Epinephelus merra (Alam and Nakamura, 2007), have proposed that the male reproductive passage within the gonad develops from slit-like structures located between stromal tissues and the tunica albuginea of the ovary and from oval spaces within the wall of the ovarian cavity (Alam and Nakamura, 2007). In contrast, there are no studies examining the reproductive tract in protandrous ﬁsh. The black porgy, Acanthopagrus schlegeli (Perciforms, Sparidae), is a marine protandrous hermaphrodite that expresses male function during the ﬁrst to second spawning periods but transforms into females during the third year (Chang et al., 1994). However, only about 40% of cultured black porgies change into females at 3 years of age, whereas the rest remain functional males during the third spawning season. Testicular tissue proliferates and proceeds to spermatogenesis (spermatogonium ! sperm) during the ﬁrst and second prespawning periods, resulting in the ﬁsh becoming male (Huang et al., 2002). During the spawning season at 3 years of age, the testicular tissue regresses, ovarian tissue, and vitellogenic oocytes develop, resulting in a sex change in the ﬁsh from a male to female (Huang et al., 2002; Lee et al., 2008). This sex pattern provides a unique model to study the mechanisms of sexual development in ﬁsh. However, the mechanisms involved in the development of male and female genital ducts in bisexual gonads are unknown. That is, how the sperm ducts and oviducts develop and differentiate to match each gonadal stage and lead to the success of sex development in the protandrous black porgy is still unclear. The objective of this study was to investigate the development and structural characteristics of the genital duct system in the protandrous black porgy using light and scanning electron microscope (SEM). 495 MATERIALS AND METHODS Black Porgy Black porgy ﬁsh raised in the tanks in the aquaculture building of the Department of Aquaculture, National Taiwan Ocean University (NTOU), were sampled at ages ranging from 6 weeks to 3 years from May, 2003 to August, 2006. The gonads and genital ducts (about from 4 to 18 mm long in the ﬁsh from 5month- to 3-year-old) with anal and urinary pores were removed by dissection and prepared for histological study. The reproductive stage of the black porgy was divided seasonally into four components: the spawning period (from January to March), the postspawning period (April to May), the intersex period (June to September), and the prespawning period (October to December) (Huang et al., 2002). The number of ﬁsh sacriﬁced at various reproductive stages and ages were as follows: (1) youngling and prespawning period: 19 ﬁsh during the 6week to 5-month period and 21 ﬁsh during the 5-month to 1-year period; (2) spawning season: 22 ﬁsh in the ﬁrst year, 17 ﬁsh in the second year, and 14 ﬁsh in the third year; and (3) postspawning and intersex period: 20 ﬁsh in the ﬁrst year, 16 ﬁsh in the second year, and 13 ﬁsh in the third year. All procedures and investigations were approved by the NTOU Institutional Animal Care and Use Committee and were performed in accordance with standard guiding principles. Gonadal Histology by Light Microscopy The gonads and genital ducts with anuses and urinary pores were preserved in Bouin’s solution for 24–72 hr (according to the size of the tissues sampled), followed by washing with 70% alcohol several times to remove as much of the picric acid as possible. The tissues were then dehydrated in an alcohol series of 80, 85, 90, 95, and 100% concentrations and then soaked in xylene. They were embedded in parafﬁn wax and sectioned at 5–6 lm thickness. Parafﬁn sections were treated with trichrome staining according to the Gomori rapid onestep trichrome method (Bancroft and Cook, 1994) to stain the connective tissue. The stained sections were sealed with Entellan (Merck, Darmstadt, Germany) and observed with a light microscope. Scanning Electron Microscopy The gonads and genital ducts were soaked in a ﬁxative solution (0.1 M buffer solution of sodium cacodylate, 1% glutaraldehyde with pH 7.4 and 3% paraformaldehyde) at 4 C immediately after removal from the ﬁsh. The desired portions were cross-sectioned into 1–5-mm thick slices and preserved in the same ﬁxative for another 24 hr. They were then washed with 0.1 