Male Gonadal Differentiation and the Paedomorphic Evolution of the Testis in Teleostei

Male Gonadal Differentiation and thePaedomorphic Evolution of the

Testis in TeleosteiTALITA SARAH MAZZONI,1,2 HARRY J. GRIER,3 AND

IRANI QUAGIO-GRASSIOTTO2,4*1Graduate Program on the Cell and Structural Biology, Instituto de Biologia, Unicamp,

Campinas-SP, Brasil2Departamento de Morfologia, Instituto de Biociencias de Botucatu, Unesp, Botucatu-SP,

Brasil3Florida Fish and Wildlife Research Institute, St. Petersburg, FL

4Centro de Aquicultura da Unesp, Caunesp, Jaboticabal-SP, Brasil

ABSTRACTTestis differentiation from representatives of the Otophysi (Cyprinus

carpio), Percomorpha (Amatitlania nigrofasciata), and Atherinomorpha(Poecilia reticulata) was comparatively described. In the undifferentiatedgonad of C. carpio, the primordial germ cells (PGCs) are scattered through-out the gonads while in A. nigrofasciata and P. reticulata the PGCs arerestricted to the ventral periphery. In the dorsal region of the developinggonads, with the exception of C. carpio, somatic cell rearrangements resultin the differentiation of the sperm duct. Pre-Sertoli cells wrap around sin-gle spermatogonia forming cysts that proliferate forming acinar-clusters.In C. carpio and A. nigrofasciata, the cysts in each acinar-cluster moveaway from each other, creating a central lumen. In C. carpio, the acinar-clusters then fuse to each other forming tubules that become lined by thegerminal epithelium. Subsequently, the tubules anastomose dorsally andcreate the sperm duct. In A. nigrofasciata, the acinar-clusters elongate,forming lobules that individually connect to the sperm duct. These arelined by the germinal epithelium. In P. reticulata, the spermatogonial cystsremain in the acinar-cluster organization. Subsequently, developing ductsconnect each cluster to the sperm duct and lobules subsequently develop.In the differentiated testis of C. carpio and A. nigrofasciata, spermatogoniaare distributed along the lengths of the anastomosing tubules or lobules,respectively. However, in P. reticulata, the spermatogonia remain restrictedto the terminal end of the lobules. Considering testis ontogeny, the sperma-togonial acinar-cluster is the adult characteristic of more derived taxa thatapproximate the early gonad developmental stages of the basal taxa. AnatRec, 297:1137–1162, 2014. VC 2014 Wiley Periodicals, Inc.

Key words: germinal epithelium; gonadal differentiation;Ostariophysi; Percomorpha; Atherinomorpha

Grant sponsor: Brazilian Agencies: FAPESP (Fundac~ao deApoio �a Pesquisa do Estado de S~ao Paulo); CNPq (ConselhoNacional de Desenvolvimento Cient�ıfico e Tecnol�ogico); CAPES/PROAP (Coordenac~ao de Aperfeicoamento de Pessoal de N�ıvelSuperior).

*Correspondence to: Irani Quagio-Grassiotto; Departamentode Morfologia, Instituto de Biociencias de Botucatu, IBB-

UNESP, Distrito de Rubi~ao Jr, s/n, 18618-970, Botucatu-SP,Brasil. E-mail: [email protected]

Received 20 October 2013; Revised 31 January 2014;Accepted 17 February 2014.

DOI 10.1002/ar.22915Published online 17 April 2014 in Wiley Online Library(wileyonlinelibrary.com).

THE ANATOMICAL RECORD 297:1137–1162 (2014)

VVC 2014 WILEY PERIODICALS, INC.

INTRODUCTION

In basal and derived taxa in Teleostei (see Nelson,2006—for review) the male gonads have a distinctiveinternal organization. The differences in basic organiza-tion of the germinal compartment in these groups of fishled Grier (1981) to classify the Teleostei testes in twodistinct structural types: anastomosing tubular typeand lobular type.

Teleostei testes can also be differentiated according tothe distribution of spermatogonia within the epitheliumthat lines the germinal compartment. Testes of theanastomosing tubular type have spermatogonia alongthe entire lengths of the tubules. Subsequently, they areinterpreted as being of the unrestricted type (Grieret al., 1980; Grier, 1993). On the other hand, the lobulartestes can be of the unrestricted or restricted types.In the lobular unrestricted type of testes, spermato-gonia are found along the entire length of the lobules. Incontrast, in the lobular restricted type, spermatogo-nia are restricted to the distal termini of the lobules(Grier, 1981; Grier, 1993).

A review by Parenti and Grier (2004) has analyzedthe organization of the germinal compartment in testes

from 136 species of fish. Among them, in all the 18 rep-resentatives from the basal taxa studied (including inthe Ostariophysi), testes were from the anastomosingtubular unrestricted type. In all the 118 representa-tives from the derived taxa studied (including the 32species of Percomorpha and the 79 species of Atherino-morpha) the testes were from the lobular type. In Per-comorpha the testes were from the lobularunrestricted type as in the Atherinomorpha testeswere from the lobular restricted type. Thus, as pro-posed by Grier (1993) and confirmed by Parenti andGrier (2004) in basal osteichthyans, including the Ostar-iophysi, the germinal compartment of the testis isformed by anastomosing tubules while in derived taxa,as in the Acanthopterygii, the germinal compartmenthas a lobular organization. Parenti and Grier (2004) alsoproposed the lobular testis type as a diagnostic or syna-pomorphic character of the Neoteleostei, and confirm theresults from Grier et al. (1980) that the restricted lobu-lar type of testis is diagnostic of atherinomorph fishes.

According to Fink (1981), evolution of the Teleostei ispaedomorphic because some characters observed inadults of more derived taxa approximate the early devel-opmental stages of the basal taxa. Based on Fink (1981),

Fig. 1. The gonadal primordia of C. carpio (30 dpf) (A–C), A. nigro-fasciata (10 dpf) (D–F), and P. reticulata (stage 3) (G–I). Light micros-copy. A–I: In parasagittal sections of the larva (A, D, and G), thegonadal primordia (arrow) are located ventral to the kidney (K) anddorsal to the gut (G) (B–C, E–F, H–I). B–C, E–F, and H–I: The gonadalprimordium, in parasagittal section, has a few layers of SC that sur-round the PGCs. The basophilic nuclei of the SC, and the cells them-

selves, are elongated. The PGCs appear isolated and scatteredamong the SCs. PGCs are large elliptical cells, each with a volumi-nous nucleus containing a prominent nucleolus. Ventral region (V), dor-sal region (D), muscle tissue (M). Staining: PAS/hematoxylin/metanilyellow. Scale bar 5 500 mm (A), 20 mm (B, E, and H), 10 mm (C, F, andI), 100 mm (D and G).

1138 MAZZONI ET AL.

Parenti and Grier (2004) proposed that “the evolutionarytransition from an anastomosing tubular to a lobulartestis in Teleostei could have resulted from the elonga-tion of the testis germinal compartment during earlydevelopment when testes enlarge prior to the breedingseason.” The restriction of spermatogonia to the distaltermini of the lobules in the Atherinomorpha, ashypothesized by Parenti and Grier (2004), might possi-bly be the result of the arrest of the maturation–regres-sion phases of the annual reproductive cycle, as theseoccur in Percomorpha.

The hypothesis regarding the evolutionary origin ofthe testicular types in Teleostei comes from the observa-tion of adult gonads. Despite the existence of severalstudies on gonadal development (Van Den Hurk, 1974;Davies and Takashima, 1980; Timmermans and Tav-erne, 1983; Colombo et al., 1984; Parmentier and Tim-mermans, 1985; Winkoop et al., 1992; Colombo andGrandi, 1996; Timmermans et al., 1996; Grandi andColombo, 1997; Meijide et al., 2005; Otani et al., 2005;Cek, 2006; Nakamura et al., 2006; Saito et al., 2007;Nakamura et al., 2008; Pandolfi et al., 2009; Guerrero-

Fig. 2. The undifferentiated gonads of C. carpio (60 dpf) (A,B), A.nigrofasciata (20 dpf) (C,D), and P. reticulata (stage 5) (E,F). Lightmicroscopy. A and B: In sagittal sections, the early, undifferentiatedgonad of C. carpio is very elongated, thin and has a major number ofSCs. The PGCs are surrounded by SCs. Ventral region (V), dorsalregion (D). C and D: In sagittal sections, the presumptive male gonadof A. nigrofasciata shows PGCs only at the ventral region of the gonad

and accumulation of SC in the dorsal region. Dorsal region (D). E andF: In longitudinal sections, the presumptive male gonad of P. reticulatashows PGCs at the caudal region of the gonad and SCs in the cranialregion (CR). Mitotic divisions (M) of the PGCs are intense. Blood ves-sels (BV). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 100mm (A), 10 mm (B, D, and F), and 50 mm (C and E).

MALE GONADAL DIFFERENTIATION IN FISH 1139

Est�evez and Moreno-Mendoza, 2010, 2012; Nakamuraet al., 2010; Kobayashi et al., 2011), none of them have afocus on the formation of the germinal compartment inthe different types of testes in Teleostei.

Thus, with the intent to provide additional informa-tion and foster a better understanding of formation ofthe different types of testes in Teleostei (under the lightof the paedomorphic theory), we have studied the forma-tion of the germinal compartment during gonadal devel-opment in representatives of both basal and derivedtaxa in the Teleostei, these being in the Otophysi, Cypri-nus carpio (Cypriniformes: Cyprinidae), in the Percomor-pha, Amatitlania nigrofasciata (Perciformes: Cichlidae),and in the Atherinomorpha, Poecilia reticulata (Cyprino-dontiformes: Poeciliidae). The data resulting from ourstudy are herein discussed in a comparative evolution-ary approach based on the available information oftestes differentiation between basal and derived taxa inTeleostei.

