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.     

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis

Summary The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.     

Early stages of testicular differentiation in the rat

Summary The initial phases of the development of the seminiferous cords (future seminiferous tubules) were studied with histological techniques and with electron microscopy. On day 14 after fertilization, seminiferous cords are well differentiated in the anterior part of the testis near the mesonephric tubules. They comprise Sertoli cells which encompass the primordial germ cells. The Sertoli cells show an expanded clear cytoplasm and microfilaments beneath the outer surface; they differentiate complex contact zones. On day 13 a few cells localized near the mesonephric tubules display the characteristics of the Sertoli cells. These cells become more and more numerous. They aggregate and they form the seminiferous cords.The primordia of male gonads explanted in vitro on the mesonephros, realize testicular organogenesis in a synthetic medium. Adding 15% fetal calf serum to the medium prevents the morphogenesis of the testicular cords, although the Sertoli cells seem to differentiate morphologically and physiologically. In these gonads differentiation of the Sertoli cells was obtained but their aggregation and the morphogenesis of the seminiferous cords were prevented. This gives new insights into testicular morphogenesis and probably provides an experimental model for a new type of gonadal anomaly.     

Testis developmental phenotypes in neurotropin receptor trkA and trkC null mutations: role in formation of seminiferous cords and germ cell survival

The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation.     

Immunolocalization of type IV collagen and laminin during rat gonadal morphogenesis and postnatal development of the testis and epididymis

The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.     

Gonadal sex differentiation in the neonatal marsupial, Monodelphis domestica

A quantitative and histological study of the gonads of newborn grey short-tailed opossums, Monodelphis domestica, is described. The pups were karyotyped, and comparisons were made within litters segregating for XX and XY sex chromosomes. A total of four litters including 25 pups were available. On the day of birth, developing testes were significantly larger than the ovaries of litter mates, and testes could be histologically distinguished by the formation of sex cords and a tunica albuginea. The data suggest that in this marsupial species gonadal differentiation may be initiated in utero.     

Intermediate filaments and epithelial differentiation of male rat embryonic gonad

The presence and distribution of desmin, vimentin, cytokeratin, and laminin in the gonads of developing male rat embryos (11-17 days) were studied by immunocytochemistry. The findings were correlated with morphological changes of the cells and with the formation of basement membranes, as determined by electron microscopy. The surface epithelial and subepithelial cells of the meesonephros in the prospective gonadal region contained desmin. At the onset of gonadal development, vimentin appeared in the somatic cells of the thickening surface epithelium, which formed the gonadal ridge. Desmin disappeared and cytokeratins appeared in the Sertoli precursor cells at the inception of their epithelial differentiation. Simultaneously, the prospective Sertoli cells became polarized during their assembly into epithelial cell aggregates; the aggregates then fused and formed elongated testicular cords. The epithelial cell differentiation was accompanied by a deposition of basement membrane material around the cords and by an increase of desmin in the cells immediately around the cords. With further differentiation of the testicular cords, some cytokeratins from the Sertoli cells, but not from the cells of the rete cords, disappeared. On the other hand, other cytokeratin polypeptides and vimentin remained in the fetal Sertoli cells. The surface cell layer slowly differentiated towards a proper epithelium after the basic formation of the testicular cords and interstitium. Desmin and vimentin persisted in the interstitial cells throughout the entire study period. The early differentiation of the gonad is apparently under a general sex-independent initiation program. The developmental changes in intermediate filaments offer an opportunity for the further analysis of their general role in early organogenesis. In light of the genetic theory of testicular differentiation, the functions of the regulatory factor(s) include specific organization of cord cells, histological organization into looping cords rather than separated follicles, and male development of the interstitium, surface epithelium and tunica albuginea.     

Steroid hormone content of the gonads of the tammar wallaby during sexual differentiation.

The gonads of the tammar wallaby, Macropus eugenii, are sexually indifferent at birth (Day 0) despite the fact that phenotypic sexual differentiation has already commenced as evidenced by the presence of a scrotum in males and mammary anlagen in females. The seminiferous cords of the testis first become clearly recognizable on Day 2 of pouch life, and ovarian differentiation is recognizable by Day 10. To monitor the endocrine development of the gonads during sexual differentiation of the urogenital tract, we measured the steroid hormone content in 92 pools of gonads from male and female tammar pouch young from the day of birth to 206 days of pouch life. Progesterone, estradiol, and dihydrotestosterone concentrations were low (less than 0.05 ng/mg protein) in both ovaries and testes at all stages examined, and testosterone concentrations were uniformly low in ovaries. Testosterone concentrations in testes were low on Days 0-4, averaging about 0.2 ng/mg protein; they rose by Days 5-10 to an average of 0.9 ng/mg protein, remained elevated until about Day 40, and thereafter fell to values similar to those in the ovaries. The phallus and urogenital sinus were able to convert testosterone to dihydrotestosterone from the earliest stages examined (Days 10 and 11). Thus in the tammar wallaby, as in eutherian mammals, testosterone is the androgen secreted by the developing testis, and dihydrotestosterone is formed in certain androgen target tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression during gonadal differentiation in the rat: A two-dimensional gel electrophoresis investigation