M buffer solution of sodium cacodylate, postﬁxed with cold 1% osmium tetroxide (OsO4) solution for 2 hr, and washed with a 0.1 M buffer solution of sodium cacodylate. The ﬁxed tissues were dehydrated in an ethanol series and put into a critical-point dryer (HCP-2 critical point dryer, Hitachi, Tokyo, Japan). The dried samples were pasted onto aluminum stubs and then gilded by E101 ion sputter (Hitachi). Finally, samples were observed using a Hitachi S-2400 SEM. 496 LEE ET AL. Fig. 1. Development of the primordial gonad and its reproductive passage. The gonadal stage of (A) and (B) were as step 1 and (D) as step 2 in Fig. 9. (A) The primordial gonads (arrow) at 6 weeks of age were located at the crossed position between the mesentery and the peritoneum. Inset: a magniﬁed primordial gonad. (B) Each primordial gonad (circle) budded out a small branch at 14 weeks of age. (C) Magniﬁed and branched primordial gonad (arrow). (D) The ovarian RESULTS The Development of the Primordial Gonad and Its Reproductive Passages in the Fingerling Period The primordial gonads of the 6-week-old ﬁsh were located in the proximal region of the dorsal wall-linked cavity served as a female reproductive passage and formed at 16–20 weeks of age. (E) Only the anus and urinary pore appeared on the surface of the caudal body before the ﬁrst spawning season. A: anus; ABC: abdominal cavity; BV: blood vessel; INT: intestine; ME: mesentery; MU: muscle; OC: ovarian cavity; PE: peritoneum; PG: primordial gonad; POT: putative ovarian tissue; PTT: putative testicular tissue; SL: scale; SB: swimbladder; UP: urinary pore. mesentery of the abdominal cavity (Fig. 1A). In the 14week-old ﬁsh, another small branch of the gonad budded out from the base of each primordial gonad (Fig. 1B,C). The gonads did not form the ovarian cavities until about 16–20 weeks of age (Fig. 1D); the genital ducts could also be found at this stage, whereas the genital pore was GENITAL DUCTS IN THE PROTANDROUS BLACK PORGY 497 Fig. 2. Formation of ‘‘ovarian cavity cracks’’ (OCC) between gonad and genital duct in 7-month-old ﬁsh. The gonadal stage was as step 3 in Fig. 9. (B), (C), and (D) were the transections of the same gonad at different locations (cranial to caudal). (A) An ovotestis consisted of mainly testicular tissue and a small portion of ovarian tissue as observed with the SEM. The thick tunica albugine separated the testicular tissue from ovarian tissue found along one side of the ovarian cavity during the ﬁrst prespawning period. (B) Paired ovotestes were located under the peritoneum. (C) The four-stranded gonads were formed due to the OCC (arrow), where the testicular main cavities linked to testicular tissue. (D) The OCC in one of the paired gonads linked back (circle). ABC: abdominal cavity; ADP: adipose tissue; OC: ovarian cavity; OCC: ovarian cavity crack; OT: ovarian tissue; PE: peritoneum; TA: tunica albuginea; TMC: testicular main cavity; TT: testicular tissue. absent from the surface of the caudal body until the ﬁrst spawning season (Fig. 1E). gonad appeared as a unique ‘‘four-stranded structure’’ (Fig. 2C). When extending spatially toward the caudal part of the ﬁsh, the two-stranded gonads were connected, and the ovarian cavities were fused (Fig. 2D). Formation of Unique Ovarian Cavity Crack in the ﬁrst Prespawning Period In the ﬁrst prespawning period, the gonads developed into an ovotestis, in which the tunica albuginea (located between testicular tissue and ovarian tissue) made up the testicular main cavity (Fig. 2A). The connective tissue within the tunica albuginea separated the testicular tissue with spermatogonia from the ovarian tissue; the ovarian tissue was distributed along one side opposite the testicular main cavity, in the ovarian cavity (Fig. 2A,B). The gonads developed cracks, which began at the testicular main cavity’s connections to the testicular tissues and ran spatially between the gonads and genital ducts; we termed these ‘‘ovarian cavity cracks.’’ Each of the paired gonads became ‘‘two-stranded,’’ and the whole Formation of the Genital Duct System The paired ovotestes coalesced together at the site of the ovarian cavities (Fig. 3A); caudally, the two ovarian cavities merged into one (Fig. 3B) and developed to form the oviduct, the inner duct of the genital ducts. Two lateral testicular main cavities beside the ovarian cavities extended and interlinked together, and the outer walls of the paired testicular tissues in the ovotestes also extended caudally to constitute the outer duct of the genital duct system, that is, the sperm duct (Fig. 3C). At the proximal genital ducts (near the gonad), the sperm duct (outer duct) encircled the oviduct (inner duct), and we termed these unique coexistent structures 498 LEE ET AL. ‘‘double cannula genital ducts’’ (DCGDs; Figs. 3C, 4A). The sperm duct and oviduct in the DCGD were adjacent during the nonspawning period, and the DCGD became exposed to the abdominal cavity running along the bottom of the urinary bladder (Fig. 4A). Later, they invaginated into the connective tissue between the mesonephric duct and the intestine (Fig. 4B), and the inferior wall of the oviduct gradually intermingled with that of the sperm duct (Fig. 4C). When the conjugated portion increased, the originally circular lumen of the sperm duct became an M-shaped canal underneath which the oviduct was located (Fig. 4D). The lateral parts of the sperm duct expanded upward and elongated, and ﬁnally the sperm duct and oviduct developed, differentiated independently and separated completely (Fig. 4E). The DCGD system including the proximal end and two detached duct structures at the distal end began to appear at about 20 weeks of age. Before the ﬁrst spawning season, neither the sperm duct nor oviduct connected to the genital pore; eventually, the oviduct became a blind end, while the sperm duct continued extending toward the urinary pore during the ﬁrst spawning period (Fig. 4F). Male Reproductive Passage in the First and Second Spawning Season Spermatozoa were released to the interspace among the mature lobules in the testis during spawning season (Fig. 5A). They were ﬁrst collected in the testicular middle cavity (Fig. 5B) and then gathered within the testicular main cavity, both of which were comprised of many blood vessels enclosed by connective tissues (Fig. 5B–E). Spermatozoa arrived continuously to the proximal region of the genital duct, which resulted in swelling of the outer sperm duct in the DCGD and compression of the inner lumen of the oviduct (Fig. 5F). They passed the distal region of the genital duct system in which the sperm duct and oviduct developed independently and separated (Fig. 5G). Finally, the sperm duct opened to the genital papilla and connected to the genital pore located between the anus and the urinary pore (Fig. 5H). Regression of the Male Reproductive Passage and Formation of the Female Reproductive Passage during Sex Change The ﬁsh has bisexual gonadal tissue including dorsal ovarian tissue and ventral testicular tissue (Fig. 6A). Testicular and ovarian tissues are separated by the tunica albuginea during the ﬁrst to third year of age, before the sex change (Fig. 6A). The testicular main cavity was situated between the regressed testicular tissue, which was composed mainly of spermatogonia and an ovary with primary oocytes during the intersex periods before sex-change (Fig. 6B, C). Entering the third spawning period, some of the nonspermiating ﬁsh underwent a natural sex-change and their gonads developed into ovarian tissue. The testicular main cavity then disappeared Fig. 3. Formation of the genital duct system in 7-month-old ﬁsh. The gonadal stage was as step 3 in Fig. 9. (A) and (B) were the transections of the same gonad at different locations (cranial to caudal). (A) Paired ovarian cavities beside the testicular main cavities moved closer at the caudal region of the paired gonads. (B) Two originally separated ovarian cavities interlinked and became the oviduct. (C) Two testicular main cavities beside the ovarian cavities also extended to connect with each other and then the sperm duct encircled the oviduct. These unique structures were termed the ‘‘double cannula genital ducts’’. ABC: abdominal cavity; ADP: adipose tissue; DCGD: double cannula genital duct; ME: mesentery; OC: ovarian cavity; OD: oviduct; OT: ovarian tissue; PE: peritoneum; SD: sperm duct; TMC: testicular main cavity; TT: testicular tissue. GENITAL DUCTS IN THE PROTANDROUS BLACK PORGY Fig. 4. Development of the ‘‘double cannula genital ducts’’ (DCGD) in 7-month-old ﬁsh. The gonadal stage was as step 3 