MATERIALS AND METHODS

The Specimens

Eggs, larvae, and juveniles were obtained fromspawns of adult C. carpio, A. nigrofasciata, and P. reticu-lata that were bred in tanks in S~ao Paulo State, Brazil.After hatching, part of each brood was sampled periodi-cally covering the period of histologically discernible sexdifferentiation. Larvae were fed with powdered, commer-cial food. The juveniles were fed with pelleted, commer-cial food. Fish were kept in 1,600 L tanks (C. carpio)and 300 L aquaria (A. nigrofasciata and P. reticulata).They were maintained under natural day length, andwater temperature was 28�C 6 2�C. The specimens wereanesthetized with 0.1% benzocaine and killed accordingto the institutional animal care protocols and approval(27/2007 and 175/2009-CEEA-IBB/UNESP). The wholelarvae, the medial region of the body from juveniles, andwhen anatomically discernible, the gonadal tissue werefixed by immersion in 2% glutaraldehyde and 4%

Fig. 3. The gonadal tissue from C. carpio (70 dpf) (A,B), A. nigrofas-ciata (20 dpf) (C–E), and P. reticulata (stage 5, 08 dpb and 10 dpb) (F–H). Light microscopy—Sagittal (A–D and F) and Longitudinal (E, G,and H) sections—immunohistochemical detection of PCNA. A–J: Pro-liferation of spermatogonia (G) and pre-Sertoli cell (PS) in C. carpio(A,B), A. nigrofasciata (C,E), and P. reticulata (F–H), as labeled byPCNA. Note the alternating proliferation that occurs between the sper-

matogonia and pre-Sertoli cell. C and D: SC proliferation as labeledby PCNA in the region corresponding to sperm duct in A. nigrofas-ciata. F and F-inset: SC proliferation as labeling by PCNA in theregion corresponding to sperm duct in P. reticulata. Ventral region (V),dorsal region (D), cranial region (CR), caudal region (CA). Staining:hematoxylin. Scale bar 5 50 mm (A and B), 20 mm (A-inset, B-inset, D,E-inset, F and H), 80 mm (C,E), 10 mm (F-inset and G).

1140 MAZZONI ET AL.

paraformaldehyde in Sorensen’s phosphate buffer (0.1M, pH 7.2) for at least 24 hr. Half of the samples werealso fixed in Bouin’s fluid.

Light Microscopy

For light microscopy, larvae and the gonadal tissuefrom juveniles were embedded in Historesin (Leica His-toResin). Serial sections (3 lm) were stained with hema-toxylin/eosin, periodic-acid-Schiff (PAS)/hematoxylin/metanil yellow (Quintero-Hunter et al., 1991), toluidineblue (pH 7.0), and with the reticulin method that enhan-ces basement membranes. Gonadal tissues were eval-uated by using a computerized image analyzer (LeicaLAS Interactive Measurements). The reticulin stain(modified from Puchtler and Waldrop, 1978; Vidal, 1988)uses an oxidizing agent, potassium permanganate, tooxidize aldehyde groups. Subsequently, the oxidizedaldehyde groups are detected by the deposition of posi-tive silver ions followed by their reduction using forma-lin. The result is a black hue of the reticulin fibers. Asreticulin fibers are part of basement membranes, themethod clearly detects basement membranes.

Immunohistochemistry for VASA-Protein,Proliferating Cell Nuclear Antigen (PCNA) and3Beta-Hydroxysteroid Dehydrogenase (3b-HSD)Enzyme

Larvae, juveniles, and gonadal tissues from juvenilespreviously fixed in Bouin’s fluid were prepared forimmunohistochemistry analysis. The immunohistochemi-cal techniques were taken in totum, not being dehy-drated in alcohol. The samples were washed inSorensen’s phosphate buffer (0.1 M, pH 7.2) for 1 hrbefore the start of techniques.

To block endogenous peroxidase, samples were treatedwith 3% hydrogen peroxide in Tris-buffered saline (TBS;pH 7.6) for 15 min. After blocking, antigen retrieval wasperformed using lead citrate (0.01 mol L21 and pH 6.0)for 15 min at 700 W in a microwave oven. Samples werewashed in TBS and submitted to a protein blocker (1%nonfat, powdered milk in TBS) for 15 min. Subsequently,the material was incubated with anti-fish polyclonalVASA antibody (Rhea Biotech—IM-0338) diluted 1:300,anti-mouse monoclonal PCNA antibody (Novocastra Lab-oratories, Newcastle, UK) diluted 1:300 or with anti-mouse monoclonal 3b-HSD antibody (Santa Cruz—SC-100466) diluted 1:100 for 1 hr in a sealed humiditychamber. The samples were rinsed in TBS and incubatedwith a biotinylated secondary antibody (Novostain SuperABC Kit—universal) (Novocastra—NCLABCu) for 30min, washed again in TBS, and treated with ABC rea-gent (avidin–biotin–peroxidase complex; NovostainSuper ABC Kit—universal) (Novocastra—NCL-ABCu)for 30 min. Finally, the peroxidase activity was revealedfor microscopy by using 0.05% 3.30-diaminobenzidine(DAB) in TBS (pH 7.6) containing 0.03% hydrogen per-oxide. For the negative control, primary antibody wasreplaced with TBS in some sections. The samples takenin totum were fixed by immersion in 2% glutaraldehydeand 4% paraformaldehyde in Sorensen’s phosphatebuffer (0.1 M, pH 7.2) for at least 24 hr and embeddedin historesin (LeicaHistoResin). The serial sections (5lm) were counterstained with hematoxylin, mounted,

and documented by using a computerized image ana-lyzer (Leica LAS Interactive Measurements).

During gonadal differentiation, VASA-protein detec-tion was performed to follow the germ cells migration,and PCNA detection was used to determine the time ofgerm and/or somatic cell (SC) proliferation. The detec-tion of 3b-HSD enzyme was performed to localize Leydigcell precursors.

RESULTS

The Gonadal Primordium in the Ostariophysiand Acanthopterygii

Initially the gonadal primordia from C. carpio (Ostar-iophysi: Otophysi: Cypriniformes: Cyprinidae), A. nigro-fasciata (Acanthopterygii: Percomorpha: Perciformes:Cichidae), and P. reticulata (Acanthopterygii: Atherino-morpha: Cyprinodontiformes: Poecilidae) are quite simi-lar (Fig. 1). The gonadal primordia are located dorsal tothe gut and ventrally to the developing kidney. Theyoccur in pairs attached to the peritoneum that lines thecoelomic cavity. Later they became attached to the coelo-mic cavity by the gonadal mesentery that becomes themesorchium in the differentiated gonad.

In parasagittal sections, the gonadal primordia fromC. carpio (Fig. 1A–C), A. nigrofasciata (Fig. 1D–F), andP. reticulata (Fig. 1G–I) are filiform, are elongated andvery thin. In C. carpio and A. nigrofasciata, they extend

Fig. 4. The undifferentiated gonads of C. carpio (75 dpf) (A,B), A.nigrofasciata (30 dpf) (C,D), and P. reticulata (10,dpb) (E,F). Lightmicroscopy—Sagittal sections—immunohistochemical detection ofVasa-protein. In C. carpio (A,B), A. nigrofasciata (C,D), and P. reticulata(E,F), the spermatogonia (G) are detected as being labeled by Vasa-protein. Note the different patterns of the spermatogonia that arelocated in the gonadal tissues. In C. carpio, the spermatogonia arelocated throughout gonadal tissue (A and B), whereas in A. nigrofas-ciata (C,D) and P. reticulata (E,F), the spermatogonia are located onlyin the ventral region of the gonadal tissue. Note that only SCs arelocated in the dorsal region (C–F). Ventral region (V), dorsal region (D),caudal region (CA), pre-Sertoli cells (PS). Staining: hematoxylin. Scalebar 5 60 mm (A), 40 mm (C and E), 20 mm (B, D, and F).


throughout the coelomic cavity. In P. reticulata, thegonadal primordium occupies a smaller area, beingrestricted to the posterior end of the coelomic cavity.

In all of these species, each primordium is composedby primordial germ cells (PGCs) and SCs (Fig. 1C,F,I).

The Undifferentiated Gonad in theOstariophysi and Acanthopterygii

The gonadal primordia develop and become undiffer-entiated gonads (Fig. 2). As cell proliferation continues,the first blood vessels appear within the gonadal tissue(Fig. 2A–D). Both, proliferating germ and SCs areresponsive to the PCNA method (Fig. 3). However, theirlabeling is not simultaneous. In other words, when thegerm cells are labeled the SCs are not and vice-versa. Inthe undifferentiated gonad, PGCs migration begins;their identity is confirmed by labeling for Vasa-protein(Fig. 4).

In C. carpio and A. nigrofasciata the undifferentiatedgonads were observed in juveniles 60 and 20 days post-fertilization (dpf), respectively. They were very long andthin, extending along the entire coelomic cavity from theurogenital papillae to the cranial region of the cavity.

In C. carpio, the undifferentiated gonads are formedby a few individual PGCs that are scattered among SCs(Fig. 2A,B). In A. nigrofasciata, the male and femalegonad tissues are distinctive because of the arrangementof the PGCs. In parasagittal sections of the presumptive

testis, the PGCs remain concentrated near the peripheryof the gonadal tissue at the ventral edge while a largenumber of SCs accumulate in the dorsal region (Fig.2C,D).

In P. reticulata, the undifferentiated gonads wereobserved in animals between 13 and 24 dpf correspond-ing to the developmental embryonic stages 3 and 4(sensu Meffe, 1985). These undifferentiated gonads havean oval and/or elongated shape, occupying the caudalend of the coelomic cavity (Fig. 2E). They are formed bysomatic and PGCs (Fig. 2F). In parasagittal and longitu-dinal sections from the presumptive testis, two distinctregions were detected in the gonadal tissue. In the cau-dal region, near the urogenital papillae, only SCs exist(Fig. 2F), whereas the remainder of the developinggonad has PGCs that are associated with SCs (Fig. 2F).

The Sexual Discernment of the Gonad

The gonads of C. carpio, A. nigrofasciata, and P. retic-ulata differentiate directly into an ovary or testis. There-fore, they have a direct gonochoristic development, withovarian differentiation preceding testiculardifferentiation.

Gonadal differentiation in C. carpio. For-mation of the testicular tubules and the germinal epi-thelium. In C. carpio, the differentiating male gonadsare observed between 75 and 85 dpf. During this period,

Fig. 5. Differentiated male gonad from C. carpio. Light micros-copy—Sagittal sections. A–C: With the proliferation of the PGCs andSCs, the gonad enlarges, and the PGCs form continuous cords (CC)that are surrounded by SCs. The PGC have a voluminous nucleus (N)with one or two nucleoli (NU), and nuages (arrowhead) close to thenuclear envelope (75 dpf). D–F: The PGCs and SCs differentiate and

become spermatogonia (G) and pre-Sertoli cells (PS), respectively.Throughout the gonad, relocation of spermatogonia forms acinarstructures (A) (85 dpf). F: The acinar structures are formed by sperma-togonia (G) and pre-Sertoli cells (PS) that are surrounded by SCs. Dor-sal region (D). Staining: PAS/hematoxylin/metanil yellow. Scalebar 5 100 mm (A and D), 20 mm (B and E), 10 mm (C and F).

1142 MAZZONI ET AL.

compact and linear structures, the continuous cords, areformed as a result of the proliferating PGCs. Thus, thePGCs proliferation, encompassed by SCs, become organ-ized into continuous cords that run parallel to thecaudo-rostral axis of the gonad (Fig. 5A–C). A basementmembrane starts to be synthesized surround the sper-matogonial cords (Fig. 7A,B). The basement membraneis initially thin and separates the interstitial and germi-nal compartments of the male gonad from one another.