Gonadal protein patterns were studied during development in the rat by two-dimensional micro-gel electrophoresis. Specific proteins were detected in both the male and the female sex at the morphologically indifferent state (two female- and one male-specific) and during differentiation. At the onset of gonadal differentiation (day 14) two additional sex-specific proteins were discovered in the male and two in the female. These proteins remained expressed during further development. One testicular protein was restricted to the cytosol of the tunica albuginea. The other one was absent from the tunica. In the female gonad, the two proteins were membrane-specific, one present in germ cells, the other in somatic cells. In the testis, one additional protein was discovered at postnatal day 1. Thus according to biochemical criteria there is no indifferent state of gonadal development. The testis and ovary express sex-specific genes both before and after the onset of gonadal differentiation.     

Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to it in vitro

To determine whether anti-Müllerian hormone (AMH) is responsible for the gonadal lesions observed in bovine genetic females united by placental anastomoses to male twins (freemartins), prospective ovaries of fetal rats were exposed to purified bovine AMH in vitro. In cultures initiated at 14 days p.c. and maintained 3 to 10 days, AMH consistently induced a characteristic 'freemartin effect', namely reduction of gonadal volume, germ cell depletion and differentiation, in the gonadal blastema, of epithelial cells with large clear cytoplasm linked by interdigitations, resembling rat fetal Sertoli cells. These cells tend to become polarized and form cords, delineated by a continuous basal membrane containing laminin and fibronectin. Such structures, resembling developing seminiferous cords, were not detected in control ovarian cultures. These data strongly suggest that AMH is the testicular factor responsible for triggering the morphological abnormalities of freemartin gonads.     

Sexual differentiation of germ cell deficient gonads in the medaka, Oryzias latipes

To determine whether germ cells perform any function in gonadal sexual differentiation, development of gonads in the medaka, Oryzias latipes, after exposure to busulfan was investigated. Busulfan suppressed proliferation of early germ cells, thus significantly reducing the number of germ cells and generating regions without germ cells in the developing gonads. Globular structures were observed in the parenchyma in these regions. The structure was male specific, developed at the same time as acinus (seminiferous tubule precursor), surrounded by the basal lamina, and contained characteristic desmosomes. These results strongly suggest that these globular structures are the precursors of seminiferous tubules devoid of germ cells. In the ovary, no follicles were observed but a well-developed ovarian cavity was evident. From these results we conclude that differentiation of gonadal parenchyma cells, except for follicular ones, is not germ cell dependent, though morphological differentiation of the somatic cells seems to follow the differentiation of germ cells.     

Differentiation and development of testis in the oviparous lizard, Calotes versicolor (Daud.)

The differentiation and development of the testis in the lizard Calotes versicolor was studied histologically and histoenzymatically from the day of oviposition (stage 27) to 2 months after hatching. The study reveals the appearance of the gonadal component as a genital ridge at stage 27. The first sign of testis differentiation is observed at stage 33, which displays a well-developed medulla consisting of seminiferous cords comprising Pre-Sertoli cells. The sex differentiation of the embryonic gonads occurs at stage 34. At this stage, seminiferous cords of the testis are prominent and extensive with many pre-Sertoli cells and few spermatogonia. The interstitial space consists of immature fibroblast-type Leydig cells. Pre-Sertoli cells of the seminiferous cords differentiate into Sertoli cells with a triangular nucleus becoming apparent around stages 36-37. The fibroblast-like Leydig cells differentiate into round matured Leydig cells at stage 40. Quantitative estimation of germ cells reveals that the number of germ cells increases in individual gonads, and in 5-day-old hatchling's, this number multiplies by manifold. Spermatogonia show reductional division in the testis of 1-day-old hatchlings.Histochemical localization of Delta5-3beta-HSDH and G-6-PDH activity appears in the seminiferous cords (medulla) of the testis after sexual differentiation (stage 36), indicating that the embryonic medulla is the site of steroidogenesis and not the cortex in C. versicolor. This study also suggests that morphological differentiation of the gonad precedes detectable steroidogenesis in this species. In 10-day-old hatchling's, Delta5-3beta-HSDH activity is seen in the interstitial cells of the testis, which, however, is not detected in the seminiferous tubules. The intensity of the enzyme activity remains more or less the same in the testis up to 10 days after hatching and begins to increase thereafter. The increase in steroidogenesis parallels the progressive post-hatching increase of the interstitial/Leydig cells.     