in Fig. 9. (A–F) were the transections of the same gonad at different locations (cranial to caudal). (A) The DCGD was beneath the urinary bladder. The sperm duct (SD, arrows) and oviduct (OD) became adjacent in the proximal region of the genital duct during the nonspawning season and the SD encircled the OD. (B) The DCGD invaginated into the connective tissue between the mesonephric duct and intestine. (C) The inferior wall of the OD (asterisk) intermingled with that of the SD. (D) The SD became M-shaped due to increasing conjugation with the wall of the OD. (E) 499 The two lateral lumen of the M-shaped SD extended upwards and the SD and OD developed independently, becoming two detached ducts in the distal end of the genital duct system. (F) The OD became a blind end and stopped extending toward the genital papilla before spawning season; only SD could be found in the connective tissue between mesonephric duct and intestine. ABC: abdominal cavity; ADP: adipose tissue; CT: connective tissue; DCGD: double cannula genital duct; INT: intestine; MD: mesonephrioc duct; OD: oviduct; SD: sperm duct; UB: urinary bladder. 500 LEE ET AL. Fig. 5. GENITAL DUCTS IN THE PROTANDROUS BLACK PORGY between the partially ovulated ovary containing the follicular remnants (Fig. 6D) and the residual testicular tissue in which the degenerated tubules and yellow bodies were present (Fig. 6E). After the regression of testicular tissue, the sperm duct remnants were compressed to the edge of the oviduct by the vitellogenic oocytes within ovarian lamella (Fig. 7A), and the previously continuous canal of the sperm duct (Fig. 7B) became degenerated in the walls of the oviduct during the postspawning period (Fig. 7C). The ﬁsh succeeded in changing sex during the third spawning period, and the ovarian cavity served as a female reproductive passage. The oviduct arrested as a blunt end at the distal region of the genital duct during male phase ﬁnally extended and connected to the genital pore at the papilla (Fig. 7D). DISCUSSION We applied histology to describe the bisexual gonads (Figs. 2, 6) and to interpret the unique structures of the genital duct system, that is, DCGD, characterized by having an oviduct contained within the lumen of the sperm duct, in the protandrous black porgy (Fig. 8). These structures differ markedly from the structure of the genital duct system in the gonochoristic teleosts (Redding and Patino, 2000), and these characteristics are the ﬁrst report in ﬁsh. This unique DCGD system is suggested to facilitate the reproductive readiness and successful reproduction when ﬁshes are in attendant on sex change. In gonochoristic teleosts, the sperm ducts and oviducts of the male ﬁsh and female ﬁsh are considered as an extension of testis and ovary, respectively (Redding and Patino, 2000). However, there is a paucity of literature detailing their morphological architecture and physiological functions. The conﬁguration of the gonad in the black porgy (Fig. 6A) belongs to the delimited type of germinal tissues in hermaphrodite ﬁsh (Sadovy and Shapio, 1987). A dorsoventral conﬁguration of the ovary-testis bisexual gonad before sex change (Figs. 6A–C), the separation of the ovary and testis by the tunica albuginea, and the ontogenetic progression from male to female contribute to the unique pattern characteristic of the coexistence of an outer sperm duct and inner oviduct (Fig. 8). In spite of their coexistence, the sperm duct did not open into the genital pore at the papilla between the urinary pore and anus until the ﬁrst spawning season. The oviduct also persisted within the lumen of the Fig. 5. The male reproductive passage in the ﬁrst and second spawning seasons (in 1- to 2-year-old ﬁsh). The gonadal stages of (A), (B), and (C) were as step 4, (D) as step 5, and (E–H) as step 7 in Fig. 9. (A) One of the mature lobules was broken, and spermatozoa were released to the interspace among them. (B) Spermatozoa were gathered to the testicular middle cavity where many blood vessels were capsulated by connective tissue. (C) Finally, a mass of spermatozoa and many blood vessels appeared in the testicular main cavity just beside the ovarian cavity. The ovarian cavity was still devoid of ovarian lamella during the ﬁrst spawning season. (D) Many blood vessels were in the testicular