In the developing testes, germ cell proliferation (fromthe now recognized spermatogonia) accompanied by theloss of the continuous cord organization, results inspherical cell clusters, having an acinar appearance

(Fig. 5D–F). Within the clusters, each spermatogoniumis surrounded by one or more pre-Sertoli cells, resultingin formation of cysts (Fig. 5F). The acinar-clusters ofspermatogonia that are scattered throughout the gonadalso become partially surrounded by the basement mem-brane (Fig. 7C–E). Around 85 dpf, numerous cysts con-taining a single spermatogonium within the acinarstructures are found throughout the developing gonadaltissue.

In each cluster, the cysts containing a single sperma-togonium move away from each other, giving rise to asmall space in the center of the acinar structure (Fig.6A,B). Neighboring clusters develop anastomoses with

Fig. 6. Differentiated male gonad from C. carpio. Light micros-copy—Sagittal sections. A: General view of the gonad (90 dpf). B andC: Details of A, showing acinar structures formed by cysts of sperma-togonia (G) that are wrapped by Sertoli cells (S). B: In the core of theacinus, the cysts move away from each other, forming a central space(*). After the fusion of two adjacent acini, the space becomes largerforming the lumen (LU) of the testicular tubules. D: General view of agonad in which the germinal epithelium is established (93 dpf). E-G:

Details of D showing the formation of new testicular tubules. Meta-phase of a spermatogonium (M). H: General view of the testis (95 dpf).I–K: Details of H showing the anastomosing tubules with a largelumen. A region of anastomosis between two tubules (arrow) is indi-cated in each micrograph. Ventral region (V), dorsal region (D), caudalregion (CA). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 100mm (A, D, and H), 10 mm (B, C, F, G, H, I, and J), and 20 mm (E).


one another (Fig. 6C–G). The anastomosis occurs in theregions devoid of basement membrane (Fig. 7F).

As a result of the fusion, the space within the clustersbecomes continuous (Fig. 6H), resulting in the develop-ment of cylindrical, hollow, and not compact structuresthat give rise to short tubules (Fig. 6I). The anastomosesprogress (Fig. 6J), and the testicular tubules become

larger and continue to anastomose with one another(Fig. 6K). These become surrounded by the same contin-uous basement membrane (Fig. 7G,H). In the dorsalregion of the testis, the testicular tubules anastomoseand form the sperm duct. The germinal compartment ofthe testis is now complete. It has an anastomosing tubu-lar structure in which the lumen is unique and

Fig. 7. The basement membrane and the formation of the germinalepithelium in C. carpio. Light microscopy—Sagittal sections—Reticulinmethod. A and B: In the undifferentiated gonad, the continuous cords(CC) of spermatogonia (G) are delimited by SCs that rest upon a base-ment membrane (arrow). Spermatogonia inside the cords are wrappedby pre-Sertoli cells (PS) and form cysts (75 dpf). C: Reorganization ofthe cysts into acinar structures (A) which are delimited by a basementmembrane (arrow) (85 dpf). D and E: Cysts containing spermatogoniaorganized as continuous cords (CC) and as acinar structures (A), bothdelimited by the basement membrane (arrow). The first spaces areobserved within the acinus (*) (90 dpf). F: Two adjacent acini sur-rounded by a continuous basement membrane (arrow). Note theabsence of a basement membrane within the acinus (90 dpf). G: Gen-

eral view of the gonad with germinal epithelium established (95 dpf).H: Detail of G, showing the basement membrane surrounding severaltesticular tubules (arrow). Note the regions of the anastomosesbetween adjacent tubules (arrowhead) and formation of the lumen (*).I: General view of the gonad in early spermatogenesis, showing thebasement membrane (arrow) surrounding several testicular tubules(TU) (130 dpf). J and K: Detail of I showing the anastomosing tubules(TA) bordered by the germinal epithelium that rests upon the basementmembrane (arrow). Note the absence of a basement membrane in theanastomosing regions (arrowhead). Blood vessel (BV), spermatocyte(C), ventral region (V), dorsal region (D). Scale bar 5 50 mm (A, D, andJ), 10 mm (B, C, E, F, and H), 100 mm (I), and 20 mm (K).

1144 MAZZONI ET AL.

continuous. The tubules are lined by a continuous germi-nal epithelium that has been formed by cysts containingsingle spermatogonia (Fig. 6K). The basement mem-brane surrounds the anastomosing tubules (Fig. 7I–K),and it is continuous with the basement membrane of thesperm duct.

The testis of C. carpio assumes its final structure. Itis a testis of the Anastomosing Tubular Type with anUnrestricted Spermatogonial Distribution (sensu Grieret al., 1980; Grier, 1981). In the caudal region of eachtestis, the testicular ducts converge into a single gono-duct that is continuous with the urogenital papillae.

The spermatogenesis. Spermatogenesis begins inmale C. carpio, 95 dpf. At this time, the cysts contain onlyspermatogonia or spermatocytes, and the lumina of thetesticular tubules do not contain any sperm (Fig. 8A–D).

In fishes that are 130 dpf, meiotic activity in thedeveloping testes is intense, resulting in a tremendousincrease of the number of cysts (Fig. 8E,F). By 150 dpf,

spermiogenesis begins (Fig. 9A–C), and spermatozoa arereleased into the testicular lumen (Fig. 9C).

In animals older than 180 dpf, there is a markedincrease in the length and the thickness of the testes(Fig. 9D). Cysts containing spermatogonia, spermato-cytes or spermatids occur in a moderate amount, andthe testicular lumen becomes progressively filled byspermatozoa (Fig. 9D–F).

After an intense spermiogenic period (Fig. 9G–I), at360 dpf, with the opening of the cysts and the releasingof the spermatozoa (spermiation), the animal is able toreproduce (Fig. 10). The anastomosing tubules are fre-quently observed to run parallel to the surface of the tes-tis (Fig. 10D,E), a characteristic of anastomosing tubulartestes in fishes wherein the tubules may also form loopsat the testis periphery (Fig. 9D–F).

Gonadal differentiation in A. nigrofasciata.Formation of the testicular lobules and the germinalepithelium. In A. nigrofasciata, testicular differentia-tion occurs about 90 dpf. By this time, as a result of anintense period of germ and SC proliferation, the testisbecomes thicker. In parasagittal sections, the ventraland dorsal regions of gonadal tissue have a distinctiveorganization (Fig. 12A,B). The PGCs have now becomespermatogonia and are mainly concentrated in theventral-region of the gonad while a few layers of thesquamous SCs are organized in the dorsal region (Fig.12A,B).

Spermatogonia (Fig. 12C,D) are always associatedwith SCs (pre-Sertoli cells), forming continuous cords atthe ventral periphery of the gonadal tissue (Fig. 12B).

In animals at 100 dpf, two adjacent layers of SCsmove away from one another forming a small space inthe dorsal region of the gonad (Fig. 12D). This is thebeginning of sperm duct formation (Fig. 12E). At thistime, the basement membrane begins to be synthesizedin the dorsal region of the gonadal tissue around thesperm duct (Fig. 13A).

Between 110 and 120 dpf, in the dorsal region of thetestis, the blood vessels become more evident. Germ cellproliferation intensifies; the cysts (containing individualspermatogonia) lose the continuous cord organizationbecoming organized in cell clusters having an acinarappearance (Fig. 12C–F). The acinar structures arelocated peripherally in the ventro-lateral region of thegonadal tissue (Fig. 12C–F).

Spermatogonia proliferation continues forming newacinar structures from the periphery toward the centralregion of the gonad. The acinar clusters of spermatogo-nia develop throughout the gonad, except in the dorsalregion where the sperm duct is located (Fig. 12C–H).

From the center of each cluster, the cysts containingsingle spermatogonia move away from each other, givingrise to a small space in the core of the acinar structure(Fig. 12G,I). A discreet basement membrane is synthe-sized around the acinar-clusters of spermatogonia (Fig.13B). At the same time, each one of the acinar-clustersof spermatogonia elongates toward the dorsal region ofthe gonad. Elongation of the acinar-clusters involvesgerm and SC proliferation. Basement membrane synthe-sis coincides with the elongation of the acinar-clustersstructures (Fig. 13C). This morphogenic change is thebeginning of testicular lobule formation (sensu Grier,

Fig. 8. Differentiating testis from C. carpio and the start of the sper-matogenesis. Light microscopy—Sagittal sections. A and B: Germinalepithelium mainly formed by spermatogonia (G), Sertoli cells and thetesticular lumen (LU) that lacks sperm (95 dpf). Blood vessel (BV). C–F: Early spermatogenic gonad (C,D: 110 dpf; E,F: 130 dpf). Note theincreasing number of spermatocytes (C), beside the large number ofspermatogonia (G) and the testicular lumen (LU) without sperm. Bloodvessel (BV), testicular tubule (TU), ventral region (V), dorsal region (D).Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 100 mm (A, C,and E), 20 mm (B, D, and F).


1981). The forming testicular lobules elongate andbecome more oval in shape (Fig. 12J,K). In the dorsalregion of the testis, each one of the elongating testicular

lobules connects to the sperm duct and the basementmembrane becomes a continuous structure from the dis-tal termini of the lobules to the sperm duct (Fig. 13D).

Fig. 9. Differentiated spermatogenic testis from C. carpio. Lightmicroscopy—Sagittal sections. A–C: Germinal epithelium is continu-ous, and the first spermatozoa (Z) are produced (150 dpf). D–F: Anas-tomoses (TA) become more intense, mainly in the dorsal region of thegonad, giving rise to the sperm duct (DU). There is a moderateamount of spermatozoa (Z) in testicular lumen (180 dpf). G–I: thesperm duct (DU) becomes larger and the germinal epithelium (GE)

shows continuous (H) and discontinuous (I) regions. There is a largeamount of the spermatozoa (Z) in testicular lumen (250 dpf). Sperma-tocyte (C), spermatid (T), blood vessel (BV), testicular tubule (TU),interstitial compartment (I), Ventral region (V), dorsal region (D), caudalregion (CA). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 250mm (A, D, and G), 20 mm (B, F, and I), 10 mm (C), 50 mm (E and H).

1146 MAZZONI ET AL.

The testicular lobules have a digitiform shape withthe blind, distal termini localized at the gonadal periph-ery (Fig. 12K). They extend from the ventro-lateral tothe dorsal region of the gonad and are lined by a contin-uous epithelium formed by cysts containing single sper-matogonia. Although the cysts containing singlespermatogonia are distributed throughout the germinalepithelium, they are primarily localized within the blindtermini of the lobules. At the blind termini of thelobules, the cysts of spermatogonia retain their acinarorganization (Fig. 12K).