Cytological characterization of the germinal line during testicular differentiation in the lizard Liolaemus gravenhorsti (gray)

The gross morphology, histology, and ultrastructure of Liolaemus gravenhorsti gonads prior to and after differntiation are described. Special emphasis has been given to characterization and changes of the germ cell line throughout intrauterine development and 3 days postpartum. During the pregonadal stage, the primordial germ cell migrates toward gonadal rudiments by way of the mesenchyme. These cells can easily be identified by their great size, voluminous and lobulated nucleus, great quantities of yolk platelets, microtubules, and numerous lipid inclusions. In the undifferentiated gonad, the germ cells (type 1 gonocytes) have an ovoid or spherical shape and autodigestion of yolk platelets, great development of Golgi complex, and mitochondrial aggregation, though fewer liposomes, pseudopodes, and microtubules were noted. Concomitantly with the beginning of mitosis, a third type of germ cell appears, the type 2 gonocytes, which are smaller, with poorly defined membranous systems in various degrees of involution. The seminiferous cords are organized when somatic cells of the medullar portion of the gonad surround type 1 gonocytes. Germinal cells are centrally localized in the cords. Near birth many gonocytes migrate toward the basal lamina of cords and differentiate into spherical prespermatogonia, with few organoids. Sertoli cells eparate them from the basal lamina. In advanced pregnancy, Leyding cells become numerous with morphology typical of androgen-producing cells.     

Effects of steroid sex hormones on chick embryo gonads in organ culture, with special reference to hormonal control of gonadal sex differentiation

At the initial stages of sex differentiation (7.5 and 8.5 days of incubation), chick embryo gonads were treated directly with testosterone or estradiol-17 beta in organ cultures. Chemically-defined media containing cholesterol as a steroid precursor were used. The differentiation of gonads in the 10 to 12-day controls, cultured in media containing no hormones, was close to that of gonads of equivalent age in ovo. Testosterone added to the medium exerted an inhibitory effect on the cortex of the female gonad and a masculinizing one on its medulla. The results of estradiol treatment confirmed the known feminizing effect of that hormone on the male gonad, the meiotic prophase in the genetically male germ cells being initiated in the induced cortex. These data may be interpreted in favour of a bihormonal theory of gonadal sex differentiation in birds, where the predominantly-synthesized male or female hormone in the gonad determines the male or female pattern of development of the corresponding gonad.     

Differential expression of acidic cytokeratins 18 and 19 during sexual differentiation of the rat gonad.

The expression of cytokeratins (CKs) 8, 18 and 19 was analyzed in male and female rat gonads from the undifferentiated stage (12.5 days of gestation) until two weeks after birth by indirect immunofluorescence, using specific monoclonal antibodies anti-CK 8 (LE41), anti-CK 19 (LP2K) and anti-CK 18 (LE65 and RGE53). In the undifferentiated blastema, the somatic cells were stained for CK 8 and CK 19, whereas no detectable immunoreactivity for CK 18 was obtained. The same staining CK pattern was observed in ovaries, in the somatic cells of ovigerous cords and in primary follicles. The staining was progressively decreasing in growing follicles after one week after birth. At the onset of testicular differentiation, when the first Sertoli cells differentiate in the gonad of 13.5-day old male fetuses, positive staining for CK 18 became evident, in addition to CK 8 and CK 19 expression. In the following days, CK 8, CK 18 and CK 19 were detected in Sertoli cells in the differentiating seminiferous cords, but progressively the reactivity for CK 19 decreased and was no longer observed after 18.5-19.5 days of gestation. In all cases, CKs were found to be coexpressed with vimentin, and germ cells were negative for both vimentin and CKs. The results reported here show first, that CKs are expressed before sexual differentiation in gonadal blastema in which no epithelial organization is observed, and second, that there is a CK 18/CK 19 shift in expression during morphogenesis of the testis which is not observed in the differentiating ovary. Future studies will have to determine whether these differences in CK expression are due to epitope-masking phenomena or to the regulation of CK synthesis.     