main cavity during the ﬁrst spawning season as observed with the SEM. Inset: a magniﬁed single blood vessel. (E) The ovarian tissue around the ovarian cavity had ovarian lamella with primary oocytes, and the testicular main cavity was full of spermatozoa 501 sperm duct at the proximal region but grew independently and became a blunt end at the distal region of the genital duct system. Like the sperm duct, the oviduct did not connect to the genital pore until the sex change occurred. We also found that in some ﬁsh the oocytes in the ovaries had developed to the vitellogenic stage, even while oviducts did not extend to the genital pores (unpublished data). We inferred that some unknown factors may be secreted from the tissues surrounding the ducts to induce the appropriate genital duct to extend and differentiate into functional ducts or undergo programmed cell death. Suzuki and Shibata (2004) investigated the developmental process of genital duct formation in the medaka, Oryzias latipes, and suggested that the formation of the distal genital duct arose from the ventral region of the nephric mesenchyme. We suggest that the development of the sperm duct and oviduct in the black porgy is derived from the somatic tissues of the testis and ovary, respectively. However, there is also the possibility of reciprocal impact between the nephric mesenchyme in the caudal region and that of genital ducts that proceeds with alternating regression and development of these two ducts. When black porgies undergo a sex change, the development of their reproductive systems includes not only male/female gametes (Chang et al., 1994) and their associated somatic cells but also the respective elongation and regression of the reproductive passages from the ovary and testis. During early gonadal development, the primordial gonad buds out a second branch (Fig. 1C) for the formation of the ovarian cavity (the female reproductive passage within the gonad), a pattern which is consistent with one of two patterns of ovarian development in teleosts (Liem et al., 2001). The putative ovarian and testicular tissues in the black porgy begin to differentiate at the regions around the ovarian cavity and ventral parts of the gonads, respectively at about 4–5 months of age (Lee et al., 2008; Fig. 9). After differentiation in the bisexual gonad, the ovary and testis are separated by the tunica albuginea (Fig. 2A) within which the male reproductive passage (we termed testicular main cavity) in the gonad is then formed. Spermatogenesis occurs within the cysts in the lobules constituting the testis. Spermatozoa are released from mature cysts and lobules and then are gathered into the interspace of the testis we termed the testicular middle cavity. Spermatozoa streams within the testis from the testicular middle cavity are gathered again into the testicular main cavity. during the second spawning season. (F) In the proximal end of the genital duct, the sperm duct in the ‘‘double cannula genital duct’’ was full of spermatozoa. This swelling cavity compressed the inner cavity of the oviduct. (G) In the distal region of the genital duct, the spermatozoa-ﬁlled SD was beside the mesonephric duct, and the oviduct was next to the intestine. (H) The sperm duct connected to genital papilla and opened to the genital pore adjacent to the urinary pore in 2year-old ﬁsh. A: anus; ABC: abdominal cavity; ADP: adipose tissue; BV: blood vessel;GP: genital pore; OC: ovarian cavity; MD: mesonephric duct; OD: oviduct; OT: ovarian tissue; PAL: papilla; PE: peritoneum; SC: spermatocyte; SD: sperm duct; SP: spermatogonium; ST: spermatid; SZ: spermatozoa; TDC: testicular middle cavity; TMC: testicular main cavity; TT: testicular tissue; UB: urinary bladder; UP: urinary pore. 502 LEE ET AL. Fig. 6. The regression of the testis during natural sex change in 3year-old ﬁsh. The gonadal stage of (A) was as step 6, (B) and (C) were as step 8, and (D) and (E) as step 9B in Fig. 9. (A) A delimited type of ovotestis structure included both testicular tissue and ovarian tissue in the gonad under SEM during the intersex season before sex change. The tunica albuginea within which the male reproductive passage (testicular main cavity) formed separates the ovarian tissue from testicular tissue. This image showed the structure’s