Evolution and phylogeny of gonad morphology in bony fishes

Gonad morphology at the gross anatomical or histological levelshas long been studied by fisheries biologists to identify annualreproductive cycles and length of breeding season, among othergoals. Comparative surveys across vertebrate taxa have not beendetailed enough, however, to describe fully the differencesand similarities among gonads of bony fishes and other vertebrates,and to use gonad morphology in phylogenetic systematic analyses.An emerging constant among vertebrates is the presence of agerminal epithelium composed of somatic and germ cells in bothmales and females. In females, the germinal epithelium linesthe ovarian lamellae. In males, arrangement of the germinalepithelium into compartments varies among osteichthyans: basaltaxa have an anastomosing tubular testis, whereas derived taxahave a lobular testis. The lobular testis is proposed as a synapomorphyof the Neoteleostei. The annual reproductive cycle is hypothesizedto be the source of morphological variation among testis types.Elongation of germinal compartments during early maturationmay result in a transition from anastomosing tubular to lobulartestes. In all male atherinomorphs surveyed, spermatogonia arerestricted to the distal termini of lobules rather than beingdistributed along the lobule; there is an epithelioid arrangementof Sertoli and germ cells rather than a germinal epithelium.Arrest of the maturation-regression phases is hypothesized tolead to formation of the atherinomorph testis. Atherinomorphsalso have a distinctive egg with fluid, rather than granular,yolk. Variation among germinal epithelia is interpreted in adeveloping phylogenetic framework to understand evolution ofgonad morphology and to propose gonad characters for phylogeneticanalyses.     

Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel,Synbranchus marmoratus Bloch 1795 (Teleostei,Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a protogynous, diandric species. During sex reversal, the ovarian germinal epithelium, which forms follicles containing an oocyte and encompassing follicle cells during the female portion of the life cycle, produces numerous invaginations, or acini, into the ovarian stroma. Within the acini, the gonia that formerly produced oocytes become spermatogonia, enter meiosis, and produce sperm. The acini are bounded by the basement membrane of the germinal epithelium. Epithelial cells of the female germinal epithelium, which formerly became follicle (granulosa) cells, now become Sertoli cells in the developing testis. Subsequently, lobules and testicular ducts form. The swamp eel testis has a lobular germinal compartment in both primary and secondary males, although the germinal compartment in testes of secondary males resides within the former ovarian lamellae. The germinal compartment, supported by a basement membrane, is composed of Sertoli and germ cells that give rise to sperm. Histological and immunohistochemical techniques were used to describe the five reproductive classes that were observed to occur during the annual reproductive cycle: regressed, early maturation, mid-maturation, late maturation, and regression. These classes are differentiated by the presence of continuous or discontinuous germinal epithelia and by the types of germ cells present. Synbranchus marmoratus has a permanent germinal epithelium. Differences between the germinal compartment of the testes of primary and secondary males were not observed.     

Annual changes in germinal epithelium determine male reproductive classes of the cobia

Five reproductive classes of cobia Rachycentron canadum , caught along the Gulf of Mexico and the south-east Atlantic coast of the U.S.A., are described during the annual reproductive cycle. These are based upon changes in the testicular germinal epithelium and the stages of germ cells that are present: early maturation, mid maturation, late maturation, regression and regressed. During early maturation, the germinal epithelium is continuous from the testicular ducts to the periphery of the testis and active spermatogenesis occurs throughout the testis. In mid maturation, the germinal epithelium near the ducts becomes discontinuous, but it remains continuous distally. In late maturation, a discontinuous germinal epithelium extends all along the lobules to the testicular periphery; lobules are swollen with sperm and there is minimal spermatogenesis. The regression class is characterized by a discontinuous epithelium throughout the testis, sperm storage and widely scattered spermatocysts. Spermatogonial proliferation also occurs along the lobule walls and at the periphery of the testis. In regressed testes, spermatogonia exist only in a continuous or discontinuous germinal epithelium, although residual sperm are nearly always present in the lobules and ducts. The presence or absence of sperm is not an accurate indicator of reproductive classes. At the periphery of the testis in the regression and regressed classes, the distal portions of lobules elongate as cords of cells containing spermatogonia and Sertoli cells. All reproductive classes can be identified in paraffin sections, although plastic sections provide better resolution. Using maturation classes defined by changes in the germinal epithelium to describe testicular development and spermatogenesis gives a more accurate picture than does using the traditional terminology.     

