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Development of the Genital Duct System in the Protandrous Black Porgy Acanthopagrus schlegeli.

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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.
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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. These data provide important information to further understand how
the protandrous black porgy optimizes reproductive success through their unique genital duct system and also
sheds light on the various strategies of reproduction in
vertebrates.
ACKNOWLEDGMENTS
The authors would like to thank the electron microscopy center (NTOU, Keelung) for providing the facilities
for these experiments. The manuscript has been edited
by the American Journal Experts.
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