dorsoventral conﬁguration in the body. (B) The testicular main cavity was located between ovary (with primary oocytes) and testis (with spermatogonia) in the ovotestis during the intersex period. Inset: magniﬁed spermatogoia in the testis. (C) Some testes in the ovotestis were apparently degenerating after the second spawning season, whereas the testicular main cavity was still present. (D) The testicular main cavity was absent and the regressed testis became residue in the ovarian wall after ovulation in the sex-changed female ﬁsh. (E) Degenerated male germ cells with yellow-brown bodies appeared in the regressed testis residues. CT: connective tissue; DCGD: double cannula genital duct; OC: ovarian cavity; OT: ovarian tissue; OW: ovarian wall; RFL: residues of follicular layer; RTT: residues of testicular tissue; SP: spermatogonium; TA: tunica albuginea; TDC: testicular middle cavity; TMC: testicular main cavity; TT: testicular tissue; YB: yellow brown bodies. GENITAL DUCTS IN THE PROTANDROUS BLACK PORGY Fig. 7. The regression of male reproductive passage and formation of the female reproductive passage during natural sex change in 3year-old ﬁsh. The gonadal stage was as step 9B in Fig. 9. (A) Abundantly vitellogenic oocytes grew toward the oviduct, which extruded the cavity of the sperm duct (arrows) to the margin of the genital duct during the spawning season. (B) Originally continuous cavities (arrows) of the sperm duct in the wall of the genital duct were still present in However, spermatozoa within the mature testis do not have the ability to swim until being spermiated out into the environment. The main vascular systems of the gonad in the black porgy were present near the ovarian tissues (Fig. 9). Small blood vessels and capillaries were distributed through every ovarian lamella (Lee et al., 2008) and cyst (Huang et al., 2002). The vascular system was revealed from partially emptied testicular tissue within the middle and main cavities (Fig. 5B–E). The space and shape of the testicular main and middle cavities were not regular. They were enlarged by a mass of spermatozoa during the spawning period and then shrunk just like cracks within the gonad during the nonspawning season. The genital duct system within the gonad of the black porgy was not observed to have deﬁnitive passage and ductal structures. That is the reason why we use ‘‘cavity’’ instead of ‘‘duct’’ (as proposed by Grier et al., 1980) to label the reproductive passages within the gonads of teleosts. In 3-year-old ﬁsh, some of the black porgies underwent natural sex changes when approaching the third spawn- 503 ﬁsh undergoing sex change. (C) Parts of the sperm duct became degenerated and replaced by connective tissue during the postspawning period. (D) The oviduct of the sex-changed ﬁsh ﬁnally extended and connected to the genital pore located at the papilla. A: anus; GP: genital pore; OD: oviduct; PAL: papilla; SL: scale; VO: vitellogenic oocytes; UP: urinary pore; WGD: wall of genital duct. Fig. 8. A schematic illustration of the anatomic structures surrounding the bisexual gonads, reproductive ducts with ‘‘double cannula genital duct’’ structure, the mesonephric duct, and other tissues in the abdominal cavity of the protandrous black porgy. A: anus; ADP: adipose tissue; CT: connective tissue; GP: genital pore; INT: intestine; MD: mesonephric duct; OC: ovarian cavity; OD: oviduct; OT: ovarian tissue; SD: sperm duct; TDC: testicular middle cavity; TMC: testicular main cavity; TT: testicular tissue; UB: urinary bladder; UP: urinary pore. 504 LEE ET AL. Fig. 9. Developmental proﬁles of the gonad and reproductive passages within the gonad during natural sex change. The developmental processes can be illustrated by the following nine steps: (1) The gonad was still undifferentiated at 6 weeks of age and budded out a branch from the base at about 14 weeks of age. (2) The gonad, in which the putative ovarian and testicular tissues had just developed, formed the ovarian cavity, which served as the female reproductive passage at about 16–20 weeks of age. (3) The connective tissue within the tunica albuginea, which surrounded the ovarian tissue, formed the testicular main cavity, which served as the male reproductive passage later in the spawning seasons, and it separated ovarian and