The testis of A. nigrofasciata assumes its final struc-ture as a testis of the Lobular Type with an UnrestrictedSpermatogonial Distribution, that is, being located alongthe lengths of the germinal epithelia lining each of thetesticular lobules (sensu Grier, 1981). The basementmembrane lining the lobules becomes conspicuous and isintensely stained by the reticulin stain (Fig. 13E–H).However, it remains discreet in the blind distal terminiof the lobules at the peripheral region of the testis (Fig.13E). In the caudal region of each one of the testes, thesperm ducts converge to form a single gonoduct that iscontinuous with the urogenital papillae. The basementmembrane forms a continuous structure around eachtesticular lobule and the sperm duct, segregating thegerminal compartment from the interstitial compart-ment (Fig. 13H,I).

The spermatogenesis. The beginning of spermato-genesis occurs in fishes about 120 dpf and is signified bythe appearance of the first cysts containing spermato-cytes at the medial region of the testis (Fig. 14A–D).

Between 130 and 150 dpf, the spermatogenic processintensifies resulting in an increase of the number ofcysts containing spermatocytes. Consequently, the size of

the testis increases as the lobules increase in length(Fig. 14E–G). Spermiogenesis starts when the first cystscontaining spermatids appear in the germinal epithe-lium. Cysts containing single spermatogonia are alsofound throughout the epithelium of the testicular lobuleamong other cysts containing spermatocytes and sper-matids (Fig. 15A–D). In animals at 150 dpf, spermiogen-esis is completed and spermatozoa are released into thetesticular lumen (Fig. 15). Each lobule is a distinctivestructure that is outlined by reticulin staining of thebasement membrane (Fig. 15E–H) that separates it fromthe interstitium. The lobules may also branch (Fig.15G), but all end blindly at the testis periphery. The ani-mals are able to reproduce (Fig. 15).

Gonadal differentiation in Poecilia reticulata.Formation of the testicular lobules and the germinalepithelium. In P. reticulata, testicular differentiationoccurs in animals at stage 5 (sensu Meffe, 1985) that cor-responds to 1 week before birth. At this time, thegonadal tissue is composed of SCs and PGCs. The latercells are localized at the periphery on the ventral regionof the differentiating testis. PGCs proliferate along withthe SCs. Proliferation of the PGCs results in the forma-tion of clusters of germ cells. In these clusters, the germcells, now recognized as spermatogonia (Fig. 17A), areindividually surrounded by cytoplasmic projections ofthe SCs, the pre-Sertoli cells (Fig. 17B). Spermatogoniaremain clustered at the periphery of the ventral regionof the gonad where they have an acinar shape. At thistime, the SCs that are localized at the dorsal region ofthe gonad proliferate (Fig. 17C,D). Until birth, the pairof differentiating testes remains completely separatedfrom one another.

Fig. 10. The testis from an adult, sexually active C. carpio (360 dpf).Light microscopy—Cross sections. A: General view of the gonadshowing the region of the sperm duct (DU) and the testicular tubules.B: Detail from A showing anastomosing tubules (TA). C: testiculartubules (TU) bordered by germinal epithelium (GE) which is formedonly by cysts containing spermatogonia (G) that are wrapped by Ser-toli cells. D: General view of the gonad showing the germinal epithe-

lium (GE) and the region of the sperm duct (DU), both supported bythe basement membrane (arrow). E and F: Details of D showing anas-tomosing tubules (TA) surrounded by a continuous basement mem-brane (arrow), which separates the germinal and the interstitialcompartments (I). Ventral region (V). Staining: PAS/hematoxylin/metanilyellow (A–C), Reticulin method (D–F). Scale bar 5 500 mm (A), 100 mm(B and F), 50 mm (C and E), and 300 mm (D).


The formation of the basement membrane and conse-quent segregation of the germinal and interstitial com-ponents of the testes can be detected from the beginningof the gonadal differentiation. Thus, the basement mem-brane is present around SCs that will give rise to thesperm duct (Fig. 19A).

After birth, spermatogonial proliferation intensifies aswell as the proliferation of the SCs at the dorsal regionof the gonad. During the period between 01 and 12 days

post-birth (dpb), the gonadal tissue increases in lengthand thickness. Then, the differentiating testes (Fig. 18A)become closer together and fuse medially, giving rise toa single testis with oval shape (Fig. 18A–E). Internally,the testes remain individualized.

At 12 dpb, the squamous SCs in the dorsal region ofthe gonad become columnar and organized in two paral-lel layers that extend throughout the total length of thetestis (Fig. 18D,E). The parallel layer of cells move away

Fig. 11. Schema showing the formation of germinal compartmentduring male gonadal differentiation in the Otophysi, C. carpio. A:Gonadal primordium. B,C: Undifferentiated gonad, with developingcontinuous cords of PGCs (C). D–F: Testicular differentiation. Note theformation of acinar structures throughout the gonadal tissue (D). E:Two adjacent acini surrounded by a basement membrane. F: Begin-ning of the formation of the testicular tubules. Note the anastomosisbetween two adjacent acini that become surrounded by a continuousbasement membrane. The germinal epithelium is established (F, F-inset). G: beginning of spermatogenesis. G-inset: Details of the ger-minal compartment, showing cysts containing gametic cells in distinct

steps of differentiation and the first production of sperm. H: Testicularstructure completely differentiated as it is found in the adult testis. H-inset: Details of the germinal and interstitial compartment, separatedfrom each other by a basement membrane. PGCs, SC, basementmembrane (BM), interstitial compartment (I), continuous cords ofPGCs (CC), acinus (A), pre-Sertoli cell (PS), spermatogonia (G), testic-ular tubule (TU), testicular lumen (LU), Sertoli cell (S), anastomosingtubules (TA), spermatocyte (C), spermatids (T), spermatozoa (Z), Ley-dig cell (LE), blood vessel (BV), metaphase (M), sperm duct (DU), andDorsal region (D).

1148 MAZZONI ET AL.

Fig. 12. Early differentiated male gonad from A. nigrofasciata. Lightmicroscopy—Sagittal sections. A and B: With the proliferation of theSC, the gonad enlarges. The PGCs and SCs differentiate becomingspermatogonia (G) and pre-Sertoli cells (PS), respectively. With theproliferation, in the ventral region of the gonadal tissue, spermatogonia(G) form continuous cords (CC) that are surrounded by SCs, formingcysts (90 dpf). C–F: In the ventral region of the gonad, the reorganiza-tion of the cysts containing spermatogonia (G) gives rise to acinarstructures (A) (100 dpf). Blood vessel (BV). D and E: The SCs in thedorsal region of the gonad reorganize (D) forming the sperm duct(DU). F: The acinar structures are formed by spermatogonia (G)wrapped by pre-Sertoli cells (PS), and externally surrounded by SCs.

G–I: In the dorsal region, the formation of the sperm duct (DU) contin-ues. In acinar structures (A) at the ventral region, the cysts containingspermatogonia (A) reorganize (110 dpf). I: Within the acini, the cystswith single spermatogonia move away from each other giving rise to acentral space (*). J and K: The acinus, which has a central lumen (*),becomes elongated and connects to the sperm duct, forming the firsttesticular lobules (LO). In the distal termini of the testicular lobule, theacinar structures (A) are maintained (115 dpf). Ventral region (V), dorsalregion (D), cranial region (CR), caudal region (CA). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 100 mm (A), 20 mm (B, D, andE), 40 mm (C, G, and J), and 10 mm (F, H, I, and K).

Fig. 13. The basement membrane and the formation of the germinalepithelium in A. nigrofasciata. Light microscopy—Sagittal sections—Reticulin method. A: Initially, the basement membrane (arrow) is pres-ent in the region corresponding to the future sperm duct (DU). Notethat the acinar structure is not completely surrounded by a basementmembrane (arrow) (100 dpf). Spermatogonia (G). B and C: With cellproliferation, the acini (A) become larger and the basement membrane(arrow) gradually surrounds each one of them. Note the basementmembrane of the sperm duct (DU) (105 and 110 dpf). Spermatogonia(G), pre-Sertoli cell (PS), blood vessel (BV). D: Gradually, the basementmembrane (arrow) from the sperm duct (DU) becomes continuouswith the basement membrane (arrow) from the testicular lobules (LO)(115 dpf). E–G: The pattern of gonadal structure remains after theentrance of spermatogonia enter into meiosis (E: 120 dpf). With the

beginning of spermatogenesis, the lobules become more elongatedand completely surrounded by basement membrane (arrow) (F,G: 125dpf). Duct (DU), lobule (LO), acinus (A), spermatocytes (C). H: Generalview of the gonad when the testicular lobules (LO) have completelydeveloped. The basement membrane becomes a continuous structuresurrounding the sperm duct (DU) and the lobules, including the distaltermini, in which the acinus (A) are localized (140 dpf). I: Detail of Hshowing the testicular lobules bordered by the germinal epithelium.Now, the germinal epithelium is supported by a basement membrane(arrow) formed by cysts containing of spermatogonia (G), or spermato-cytes (C) or spermatids. Acinus (A), spermatozoa (Z), ventral region(V), dorsal region (D). Scale bar 5 10 mm (A, B, and C), 20 mm (D, G,and I), 30 mm (E), 50 mm (F), and 100 mm (H).

from each other and a space is created between them.This space will give rise to the sperm duct, in the dorsalregion of the testis (Fig. 18E). Gradually, a basementmembrane is synthesized by the pre-Sertoli cells thatsurround the acinar-peripheral clusters of spermatogo-nia (Fig. 19B). The synthesis progresses in a direction tothe dorsal region of the gonad delineating a sacculatestructure (Fig. 19B).

Several extensions protrude from the forming spermduct toward the acinar-clusters of spermatogonia (Fig.18B,C). At the same time, a reallocation of SCs, accumu-lated around the acinar-clusters, gives rise to small lobu-lar extensions (Fig. 18B,C). These extend toward thesperm duct. Thus, a lobular connection (a secondaryduct) is established between the acinar-clusters of sper-matogonia and the sperm duct (Fig. 18C). From now on,in each testis, the individual lobules (containing theacinar-clusters of spermatogonia in their distal termini)are connected to the sperm duct. The basement

membrane that surrounds each testicular lobule is con-tinuous with the basement membrane of the secondaryduct and of the sperm duct (Fig. 19C,D). Each one ofthese lobules becomes individually delimited by a base-ment membrane (Fig. 19D). The sperm duct of each ofthe fused testes converges caudally to form a single gon-oduct that it is continuous with the urogenital papillae.