Ultrastructure of the testis in Synbranchus marmoratus (Teleostei,Synbranchidae): the germinal compartment

Synbranchus marmoratus, is a protogynic diandric species in which two types of males, primary and secondary, are found. In both types, the germinal compartment in the testes is of the unrestricted lobular type, but in secondary (sex reversed females) males the lobules develop within the former ovarian lamellae. In the present study, the germinal compartment was examined in both types of males using light microscopy as well as scanning and transmission electron microscopy. Germinal compartment is limited by a basement membrane and contains Sertoli and germ cells. During maturation, processes of Sertoli cells form the borders of spermatocysts containing isogenic germ cells. Characteristically, type A and type B spermatogonia have a single nucleolus and grouped mitochondria associated with dense bodies or nuage. Type B spermatogonia, spermatocytes and spermatids are joined by cytoplasmatic bridges and are confined within spermatocysts. Secondary spermatocytes are difficult to find, indicating that this stage is of short duration. Biflagellated spermatozoa have a rounded head, no acrosome, and possess a midpiece consisting of two basal bodies, each of which produces a flagellum with a typical 9+2 microtubular composition. No associations occur between sperm and Sertoli cells. There were no differences between spermatogenesis in primary and secondary males in this protogynic, diandric fish.     

Gonad development and evidence of protogyny in the red-throat emperor on the Great Barrier Reef

The gonad development in the red-throat emperor Lethrinus miniatus is described and the first detailed evidence for protogyny in this species provided. The identification of transitional individuals, bimodal sex-specific size-frequency distributions and female biased sex ratios suggest that L. miniatus is most likely a protogynous hermaphrodite. Transitional L. miniatus gonads were characterized by the concurrent degeneration of all oocytes and the proliferation of spermatocysts near the edge of the lamellae, an increase in blood vessels along strands of stromal tissue within the lamellae and the formation of multiple sperm sinuses. The sites of oocyte degeneration and proliferation of spermatocysts were spatially segregated. An increase in blood vessels along strands of stromal tissue within the lamellae of transitional phase gonads is likely to assist in the breakdown of oocytes and the proliferation of spermatocysts. Most mature resting females containing spermatocysts occurred within the transitional size-frequency distribution, suggesting that the presence of spermatocysts in these females may be an early sign of sex change. Oocytes within female gonads were interrupted by filamentous strands of stromal tissue within the lamellae. The testis contained a remanent ovarian lumen but no residual oocytes. Three characteristics of transitional L. miniatus gonads were found to be unusual and described for few other species of coral reef fishes. These included the absence of oocytes within testes, increased numbers of blood vessels, and the presence of strands of stromal tissue within the lamellae.     

长吻鮠精巢及精子结构的研究

长吻鮠精巢高度分支呈指状。后1/3紫红色,由上皮细胞组成,既不产生精子,也不贮存精子。精巢的内部结构为叶型,由体细胞和生殖细胞构成,小叶的基本单位是小囊。精子头短而圆,主要为核占据,无顶体,核凹窝十分发达,有中心粒帽;尾极长,具侧鳍,轴丝基部有发达的囊泡状结构和线粒体。     

Gonadal morphogenesis and sex differentiation in the viviparous fish Chapalichthys encaustus (Teleostei, Cyprinodontiformes, Goodeidae)

This study describes the structural and ultrastructural characteristics of gonadal sex differentiation and expression of Vasa, a germline marker, in different developmental stages of embryos and newborn fry of the barred splitfin Chapalichthys encaustus, a viviparous freshwater teleost endemic to Mexico. In stage 2 embryos, the gonadal crest was established; gonadal primordia were located on the coelomic epithelium, formed by scarce germ and somatic cells. At stage 3, the undifferentiated gonad appeared suspended from the mesentery of the developing swimbladder and contained a larger number of germ and somatic cells. At stages 4 and 5, the gonads had groups of meiotic and non-meiotic germ cells surrounded by somatic cells; meiosis was evident from the presence of synaptonemal complexes. These stages constituted a transition towards differentiation. At stage 6 and at birth, the gonad was morphologically differentiated into an ovary or a testis. Ovarian differentiation was revealed by the presence of follicles containing meiotic oocytes, and testicular differentiation by the development of testicular lobules containing spermatogonia in mitotic arrest, surrounded by Sertoli cells. Nuage, electron-dense material associated with mitochondria, was observed in germ cells at all gonadal stages. The Vasa protein was detected in all of the previously described stages within the germ-cell cytoplasm. This is the first report on morphological characteristics and expression of the Vasa gene during sexual differentiation in viviparous species of the Goodeidae family. Chapalichthys encaustus may serve as a model to study processes of sexual differentiation in viviparous fishes and teleosts.     