testicular tissues during the prespawning season. (4) The swelling testicular main cavity (TMC) full of spermatozoa extruded the ovarian cavity to the margin of the gonad, in which ovarian tissue grouped as a cell nest distributed along one side opposite to the TMC during the ﬁrst spawning period. (5) Ovarian lamella started to develop and testicular tissue regressed after the ﬁrst spawning season, whereas the TMC and testicular middle cavity (TDC) gradually shrank. (6) Ovarian tissue proliferated during the intersex period but female germ cells were still at the primary oocyte stage. The TMC and TDC became regressed within the testic- ular tissue. (7) Testicular tissue proliferated again and ovarian tissue regressed before the second spawning period. Fish became functional males during the second spawning season. The TMC and TDC swelled from the mass of spermatozoa. (8) Testicular tissue regressed and ovarian tissue grew after the second spawning period. The TMC and TDC shrank. (9A) The ﬁsh remained as functional males (no sex change) when testicular tissue grew and became the functional testis during the third spawning period. (9B) Testicular tissue further regressed and became residue during the intersex period; the TMC and TDC were already absent. Primary oocytes proceeded to vitellogenesis during the prespawning period and developed into vitellogenic oocytes. These ﬁshes succeeded in changing sex during the third spawning period, and the ovarian cavity served as a female reproductive passage. BG: branched gonad; MAV: main artery and vein; MOT: mature ovarian tissue; MTT: mature testicular tissue; OC: ovarian cavity; OL: ovarian lamella; OT: ovarian tissue; PGC: primordial germ cells; PO: primary oocytes; POT: putative ovarian tissue; PTT: putative testicular tissue; RTT: residues of testicular tissue; TDC: testicular middle cavity; TMC: testicular main cavity; TT: testicular tissue; UDG: undifferentiated gonad; VO: vitellogenic oocytes. ing season. This occurred when ovarian tissues proliferated and testicular tissue degenerated (Lee et al., 2008; Wu et al., 2008). During natural sex change, tissues of the sperm duct either became totally degenerated along the genital duct or left remnants near the urinary pores. Long-term estradiol administration resulted in the development of the ovary and successful sex change (Lee et al., 2000, 2001; Du et al., 2003; Lee et al., 2004). We also observed that estradiol administration induced oviduct development but inhibited sperm duct development in the ﬁsh (unpublished data). Based on these results during early gonadal development and later sex change, we suggest that the development and regression of the male and female reproductive passages in the black porgy is dependent on that of the testicular and ovarian tissues. We also made the novel observation that a unique ‘‘ovarian cavity crack’’ structure appeared between the gonads and the genital ducts. Temporally, the ‘‘fourstranded gonad’’ structures only appeared before the ﬁrst spawning period and existed in a very small area. We suggest that the role of the ovarian cavity crack is to balance the inner pressure among the two blindended genital ducts, the abdominal cavity and the microenvironment. In conclusion, this is the ﬁrst detailed investigation of the histological characteristics and development of the genital duct system in the protandrous black porgy. Their unique DCGD structures, which include an oviduct contained within the lumen of the sperm duct in the proximal region and two detached ducts in the distal region, greatly differ from the known conﬁguration of GENITAL DUCTS IN THE PROTANDROUS BLACK PORGY the genital ducts in the gonochoristic teleosts. The dorsoventral conﬁguration of the ovary-testis bisexual gonad before sex change, the separation of ovary and testis by the tunica albugenia, and the ontogenetic expression ﬁrst as male and later as female result in the coexistence of an outer sperm duct and inner oviduct. The oviduct and sperm duct extend and develop from the ovary and testis, respectively. The success of the sex change in the protandrous porgy relies not only on the changes of alternate germ cells but also on the development of the somatic cells of the genital duct systems. 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