The testis of P. reticulata then assumes its final struc-ture. It is a testis of the Lobular Type with a RestrictedSpermatogonial Distribution (sensu Grier et al., 1980;Grier, 1981).

The spermatogenesis. Spermatogenesis in P. reticu-lata starts in animals at 14 dpb, the time at which thetestis structure is established (Fig. 20A,B). At the blinddistal termini of each testicular lobule, a single primaryspermatogonium that is enveloped by the cytoplasmicprocesses of Sertoli cells, enters into mitosis, formingcysts of secondary spermatogonia. Inside the cyst, thesecondary spermatogonia, after a number of mitotic

Fig. 14. Differentiating testis and the start of the spermatogenesis inA. nigrofasciata. Light microscopy—Sagittal sections. A–D: Early sper-matogenic gonad (120 dpf). The testicular lumen (LU) and duct (DU)are without sperm. The germinal epithelium in the testicular lobuleshas spermatocysts containing spermatogonia (G) or spermatocytes(C). Testicular lobule (LO), Sertoli cell (S). E–G: Germ cells in the ger-minal epithelium are primarily spermatogonia (G); the germinal epithe-

lium is continuous in the testicular lobule (LO). The first spermatozoa(Z) are produced. Spermatogonia (G) primarily accumulate in the aci-nar structures (A) at the distal termini of the testicular lobules (130dpf). Sertoli cell (S). Dorsal region (D). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 60 mm (A and E), 50 mm (B), 20 mm (C andF), and 10 mm (D and G).


cycles, enter into meiosis resulting in a cyst of the sper-matocytes (Fig. 20A,B). The cyst containing spermato-cytes is larger and occupies the entire width of thetesticular lobule.

The spermatogenesis process intensifies, and newcysts are produced from the distal termini of the lobuleswhere the spermatogonia remain restricted (Fig. 20B).Concurrent with the formation of the cysts, the Sertolicells proliferate. With the production of new cysts andtheir entrance into meiosis, the testicular lobules elon-gate (Fig. 20B). Between 14 and 18 dpb, the cysts con-taining spermatocytes increase in number (Fig. 20C).With this increasing number of cysts, the testis enlargesbecoming more oval and less elongated (Fig. 20C). Insideeach one of the lobules, the cysts are arranged in a rowaccording to the order in which they were produced.Consequently the cysts that were first produced arethose that are closer to the sperm duct (Fig. 20D). Eachone of the cysts is supported by the cytoplasmic projec-tions from the Sertoli cells, which rest on the basementmembrane of the lobule wall (Fig. 19F,G). However,between the cysts within the same lobule there is no abasement membrane (Fig. 19G).

After 18 dpb, the first cysts with spermatids appear(Fig. 20E). Spermiogenesis starts within the cysts thatwere produced first. With this, the cysts of spermatidsare closer to the sperm duct, while the cysts of the sper-matocytes occupy an intermediate position in the testicu-lar lobules (Fig. 20E,F). Cysts containing spermatogoniacontinue to be produced at the blind termini of thelobules (Fig. 20F).

Beginning at 26 through 30 days and extending until80 dpb the number of cysts containing spermatidsincreases. At this time, a glycoprotein secretion (positivein the PAS and eosin stain) is produced and fills thelumina of the testicular ducts.

At 90 dpb (Figs. 21C,H and 22), spermiogenesis iscompleted (Fig. 21A,B), and the cysts open releasingthe first spermatozoa into the lumina of the ducts(Figs. 21C and 22). With the opening of the cysts, thesquamous Sertoli cells gradually become columnar,becoming part of the duct epithelium (Figs. 21D and22D,E).

Sperm of the P. reticulata are organized into sphericalpackets, known as spermatozeugmata (Fig. 21H). In thespermatozeugmata, the elongate nuclei of the

Fig. 15. Differentiated spermatogenic testes of A. nigrofasciata (150dpf). Light microscopy—Longitudinal sections. A: General view of thegonad showing the region of the testicular lobules (LO) and the spermduct (DU). B: Detail of A showing the sperm duct (DU) filled with sper-matozoa. Note the acinar structures (A) at the ventral region of thegonad. C: Testicular lobules (LO) bordered by the germinal epitheliumand filled with spermatozoa (Z). Note the presence of acinar structures(A) at the distal termini of the lobules. D: Detail of the distal termini ofthe lobule showing the acinar structure formed by cysts containingspermatogonia (G) that are associated with Sertoli cells (S). The germi-

nal epithelium has cysts containing spermatogonia, spermatocytes (C)and spermatids (T). Spermatozoa (Z). E: General view of the gonad,showing the region of the sperm duct (DU) and the testicular lobules(LO). F and G: Details of E showing the testicular lobules (LO) sur-rounded by the basement membrane (arrow), which segregates thegerminal from the interstitial compartment. H: Testicular lobule (LO)bordered by germinal epithelium (GE) that rests on the basementmembrane (arrow). Cranial region (CR). Staining: PAS/hematoxylin/metanil yellow (A–D), Reticulin method (E–H). Scale bar 5 300 mm (Aand E), 100 mm (B and F), 40 mm (C and G), and 10 mm (D and H).

1152 MAZZONI ET AL.

spermatozoa are circumferentially aligned while theflagella are arranged in the central region (Fig. 21H).During spermatogenesis and production of spermatozoa,

the basement membrane surrounding the lobulesremains continuous with the testicular ducts (Fig. 22F–J) as well as in the gonads of fish able to reproduce.

Fig. 16. Schema showing the formation of the germinal compart-ment during the male gonadal differentiation in the percomorph, A.nigrofasciata. A: Gonadal primordium. B: Undifferentiated gonad withPGCs located at the ventral region. C: Differentiating gonad with theformation of continuous cords by the PGCs that remain at the ventralregion. Note the reorganization of SCs at the dorsal region of thegonad giving rise to the testicular duct. D: Formation of acinar struc-tures at the ventral region of the gonad by cysts containing spermato-gonia wrapped by the Sertoli cells. Note the formation of thebasement membrane around each acinus. E-F: Formation of the tes-ticular lobules. E: Elongation of the acinar structures and the connec-tion to the testicular duct. The germinal epithelium is established (E-inset), it is composed by cysts containing spermatogonia resting on abasement membrane. F: The formation of the testicular lobules com-

pleted. Now the testicular lumen is connected to testicular duct. G:Testicular structure completely differentiated as it is found in the adulttestis. Spermatogenesis has started. G-inset: Testicular lobules show-ing details of the germinal and interstitial compartment, separatedfrom one another by a basement membrane. In the germinal epithe-lium, the cysts contain gametic cells in distinct steps of differentiation,and the production of sperm begins. Note the spermatogonia in theacinar structure at the termini of the lobules and also randomly distrib-uted along of them. PGCs, SC, basement membrane (BM), interstitialcompartment (I), continuous cords of PGCs (CC), acinus (A), pre-Sertoli cell (PS), spermatogonia (G), testicular lobule (LO), testicularlumen (LU), Sertoli cell (S), spermatocyte (C), spermatids (T), sperma-tozoa (Z), Leydig cell (LE), blood vessel (BV), metaphase (M), espermduct (DU), Ventral region (V), dorsal region (D).


The Leydig cells

In all the species studied, C. carpio, A. nigrofasciata,and P. reticulata, beginning with the formation of thespermatogonial, acinar-clusters, and throughout gonadaldifferentiation, SCs close to the blood vessels express 3b-HSD enzymes. These cells, the presumptive Leydig cellsprecursors, are located in the interstitial compartmentand express the 3b-HSD enzymes (Fig. 24).

A summary and scheme of the gonadalmorphogenesis and testis differentiation

The cellular events that occur during gonadal morpho-genesis in C. carpio, A. nigrofasciata, and P. reticulataare summarized and can be appreciated in the schemesof the Figures 11, 16, and 23, respectively.

DISCUSSION

The gonadal primordia and theundifferentiated gonads

The formation of the gonadal primordium is a con-served process. In the species herein studied, it does notdiffer from the available information on basal or derivedtaxa in Teleostei (Satoh, 1974; Hamaguchi, 1982; Tim-mermans and Taverne, 1989; Winkoop et al., 1992;

Flores and Burns, 1993; Parmentier and Timmermans,1985; Timmermans et al., 1996; Nakamura et al., 2006;Saito et al., 2007; Nakamura et al., 2008; Guerrero-Est�evez and Moreno-Mendoza, 2010, 2012; Mazzoniet al., 2010). In all species, the gonadal primordium iscomposed of a few larger PGCs that are scattered amongsquamous SCs located in the mesothelium of gonadalridges (Devlin and Nagahama, 2002 for review). Gonadalprimordia are paired structures that should give rise totwo individual testes. However, as reported here, inderived taxa in which viviparity has evolved, as in P.reticulata, the gonadal primordia may fuse to oneanother giving rise to a single gonad in the adult. How-ever, the testes remain individualized internally. Thisseems to be a recurrent morphological pattern in differ-ent viviparous fishes. For examples, see the reports onother viviparous Atherinomorpha, Mollienisia latipinna(Van Den Hurk, 1974), P. reticulata (Cyprinodontiformes:Poeciliidae) (Nagahama, 1983; Grove and Wourms, 1994;Potter and Kramer, 2000; present study), Chapalichthysencaustus (Cyprinodontiformes: Goodeidae) (Guerrero-Est�evez and Moreno-Mendoza, 2012) and Percomorpha,Zoarces viviparus (Perciformes: Zoarcidae: Zoarcinae)(Rasmussen et al., 2006).

Intense cell proliferation signals the transition fromthe gonadal primordia to the undifferentiated gonad(Winkoop et al., 1992; Hliwa et al., 2003; present study).

Fig. 17. Early differentiated male gonad from P. reticulata. Lightmicroscopy—Longitudinal (A) and sagittal sections (B–D). A: Thegonadal tissue is composed of acinar structures (A) in the cranialregion and only by SCs in the caudal region (early stage 5). Spermato-gonia (G). B: As the gonad enlarges, the acinar structures (A) occupythe ventral region as only SCs are found in the dorsal region of the

gonad. The acini are formed by spermatogonia (G) associated withpre-Sertoli cells (PS) (early stage 5). C and D: With the proliferation ofthe SCs and spermatogonia (G), the gonad enlarges (late stage 5).Acinar structure (A), dorsal region (D), cranial region (CR), caudalregion (CA). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 20mm (A and D), 10 mm (B and C).

1154 MAZZONI ET AL.

As in C. carpio or A. nigrofasciata or P. reticulata, thePCNA method clearly demonstrates that germ and SCproliferation occurs in alternate cycles. This alternationof proliferation cycles continues to occur from the undif-ferentiating to the differentiated gonad mainly related tothe pre-Sertoli cells. Alternating proliferation of thegerm and Sertoli cells remains in the adult male gonads(Lo Nostro et al., 2003; Schulz et al., 2005).