The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus)

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.     

Ontogenetic testicular development and spermatogenesis in rays: the Cownose Ray,Rhinoptera bonasus,as a model

An understanding of testicular anatomy, development, and seasonality has implications for studies of morphology, behavior, physiology, and bioenergetics of males. Ontogenetic testicular development and spermatogenesis is essentially unknown for chondrichthyans. We examined embryo, juvenile, and adult male Cownose Rays (Rhinoptera bonasus) during development and throughout the annual reproductive cycle. Spermatogonia and Sertoli cells originated from germ cells and somatic cells, respectively, in the embryonic testicular germinal epithelium. In embryos and small juveniles, discrete regions of spermatocyst production appeared within a series of papillae that projected from the dorsal surface of each testis. Because these papillary germinal zones appeared to proliferate through ontogeny, we hypothesize that (1) the germinal zones of juvenile and adult testes are derived from embryonic testicular papillae that form from the germinal epithelium and (2) the papillae become the dorso-central portion of the distinct testicular lobes that form at maturation due to increased spermatocyst production. Our observations indicate that testicular development and the process of spermatogenesis began during embryonic development and increased in scale through ontogeny until maturation, when distinct testicular lobes formed and began enlarging or shrinking based on the annual reproductive cycle. Gonadosomatic indices peaked corresponding to seasonal increased sperm production between January and April, just prior to the April–June mating period. In all life stages, spermatocysts had efferent ducts associated with them from their formation through all stages of development. Year-round presence in the Charlotte Harbor estuarine system, Florida made R. bonasus a good model for beginning to understand ontogenetic gonad development and spermatogenesis in chondrichthyans, especially viviparous rays.     

Organization of the male gonad in a protogynous fish,Thalassoma bifasciatum (Teleostei: Labridae)

Although the testis in teleosts has been investigated for many years, little attention has been paid to the structure of the outer layers that enclose the testis and to their possible contributions to its organization. The present study in a protogynous male labrid, Thalassoma bifasciatum (bluehead wrasse), describes the arrangement and cytology of these tissues (for convenience, referred to collectively as the outer wall, OW) which include: the outer peritoneal layer and subjacent collagen fibers, myoid cells and diverse other cells and tissues, e.g., fibrocytes, presumptive mesenchyme, macrophages, granulocytes, nerves, and blood vessels. Beneath the OW are two compartments; one is the gamete-laden spermatocysts, the other the interstitium, which is composed of cells and tissues that lie between the spermatocysts. Both OW and interstitium contain similar kinds of tissues and cells. Moreover, the layers of the OW immediately subjacent to the peritoneum are continuous with that in the interstitium. It is suggested that the continuity between these two areas provides opportunities for the exchange of cells that could aid in the maintenance and reorganization of the testis and with the myoid and neural tissue to establish an extensive, coordinated motile system that aids movement of sperm from spermatocysts to the ducts. A recent report on the reexamination of the germinal epithelium concept and its identification in the common snook, Centropomus undecimalis, stimulated us to examine the feasibility of applying this concept to gonad organization and gamete development in T. bifasciatum. In addition, the ultrastructure of the Sertoli cell and formation of spermatocysts are described. Spermatocysts increase in size during the development of gametes. Observations and discussion are presented suggesting how Sertoli cells may accommodate this growth and how new populations of these cells may arise in the mature adult. Finally, ultrastructural characteristics for each stage of spermatogenesis are presented and, using (3H)thymidine and autoradiography, data on the chronology of spermatogonia-sperm cycle are included.     

Ameboid cells in spermatogenic cysts of caecilian testis

Sertoli cells constitute a permanent feature of the testis lobules in caecilians irrespective of the functional state of the testis. The developing germ cells are intimately associated with the Sertoli cells, which are adherent to the basal lamina, until spermiation. There are irregularly shaped cells in the cores of the testis lobules that interact with germ cells at the face opposite to their attachment with Sertoli cells. These irregularly shaped (ameboid) cells first appear in the lumen of the cysts containing primary spermatocytes and are continually present until spermiation. We did not observe any cytoplasmic continuity between a Sertoli cell and an ameboid cell. Both light microscopic and TEM observations reveal a phagocytic role for the ameboid cells: they scavenge the residual bodies shed by spermatozoa. Organization of the ameboid cells is grossly different from that of the spermatogenic and Sertoli cells. They appear to develop from the epithelium at the juncture of the collecting ductule with the testis lobule.     