The gonadal differentiation and the earliestsexual discernment

In the ostariophysan, C. carpio, the cellular arrange-ment of the early, undifferentiated gonad has a patternvery similar to the previous reports on basal taxa in Tele-ostei (Davies and Takashima, 1980; Timmermans and Tav-erne, 1983; Colombo et al., 1984; Parmentier andTimmermans, 1985; Winkoop et al., 1992; Colombo andGrandi, 1996; Timmermans et al., 1996; Grandi and

Colombo, 1997; Otani et al., 2005; Cek, 2006; Mazzoniet al., 2010); it is not possible to distinguish between thepresumptive male and the presumptive female. Howeverin the Acanthopterygii, here studied, in the percomorph,A. nigrofasciata and in the atherinomorph, P. reticulata,the early cellular arrangement of the undifferentiatedgonads is distinctly different from C. carpio, differing inregard to the presumptive males and presumptive females.

In presumptive male representatives of the Acanthop-terygii, the germ cells accumulate at the ventral periph-ery of the early, undifferentiated gonad while in thepresumptive females the germ cells are centrally local-ized. The presumptive male can also be distinguished bythe organized layers of SCs at the dorsal region of theundifferentiated gonad that were not found in the pre-sumptive females. Apparently, these differences were notdetected in previous reports regarding the gonadal differ-entiation in more derived taxa of Teleostei as in the per-comorph, Cichlasoma dimerus (Meijide et al., 2005;

Fig. 18. Differentiated male gonad from P. reticulata. Light micros-copy—Longitudinal sections. A: The reorganization of the SCs at thecentral region of the gonadal tissue (A) gives raise to the future spermduct (1 dpb). Pre-Sertoli cells (PS), spermatogonia (G). B. The acinarstructures (A) formed by clusters of cysts containing spermatogonia(G) wrapped by the pre-Sertoli cells remain at the distal region of thegonad (12 dpb). Sperm duct (DU). C: Sperm duct (DU) and the acinar

structure (A) connects to one another forming the first testicularlobules (12 dpb). D and E: In the central region of the gonad, the SCsthat form the sperm ducts (DU) become cuboidal and reorganize inparallel rows (12 dpb). Acinus (A), spermatogonia (G), cranial region(CR), caudal region (CA). Staining: Toluidine blue. Scale bar 5 20 mm(A), 40 mm (B), 10 mm (C and E), and 30 mm (D).


Pandolfi et al., 2009) and in the atherinomorphs, Mollie-nisia latipinna (Van Den Hurk, 1974) and Oryzias latipes(Nakamura et al., 2006, 2008, 2010; Saito et al., 2007).

The first morphological signals of gonadal differentia-tion are related to the formation of the testicular ductsin males or by the entrance into meiosis in females.Until now, these are the parameters that allow the mor-phological discernment from the sexual fate of thegonads in the gonochoristic species (Nakamura andTakahashi, 1973; Nakamura and Nagahama, 1989;Pati~no and Takashima 1995; Str€ussmann et al., 1996;Nakamura et al., 1998; Meijide et al., 2005; Komatsuet al., 2006; Rasmussen et al., 2006; Pandolfi et al.,2009; Guerrero-Est�evez and Moreno-Mendoza, 2010,2012; Kobayashi et al., 2011).

As first reported by Mazzoni et al. (2010) and con-firmed here, the morphological discernment of the sexualfate of the gonads could be determined during earlydevelopment. However, even if the distinct arrangementof the PGCs and SCs allow recognizing the sexual fate ofthe gonads, we have to consider that even in this periodthe germ cells are potentially able to differentiate asspermatogonia or oogonia (Devlin and Nagahama, 2002;Rasmussen et al., 2006).

In the presumptive testes from the Otophysi, C. car-pio, and from the Percomorpha, A. nigrofasciata, therelocation of the initial, continuous cords of PGCs toform acinar-clusters is the first morphological trait sig-naling sexual differentiation of the gonad. In the pre-sumptive testes of the atherinomorph, P. reticulata,following the initial aggregation in a clustered shape,the PGCs also assume an acinar-cluster arrangement.

The formation of the acinar-clusters of spermatogoniaduring gonadal differentiation has been reported inother species closely related to those here studied, suchas: the Percomorpha, Cichlasoma dimerus (Perciformes:Cichlidae) (Meijide et al., 2005) and in the Atherinomor-pha Oryzias latipes (Beloniformes: Adrianichthyidae)(Shinomiya et al., 2001). Shinomiya et al. (2001) sug-gested that these acinar-clusters would be the precur-sors of the testicular lobules. These reports reinforce ourfindings regarding the arrangement of spermatogonia inacinar-clusters, that is, this could be the first morpholog-ical characteristic of the male fate of the gonad in Tele-ostei. Other evidence comes from the protogynousspecies, the Synbranchiformes, Synbranchusmarmoratus, in which the first morphological expressionof sex reversal is the detection of acinar-clusters of

Fig. 19. The basement membrane and the formation of the germinalepithelium in P. reticulata. Light microscopy—Longitudinal sections—Reticulin method. A: The basement membrane (arrow) is present ini-tially in the region corresponding to sperm duct, supporting the SCs.Note the absence of the basement membrane around the acinarstructures (A) (stage 5). B–D: With cell proliferation, the gonadal tissueincreases in size and the basement membrane (arrow) is synthesizedaround the acini (A) (B: 6 dpb). C and D: The basement membrane(arrow) from the duct (DU) becomes continuous with the basementmembrane (arrow) of each acinus (A) (12 dpb). E and F: General view

of the gonad with the testicular lobules (LO) completely developed.Note the basement membrane (arrow) is quite developed in the mainsperm duct (DU) and also around the testicular lobules (LO) (E: 15dpb, F: 16 dpb). G: Detail of F showing the basement membrane(arrow) continuous around testicular lobule. Note the absence of thebasement membrane (arrowhead) within of the lobule (LO), betweenthe adjacent cysts. Spermatocytes (C), ventral region (V), cranialregion (CR). Scale bar 5 10 mm (A, B, D, and G), 40 mm (C), 60 mm (E),and 80 mm (F).

1156 MAZZONI ET AL.

spermatogonia in the ovarian germinal epithelium (LoNostro et al., 2003).

In the species here studied (C. carpio or A. nigrofas-ciata and P. reticulata), the early expression of the 3b-HSD enzyme that was detected in the interstitial SCs

(the presumptive Leydig precursors), near the spermato-gonia acinar-clusters. This is an indication that thesecells are associated with early spermatogenesis. The 3b-HSD detection, as earlier used by Van Der Hurk in fish(1974), is a universal marker for the precursors and

Fig. 20. The differentiating testis from P. reticulata and the start ofthe spermatogenesis. Light microscopy—Longitudinal sections. A andB: Early spermatogenic gonad (14 dpb). Now, the testicular lobules(LO) are completely formed. The acinar structures formed by the cystsof spermatogonia (G) remain at the blind termini of the lobules. Asspermatogonia proliferate and enter into meiosis, the cysts of sperma-tocytes (C) are formed. There are no sperm within sperm duct (DU). C:The testicular lobules (LO) and the entire testis increase in length dueto the formation of a larger number of cysts, mainly cysts of sperma-tocytes (16 dpb). Sperm duct (DU). D: Detail of the connectionbetween the sperm duct (DU) and the testicular lobule. Spermatocytes

(C). E: The first cysts of spermatids (T) appear close to the spermduct (DU) (18 dpb). Note the acinar structures (A) formed by cysts ofsingle spermatogonia (G) at the distal termini of the lobules (LO). Thereare no spermatozoa. F: Detail of a testicular lobule, showing the orga-nization of the cysts, with single spermatogonia (G) being wrapped bySertoli cells (S) and restricted to the distal termini of the lobules. Sper-matocytes (C), sperm duct (DU), cranial region (CR), caudal region(CA). Staining: Toluidine blue (A and B), PAS/hematoxylin/metanil yel-low (C–F). Scale bar 5 60 mm (A), 20 mm (B, D), 100 mm (C and E), and30 mm (F).


differentiated Leydig cells (see Haider, 2004; Chen et al.,2009 for review). In animals in general, and as expectedin Teleostei, steroidogenesis, sexual determination,gonadal differentiation and spermatogenesis are inti-mately related processes (see Hofsten and Olsson, 2005;Nagahama, 2005; Schulz and Miura, 2002—for review).

The formation of the sperm duct preceding the initia-tion of germ cell meiosis (Pati~no and Takashima 1995;Nakamura et al., 1998; Meijide et al., 2005; Rasmussenet al., 2006; Pandolfi et al., 2009) was established for theAcanthopterygii, as can be seen in the Percomorpha(Meijide et al., 2005; present study) and Atherinomorpha(Van Den Hurk, 1974; Nakamura et al., 1998; presentstudy), but not for the Otophysi. In Otophysi the forma-tion of the sperm duct is the last structure to be formedresulting from the anastomoses of the testicular tubules(present study).

The testicular differentiation

In a comparative view, the early steps of the gonadal dif-ferentiation are quite similar in the Otophysi, C. carpio andin the Percomorpha, A. nigrofasciata. Mainly, they sharethe formation of the continuous cords being formed by thePGCs prior to the arrangement of the spermatogonia intoacinar-clusters structures. However, in the Atherinomor-pha, P. reticulata despite the presence of acinar-clusters ofspermatogonia, continuous cords of PGCS never form amajor difference between this and the other two species.

Primarily from this fact, gonadal differentiation takesdifferent pathways between the representatives from theOtophysi and from the Percomorpha than it does in therepresentative from the Atherinomorpha. In the Otophysi,C. carpio, the acinar-clusters of spermatogonia form anas-tomoses with one another and subsequently give rise to thefirst testicular tubules. This fusion occurs after the central

Fig. 21. Differentiated spermatogenic testis from P. reticulata. Lightmicroscopy—Longitudinal sections. A and B: The testicular lobulesbecome more elongated due to the increasing of number of cysts con-taining spermatids (T) and spermatocytes (C) (30 dpb). A PAS-positivesecretion is produced by the epithelial cells of the sperm duct (DU) andsurrounds the spermatozeugmata. Spermatogonia (G). C and D: Theproduction of spermatozoa (Z) begins (90 dpb). D: Spermatoza occur inspermatozeugmata (ZG) which occupy the lumen of the sperm duct(DU). Testicular lobules (LO), spermatocytes (C), spermatids (T). E–H:

Details of D. E: Cyst of spermatogonia (G) wrapped by Sertoli cells (S).F: A cyst containing metaphase spermatogonia (M) and delimited by apink-staining basement membrane. Spermatocytes (C). G: Cysts ofspermatocytes (C) and cysts of spermatids (T), each one of themwrapped and separated from one another by Sertoli cells (S). H: In theSpermatozeugmata, the sperm nuclei (N) are circumferentially alignedwhile the flagella (F) are arranged in the central region. SCs, Ventralregion (V). Staining: PAS/hematoxylin/metanil yellow. Scale bar 5 50 mm(A), 20 mm (B), 100 mm (C and D), 10 mm (E, F, G, and H).