Ultrastructure and histochemistry of the testicular efferent duct system and spermiogenesis in Opistognathus whitehurstii (Teleostei, Trachinoidei)

 Testis organization and spermatogenesis, with the emphasis on spermiogenesis, in Opistognathus whitehurstii are described by ultrastructural and histochemical methods. The germinal epithelium is extremely reduced and restricted to the periphery of the testis, while most of the organ is occupied by a highly developed system of testicular efferent ducts. A semicystic type of spermatogenesis is observed and in the germinal epithelium spermatogenesis occurs only until the spermatidal stage. Young spermatids are released into the lumen of the testicular lobules and mature to sperm within the efferent duct system. The epithelial cells of these ducts are involved in protein and glycogen secretion and in phagocytosis of degenerating germ cells and residual bodies cast off by developing spermatids. On the basis of these functions, the testicular efferent duct system cells are considered to be homologous to the Sertoli cells. A correlation between a highly developed testicular efferent duct system and semicystic spermatogenesis is examined and a possible functional meaning of this apparently unusual mode of sperm production is proposed. Accepted: 18 March 1997     

A sex cord‐like structure and some remarkable features in early gonadal sex differentiation in the marine teleost Siganus guttatus(Bloch)

Several notable features of early gonadal sex differentiation in the golden rabbitfish Siganus guttatus are described including the first report among teleosts of a distinctive dual structure, consisting of somatic cells directly enclosing germ cells (sex cord‐like structure, SCS) and outer somatic tissue surrounding the SCS, in both undifferentiated and early differentiated gonads. Germ cells occurred and proliferated exclusively in the SCS during the process of ovarian and testicular differentiation. A second remarkable characteristic was the delayed germinal cell proliferation for oogenesis in the ovary, that commenced simultaneously with that in the testis, a relatively long time after the onset of somatic development. These observations suggest the possibility that sex differentiation of germ cells is preceded by some sex specific changes in somatic components of the SCS that are light‐microscopically indistinguishable between the sexes. The third unique feature was the detachment of gonadal tissue, including both somatic and germ cells, into the ovarian cavity in the ovary and into the seminiferous lobules and main seminal duct in the testis. This phenomenon occurred in the testis, forming the efferent duct network after 73 days post‐hatch (DPH), and in the ovaries, forming the ovigerous lamellae and regulating the number of oocytes attaining full maturation at c . 129 DPH.     

Phagocytosis of sperm by follicle cells of the carnivorous sponge Asbestopluma occidentalis (Porifera, Demospongiae)

During spermatogenesis of the carnivorous sponge Asbestopluma occidentalis, follicle cells that lined the spermatocysts phagocytosed unreleased mature sperm. Such follicle cells are part of the complex envelope that limits spermatocysts of A. occidentalis, which is also comprised of a collagen layer, a thick layer of intertwined cells, and spicules. Follicle cells showed vesicles containing single phagocytosed spermatozoa within their cytoplasm. Additionally, lipids and other inclusions were observed within the cytoplasm of follicle cells. It is likely that follicle cells recapture nutrients by phagocytosing spermatozoa and use them to form lipids and other inclusions. Such sperm phagocytosis is usually performed in higher invertebrates and vertebrates by Sertoli cells that are located in the testis wall. While Sertoli cells develop a wide range of functions such as creating a blood-testis barrier, providing crucial factors to ensure correct progression of spermatogenesis, and phagocytosis of aberrant, degenerating, and unreleased sperm cells, sponge follicle cells may only display phagocytotic activity on spermatogenic cells.     

Histological and ultrastructural studies on the effects of hCG on sex inversion in the protogynous teleost Coris julis

In the protogynous diandric teleost Coris julis , sex inversion can be induced by injection of human chorionic gonadotropin (hCG). The histological changes of the gonad are accompanied by a transformation of the female livery to that of secondary males. At the ultrastructural level, the presence of intragonadal primordial germ cells in spermatogonial nests and within the newly forming seminiferous lobules is shown. Leydig cells and granulocytes appear in the inverting gonads. The results support the assumption that gonadotropic hormone has a key role in protogynous sex change.     