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lumen and basement membrane around the acinar struc-tures is formed. It is also the moment in which the germi-nal epithelium that lines the testicular tubules begins to beformed resulting in the unrestricted distribution of sper-matogonia along the tubule lengths. On the other hand, inthe Percomorpha, A. nigrofasciata, each one of the acinar-clusters of spermatogonia lengthens and gives rise to indi-vidual lobules. The elongation occurs after the basementmembrane is present surrounding the acinar structure andit occurs simultaneously with formation of the centrallumen. As elongation occurs, spermatogonia become dis-tributed along the entire length of the developing lobule.Simultaneously, the germinal epithelium that lines the tes-ticular lobules begins to be formed resulting in an unre-stricted distribution of spermatogonia. Thus, in thegerminal epithelium of the Otophysi, C. carpio, and in thePercomorpha, A. nigrofasciata, despite their being formedin a different ways, spermatogonia have an UnrestrictedDistribution (sensu Grier et al., 1980).

In the Otophysi, C. carpio, the fusion among theacinar-clusters of spermatogonia occurs throughout thegonad forming elongated tubules that anastomose withone another and to the sperm duct. Consequently thetestes in the Otophysi are of the Anastomosing Tubu-lar Type (sensu Grier et al., 1980).

In the Percomorpha, A. nigrofasciata, the elongationof the acinar-clusters of spermatogonia begins at theventral periphery of the gonad and extends toward thedorsal region. As these germinal compartments endblindly at the developing testis periphery, they are con-sidered lobules (sensu Grier, 1981). The lobules becomeradially arranged and each one of them connects to thesperm duct. Consequently the testes in the Percomorphaare of the Lobular Type (sensu Grier, 1981).

In the Atherinomorpha, P. reticulata, developing ductsrun from each one of the acinar-clusters of spermatogonia,located on the ventral periphery of the gonad, connectingthem to the sperm duct. The clusters of spermatogoniaremain restricted to the distal termini of the forminglobules and a true germinal epithelium is not formed(sensu Grier et al., 1980; Grier, 1981). Thus, the testes inthe Atherinomorpha are of the Lobular Type (sensuGrier, 1981), and in it spermatogonia have a RestrictedDistribution (sensu Grier et al., 1980; Grier, 1981).

The ontogeny and the paedomorphichypothesis of the testis evolution in Teleostei

The ontogenic process that gives rise to the distincttesticular types in Teleostei shows that the first

Fig. 22. The testis from a sexually active adult of P. reticulata (90dpb). Light microscopy—Longitudinal sections. A: General view of thegonad showing the region of the sperm duct (DU) and the testicularlobules (LO). B: Detail of the A showing the testicular lobules (LO) withcysts containing spermatocytes (C) and spermatids (T). C: Detail ofthe cyst of spermatocyte (C). Sertoli cells (S). Interstitial compartment(I). D: Region of the sperm duct (DU) filled with spermatozeugmata(ZG) and PAS-positive secretion. SCs of the duct, spermatids (T),spermatocytes (C). E: Spermatozeugmata (ZG) released inside theduct (DU). Interstitial compartment (I). F: General view of the gonadshowing the sperm duct (DU) and the testicular lobules (LO). G and H:The testicular lobules (LO) are surrounded by a basement membrane(arrow), which supports and separates the germinal epithelium from

the interstitial compartment (I). Duct (DU). H: Detail of G. Note theabsence of the basement membrane (arrowhead) between two cystsof spermatocytes within the same lobule (C). I: Region of the spermduct (DU) filled with spermatozeugmata (ZG). Note the basementmembrane (arrow) that supports the epithelium of the sperm ducts. J:see the connection between the sperm duct (DU) and the testicularlobule. The basement membrane (arrow) is continuous between them.Note the absence of the basement membrane (arrowhead) betweenthe sperm duct and the testicular lobule. Spermatids (T), cranial region(CR). Staining: PAS/hematoxylin/metanil yellow (A–E), Reticulin method(F–J). Scale bar 5 200 mm (A and F), 50 mm (B, D, G, and I), and 10mm (C, E, H, and J).


Fig. 23. Schema showing the formation of the germinal compart-ment during male gonadal differentiation in a viviparous atherino-morph, represented by P. reticulata. A: Gonadal primordium. B:Undifferentiated gonad with the PGCs located at the ventral region. C:Differentiating gonad defined by the formation of clustered structuresof PGCs at the ventral region. Reorganization of the SCs at the dorsalregion will give raise to the future sperm duct. Note that it already hasa basement membrane in formation. D: Formation of the testicularduct at the dorsal region of the gonad as acinar structures of cysts,containing spermatogonia wrapped by the pre-Sertoli cells, are local-ized at the ventral region of the gonad. Each one of the acini is sur-rounded by a continuous basement membrane and is surrounded byneighboring SCs (arrow). E: Formation of testicular lobules. A rear-rangement of neighboring SCs in lobular-like structures connects eachindividual acinus to the sperm duct, forming the first testicular lobules.Note the individualization of each lobule by the basement membrane,which is continuous with the basement membrane of the duct. The

acinar-structures formed by cysts containing single spermatogoniawrapped by Sertoli cells remain restricted to the distal termini of thelobule. F: The testicular lobules are formed. Spermatogenesis pro-gresses in a centripetal direction. Thus, with the formation of newcysts the lobules elongate. The lumen of the sperm duct becomesfilled by a secretion, and spermatozoa are arranged in spermatozeug-mata. F-inset: Testis with a restricted lobular type. Note the spermato-gonia located only in the distal portion of the lobules. Details of thegerminal and interstitial compartments. Detail of the spermatozeug-mata showing the position of the spermatozoa therein. PGCs, SCs,basement membrane (BM), interstitial compartment (I), acinus (A), pre-Sertoli cell (PS), spermatogonia (G), testicular lobules (LO), testicularlumen (LU), Sertoli cell (S), spermatocytes (C), spermatids (T), sperma-tozoa (Z), interstitial compartment (IC), Leydig cell (LE), blood vessel(BV), metaphase (M), sperm duct (DU), nuclei (N), flagellum (F), sper-matozeugmata (ZG), ventral region (V), dorsal region (D), cranial region(CR), caudal region (CA).

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structure common to all of them is the arrangement ofthe spermatogonia into acinus-like clusters. The Anasto-mosing Tubular testis develops as a result of the initialanastomoses among the acinar-clusters of spermatogoniaand then from the newly formed tubules. The Anasto-mosing Tubular Testis with an Unrestricted Spermatogo-nial Distribution (sensu Grier et al., 1980) ischaracteristic of basal groups in Teleostei, such as theOstariophysi. On the other hand, the lobules within theLobular Testis having an Unrestricted SpermatogonialDistribution, characteristics of the Percomorpha (sensuGrier et al., 1980), are the result of the elongation of thespermatogonial acinar-clusters themselves. Lastly theLobular Testis with a Restricted Spermatogonial Distri-bution, a characteristic of the Atherinophorma (sensuGrier, 1981), results from a lobular connection that isestablished between each one of the acinar-clusters and

the sperm duct. In this group, the acinus-like cluster ofspermatogonia remains as also occurs in the differenti-ated adult gonad.

Considering ontogenic processes, the spermatogonial,acinar-like clusters are the characteristic structuralunits observed in adult testes of more derived taxa thatapproximate the early gonadal developmental stages ofthe basal vertebrate taxa. The acinar-like clusters appa-rently presage the development of spermatocysts inbasal taxa prior to the evolution of Vertebrata; thesebasal taxa include the agnathans and elasmobranchs,both groups having polyspermatocystic testes (Grier,1992, 1993). Each spermatocyst is separated from inter-stitial tissue by a basement membrane, a reflection onlater evolution of the anastomosing tubular testes in C.carpio, the unrestricted lobular testes in A. nigrofasciataor the restricted lobular testes in P. reticulata, groupswhere larger morphological units, anastomosing tubulesor lobules, are bounded by basement membranes. Theinitial associations of SCs and PGCs, as acinar-clusters,establish the patterns in which the extracellular matrix(Grier, 1992) is formed, establishes the separationbetween the germinal and interstitial gonadal compart-ments and importantly establishes different gonadaltypes between basal and derived fish taxa.

LITERATURE CITED

Cek S. 2006. Early gonadal development and sex differentiation inrosy barb (Puntius conchonius). Anim Biol 56:335–350.

Chen H, Ge RS, Zirkin BR. 2009. Leydig cells: From stem cells toaging. Mol Cell Endocrinol 306:9–16.

Colombo G, Grandi G, Rossi R. 1984. Gonad differentiation andbody growth in Anguilla anguilla L. J Fish Bio 24:215–228.

Colombo G, Grandi G. 1996. Histological study of the developmentand sex differentiation of the gonad in the European eel. J FishBiol 48:493–512.

Davies PR, Takashima, F. 1980. Sex determination in common carp,Cyprinus carpio. J Tokyo Univ Fish 66:191–199.

Devlin RH, Nagahama Y. 2002. Sex determination and sex differen-tiation in fish: an overview of genetic, physiological, and environ-mental influences. Aquaculture 208:191–364.

Fink WL. 1981. Ontogeny and phylogeny of tooth attachment modesin actinopterygian fishes. J Morphol 167:167–184.

Flores JA, Burns JR. 1993. Ultrastructural study of embryonic andearly adult germ cells, and their support cells, in both sexes ofXiphophorus (Teleostei: Poecillidae). Cell Tissue Res 271:263–270.

Grandi G, Colombo G. 1997. Development and early differentiationof gonad in the European eel (Anguilla Anguilla [L], Anguilli-formes, Teleostei): a cytological and ultrastructural study. J Mor-phol 231:195–216.

Grier HJ, Linton JR, Leatherland JF, De Vlaming VL. 1980. Struc-tural evidence for two different testicular types in teleost fishes.Am J Anat 159:331–345.

Grier HJ. 1981. Celular organization of the testis and spermatogen-esis in fishes. Am Zool 21:345–357.

Grier HJ. 1992. Chordate testis: the extracellular matrix hypothe-sis. J Exp Zool 261:151–160.

Grier HJ. 1993. Comparative organization of Sertoli cells includingthe Sertoli cell barrier. In: Russel LD, Griswold MD, editors. TheSertoli cell. Clearwater: Cache River Press. p 704–730.

Grove BD, Wourms JP. 1994. Follicular placenta of the viviparousfish, Heterandria formosa. II. Ultrastructure and development ofthe follicular placenta. J Morphol 220:167–184.

Guerrero-Est�evez S, Moreno-Mendoza N. 2010. Sexual determina-tion and differentiation in teleost fish. Rev Fish Biol Fisher 20:101–121.

Fig. 24. Differentiated male gonads from C. carpio (90 dpf) (A–C), A.nigrofasciata (115 dpf) (D,E) and P. reticulata (12 dpb) (F,G). Lightmicroscopy—Sagittal (A–E) and longitudinal (F,G) sections—immuno-histochemical detection of 3b-HSD enzyme. A–C: gonad of the C. car-pio with lumen (LU) formed and germinal epithelium composed byspermatogonia (G) only. D and E: gonad of the A. nigrofasciata withlobules (LO) formed and a germinal epithelium only having spermato-gonia (G). F and G: gonad of the P. reticulata with lobules (LO) com-posed by spermatogonia (G) only Leydig cell precursors (arrow). C, Eand G: In the interstitial compartment (I), some cells were detected aslabeling by 3b-HSD. These cells are Leydig cell precursors (L; arrow).They have a spherical shape, spherical or oval nucleus and evidentnucleolus. Lumen (LU), Sperm duct (DU). Ventral region (V), dorsalregion (D), caudal region (CA). Staining: hematoxylin. Scale bar 5 100mm (A), 50 mm (B, D, and F), 15 mm (C, E, and G).


Guerrero-Est�evez S, Moreno-Mendoza N. 2012. Gonadal morpho-genesis and sex differentiation in the viviparous fish Chapalich-thys encaustus (Teleostei, Cyprinodontiformes, Goodeidae). J FishBiol 80(3):572–594.

Haider SG. 2004. Cell biology of leydig cells in the testis. Int RevCytol 233:181–241.

Hamaguchi S. 1982. A light- and electron-microscopic study on themigration of primordial germ cells in the teleost, Oryzias latipes.Cell Tissue Res 227:139–151.

Hliwa P, Demska-ZakeR�s K, Martyniak A, Kr�ol J. 2003. Gonadal dif-ferentiation in Vimba vimba (L. 1758). Czech J Anim Sci 48(11):441–448.

Hofsten JV, Olsson PE. 2005. Zebrafish sex determination and dif-ferentiation: involvement of FTZ-F1 genes. Reprod Biol Endocrin3:63.

Kobayashi T, Ishibashi R, Yamamoto S, Otani S, Ueno K, MurataO. 2011. Gonadal morphogenesis and sex differentiation in cul-tured chub mackerel, Scomber japonicas. Aquacult Res 42:230–239.

Komatsu T, Nakamura S, Nakamura M. 2006. Sex cord-like struc-ture and some remarkable features in early gonadal sex differen-tiation in the marine teleost Siganus guttatus (Bloch). J Fish Biol68:236–250.

Lo Nostro F, Grier H, Andreone L, Guerrero, GA. 2003. Involvementof the gonadal germinal epithelium during sex reversal and sea-sonal testicular cycling in the prtogynous swamp eel, Synbran-chus marmoratus Bloch 1795 (Teleostei, Synbranchidae). JMorphol 257:107–126.

Mazzoni TS, Grier HJ, Quagio-Grassiotto I. 2010. Germline cystsand the formation of the germinal epithelium during the femalegonadal morphogenesis in Cyprinus carpio (Teleostei: Ostario-physi). Anat Rec 293(9): 1581–1606.

Meffe GK. 1985. Life history patterns of Gambusia marshi (Poecilii-dae) from Cuatro Ci�enegas, Mexico. Copeia 4:898–905.

Meijide FJ, Lo Nostro F, Guerrero GA. 2005. Gonadal developmentand sex differentiation in the cichlid fish Cichlasoma dimerus(Teleostei, Perciformes): a light- and electron-microscopic study. JMorphol 264:191–210.

Nagahama Y. 1983. The functional morphology of teleost gonads.In: Hoar WS, Randall DJ, Donaldson EM, editors. Fish physiol-ogy. New York: Academic Press. V. IX, chap. 6.

Nagahama Y. 2005. Molecular mechanisms of sex determinationand gonadal sex differentiation in fish. Fish Physiol Biochem 31:105–109.

Nakamura M, Takahashi H. 1973. Gonadal sex differentiation inTilapia mossambicus with special regard to the time of estrogentreatment effective in inducing complete feminization of geneticmales. Bull Fac Fish Hokkaido Univ 24:1–13.

Nakamura M, Nagahama Y. 1989. Differentiation and developmentof Leydig cells, and change of testosterone levels during testiculardifferentiation in tilapia Oreochromis niloticus. Fish Physiol Bio-chem 7:211–219.

Nakamura M, Kobayashi T, Chang XT, Nagahama Y. 1998. Gonadalsex differentiation in teleost fish. J Exp Zool 281:362–372.

Nakamura S, Kokayashi D, Aoki Y, Yokoi H, Ebe Y, Wittbrodt J,Tanaka M. 2006. Identification and lineage tracing of two popula-tions of somatic gonadal precursors in medaka embryos. Dev Biol295:678–688.

Nakamura S, Aoki Y, Saito D, Kuroki Y, Fujiyama A, Naruse K,Tanaka M. 2008. Sox9b/sox9a2-EGFP transgenic medaka revealsthe morphological reorganization of the gonads and a commonprecursor of both the female and male supporting cells. MolReprod Dev 75:472–476.

Nakamura S, Kobayashi K, Nishimura T, Higashijima S, TanakaM. 2010. Identification of germline stem cells in the ovary of theteleost medaka. Science 328:1561–1563.

Nelson JS. 2006. Fishes of the world. 4nd ed. New York: John Wiley& Sons. 601 p.

Otani S, Kitauchi T, Saito T, Sakao S, Maegawa S, Inoue K, YamahaE. 2005. The formation of primordial germ cells from germline cells

in spherical embryos derived from the blastodisc of 2-cell embryosin goldfish, Carassius auratus. Int J Dev Biol 49:843–850.

Parenti LR, Grier HJ. 2004. Evolution and phylogeny of gonad mor-phology in bony fishes. Integr Comp Biol 44:333–348.

Parmentier HK, Timmermans LPM. 1985. The differentiation ofgerm cells and gonads during development of carp (Cyprinus car-pio L.). A study with anti-carp sperm monoclonal antibodies. JEmbryol Exp Morphol 90:13–32.

Pandolfi M, Canepa MM, Meijide FJ, Alonso F, V�azquez GR,Maggese MC, Vissio PG. 2009. Studies on the reproductive anddevelopmental biology of Cichlasoma dimerus (Percifomes, Cichli-dae). Biocell 33(1):1–18.

Pati~no R, Takashima F. 1995. In: Takashima F, Hibiya T, editors.An atlas of fish histology: Normal and pathological features. 2nded. New York: Stuttgart Kodansha-Gustav Fisher. p 128–153.

Potter H, Kramer CR. 2000. Ultrastructural observations on spermstorage of the platyfish, Xiphophorus maculates (Teleostei: Poeci-liidae): the role of the duct epithelium. J Morphol 245:110–129.

Puchtler H, Waldrop FW. 1978. Silver impregnation methods forreticulum fibers and reticulin: a re-investigation of their originsand specificity. Histochemistry 57(3):177.

Quintero-Hunter I, Grier H, Muscato M. 1991. Enhancement of his-tological detail using metanil yellow as counterstain in periodicacid/Schiff0s hematoxylin staining of glycol methacrylate tissuesections. Biotech Histochem 66:169–172.

Rasmussen TH, Jespersen A, Korsgaard B. 2006. Gonadal morpho-genesis and sex differentiation in intraovarian embryos of theviviparous fish Zoarces viviparus (Teleostei, Perciformes, Zoarci-dae): a histological and ultrastructural study. J Morphol 267:1032–1047.

Saito D, Morinaga C, Aoki Y, Nakamura S, Mitani H, Furutani-Seiki M, Kondh H, Tanaka M. 2007. Proliferation of germ cellsduring gonadal sex differentiation in medaka: insights from germcell-depleted mutant zenzai. Dev Biol 310:280–290.

Satoh N 1974. An ultrastructural study of sex differentiation in theteleost Oryzias latipes. J Embryol Exp Morphol 32:195–215.

Schulz RW, Miura T. 2002. Spermatogenesis and its endocrine regu-lation. Fish Physiol Biochem 26:43–56.

Schulz RW, Menting S, Bogerd J, Franca LR, Vilela DAR, GodinhoHP. 2005. Sertoli cell proliferation in the adult testis - evidencefrom two fish species belonging to different orders. Biol Reprod73:891–898.

Shinomiya A, Hamaguchi S, Shibata N. 2001. Sexual differentiationof germ cell deficient gonads in the Medaka, Oryzias latipes. JExp Zool 290:402–410.

Str€ussmann CA, Takashima F, Toda K. 1996. Sex differentiationand hormonal feminization in pejerrey Odontesthes bonariensis.Aquaculture 139:31–45.

Timmermans LPM, Taverne N. 1983. Origin and differentiation ofprimordial germ cells (PGCs) in the rosy barb, Barbus conchonius(Cyprinidae, Teleostei). Acta Morph Neerl Scan 21:182.

Timmermans LPM, Taverne N. 1989 Segregation of primordialgerm cells: their numbers and fate during early development ofBarbus conchonius (Cyprinidae, Teleostei) as indicated by 3H-thymidine incorporation. J Morphol 202:225–237.

Timmermans LPM, Wang J, Taverne N, Schipper H, ParmentierHK. 1996. Characterization of antigens specific for spermatozoaand germ cells during early gonadal development of carp (Cypri-nus carpio, Cyprinidae, Teleostei) with monoclonal antibodies.Int. J Dev Biol 1:221S–222S.

Van Den Hurk R. 1974. Steroidogenesis in the testis and gonado-tropic activity in the pituitary during postnatal development ofthe black molly (Mollienisia latipinna). Endocrinol C 77(2):193–200.

Vidal BC. 1988. Histochemical and anisotropical properties character-istics of silver impregnation: the differentiation of reticulin fibersfrom the other interstitial collagens. Zool Jb Anat 117:485–494.

Winkoop A, Booms GHR, Dulos GJ, Timmermans LPM. 1992.Ultrastructural changes in primordial germ cells during earlygonadal development of the common carp (Cyprinus carpio L. Tel-eostei). Cell Tissue Res 267:337–346.

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Male Gonadal Differentiation and the Paedomorphic Evolution of the Testis in Teleostei

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