A scanning electron microscopic study of germ cell maturation in the reproductive tract of the male soupfin shark (Galeorhinus galeus)

Germ cell maturation in the reproductive tract of the soupfin shark (Galeorhinus galeus) was studied using scanning electron microscopy (SEM). The SEM showed changes in Sertoli cytoplasm volume during spermatogenic development. In immature spermatocysts in the germinal zone, spermatogonia were embedded in Sertoli cytoplasm. In spermatogonial spermatocysts, Sertoli cells were adluminally located in the spermatocyst, with spermatogonia enveloped in the basal portions of the cytoplasm. During the round spermatid stage, Sertoli cytoplasm was very scanty. Spermatid elongation was accompanied by a progressive increase in the volume of Sertoli cytoplasm, notably around the spermatid heads. In the mature spermatocyst, bundles of spermatozoa are totally enveloped by Sertoli cytoplasm. Spermatozoa occurred randomly in the epididymis. However, in the ampulla ductus deferentis, spermatozoa reaggregated and were embedded in a mucoid substance to form highly ordered spherical bundles. In the sperm bundle, the spermatozoa heads were arranged such that the helical turns of adjacent spermatozoa were precisely aligned, and all the heads in the bundle formed a distinct apex. This study demonstrates the utility of exploring the relationship between germ cells and Sertoli cells in an evolutionarily ancient vertebrate, such as the shark.     

Germ cell deficiency causes testis cord differentiation in reconstituted mouse fetal ovaries

Sex-reversal in fetal ovaries was studied by using a dissociation-reconstitution technique. Gonads of 12.5 gestation-day male and female mouse fetuses were dissociated into single cells. To eliminate germ cells, the dissociated cells were cultured for 14 h, and then somatic cells attached to culture dishes were harvested and aggregated by gyratory culture for 24 h. The aggregates were then transplanted into ovarian bursa in ovary-ectomized nude mice. The recovered explants were examined histologically. Male somatic cells developed into testes containing Sertoli cells, Leidig cells, and tunica albuginea. Female somatic cells formed testis cords and differentiated into Sertoli cells, but they did not differentiate into other testis components or ovarian tissues. However, aggregates consisting of both female and male somatic cells differentiated into well-developed testes containing Leidig cells and tunica albuginea as well as Sertoli cells. Enzyme marker analysis showed significant contributions of female cells in these organized testes. In contrast, aggregates containing both female germ cells and somatic cells developed into ovaries and did not differentiate into any testicular tissues. The results indicate that female somatic cells in fetal gonads at 12.5 gestation day have the potency to form testis cords and differentiate into Sertoli cells. The subsequent steps in testis development require the contributions of male cells. The present study also suggests that testicular differentiation is independent of germ cells but ovarian development involves the interaction between germ cells and somatic cells.     

Histological process and dynamics of germ cell degeneration in pejerrey Odontesthes bonariensis larvae and juveniles during exposure to warm water

Elevated temperature causes degeneration and disappearance of the germ cells in the males of scrotal mammals. It was recently shown that heat-induced germ cell degeneration occurs also in fish but, unlike in mammals, it occurs not only in males but also in females. The purpose of this study was to clarify the histological process and dynamics of heat-induced germ cell disappearance in pejerrey Odontesthes bonariensis larvae and juveniles. Monosex and mixed-sex fish produced by thermal manipulation of sex (temperature-dependent sex determination) were subjected to 29 degrees C for periods between 1 and 12 weeks, and used to analyze, by histological methods, the changes in gonadal size and the number of normal and degenerating germ cells. Groups exposed to 29 degrees C for 8-12 weeks were subsequently transferred to 24 degrees C to verify if any gonadal damage would be permanent. Germ cell degeneration, histologically characterized by nuclear pyknosis or eosinophilia and cytoplasmic eosinophilia, was observed with increasing frequency at higher temperatures (29>24> 17 degrees C) and more in males than in females. Clear degenerative changes in the germinal epithelium usually began within one week of exposure to 29 degrees C and appeared clearer in females than in males. Complete loss of germ cells was observed only in individuals exposed for periods of 8-12 weeks to 29 degrees C but no treatment produced 100% sterile fish. Germ cells that remained in the gonads after exposure to 29 degrees C retained the capacity to rapidly recolonize germ cell-depleted areas, suggesting that the associated somatic cells in the gonads are little or not affected by this temperature.