Posthatching development of Alligator mississippiensis ovary and testis

We investigated ovary and testis development of Alligator mississippiensis during the first 5 months posthatch. To better describe follicle assembly and seminiferous cord development, we used histochemical techniques to detect carbohydrate‐rich extracellular matrix components in 1‐week, 1‐month, 3‐month, and 5‐month‐old gonads. We found profound morphological changes in both ovary and testis. During this time, oogenesis progressed up to diplotene arrest and meiotic germ cells increasingly interacted with follicular cells. Concomitant with follicles becoming invested with full complements of granulosa cells, a periodic acid Schiff's (PAS)‐positive basement membrane formed. As follicles enlarged and thecal layers were observed, basement membranes and thecal compartments gained periodic acid‐methionine silver (PAMS)‐reactive fibers. The ovarian medulla increased first PAS‐ and then PAMS reactivity as it fragmented into wide lacunae lined with low cuboidal to squamous epithelia. During this same period, testicular germ cells found along the tubule margins were observed progressing from spermatogonia to round spermatids located within the center of tubules. Accompanying this meiotic development, interstitial Leydig cell clusters become more visible and testicular capsules thickened. During the observed testis development, the thickening tunica albuginea and widening interstitial tissues showed increasing PAS‐ and PAMS reactivity. We observed putative intersex structures in both ovary and testis. On the coelomic aspect of testes were cell clusters with germ cell morphology and at the posterior end of ovaries, we observed “medullary rests” resembling immature testis cords. We hypothesize laboratory conditions accelerated gonad maturation due to optimum conditions, including nutrients and temperature. Laboratory alligators grew more rapidly and with increased body conditions compared with previous measured, field‐caught animals. Additionally, we predict the morphological maturation observed in these gonads is concomitant with increased endocrine activities. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Immunocytochemical and quantitative study of the tunica albuginea testis in young and ageing men

 A light and electron microscope immunohistochemical study of the tunica albuginea from both young and elderly men was carried out to determine the distribution of the cells that contain actin, vimentin and/or desmin, and to evaluate the possible variations with ageing by means of quantitative studies. Testicular volume and testicular parenchyma volume decreased significantly with age whereas the tunica albuginea volume remained unchanged. These results agree with the scanty quantitative changes observed in the testicular connective tissue with age, and the notion that age-related changes in testicular volume are principally restricted to the seminiferous tubules. Three connective tissue layers could be distinguished in the tunica albuginea in both young and elderly men. The middle and inner layers increased in width with age while the width of the outer layer decreased. The average width of the tunica albuginea increased significantly with ageing. The tunica albuginea of young men and elderly men presented two types of fusiform cells: (1) fibroblast-like cells, which immunoreacted to actin and vimentin, but not to desmin; and (2) myoid cells, which immunoreacted to actin, vimentin and desmin. In both young men and elderly men, the total number of desmin-positive cells (myoid cells) was significantly lower than that of fibroblasts. However, the total number of desmin-positive cells was significantly increased in ageing men. In young testes, desmin-positive cells were more abundant in the outer layer of the tunica albuginea, whereas in elderly men these cells predominated in the middle layer. The increased desmin immunoexpression in the tunica albuginea of ageing men contrasts with the decrease in desmin immunoreaction in other myoid cells of the testis, the peritubular myoid cells, but only in seminiferous tubules that showed severe germ cell depletion. This suggests that changes in intermediate filament immunoexpression in peritubular cells are focalised, and thus, under local control, whereas changes in the tunica albuginea cells are generalised and possibly related to factors also affecting the connective tissue in other organs Accepted: 15 January 1997     

The presence of a common embryonic blastema for ovarian and testicular parenchymal (follicular,interstitial and tubular) cells in cattle,Bos taurus

Summary The early embryonic gonadal development in the cattle is characterized by the appearance of an alkaline phosphatase positive blastema. Its derivatives in gonads of both sexes, follicular cells in the female and interstitial cells in the male, also show positive alkaline phosphatase reaction. Primordial germ cells are equally alkaline phosphatase positive, but loose this activity when they later transform to oögonia and oöcytes, or to spermatogonia respectively. Using the enzyme activity as label to trace these constituents in the developmental steps of the bovine gonads, the following results were obtained.Differentiation processes leading to the appearance of the sex cords take place in situ within the gonadal blastema which occupies the main central part of the gonadal fold. It is essentially a segregation process of the follicular cell cords or of the interstitial cells and the tubular primordia from the undifferentiated common anlage.The so-called germinal epithelium is not involved in the differentiation of sex cords. Its participation — if any — in the gonadal development is restricted to a very short and rather early period. Secondary sex cords (Pflügers cords) do not occur. In the cattle there is no reason to assume a cortico-medullary antagonism in the sex determined gonadal development.It can be assumed that the follicular cells in the ovary and the interstitial cells in the testis are homologous. This applies possibly also to the tubular cells of the testis. Homology should be admitted also for the rete structures, which remain small and undeveloped in the ovary while in the male they show considerable development.In the ovary the follicular cell cords differentiating within the central blastema match in a junctional zone with the peripheral layer of oögonia. These are taken up by the most peripheral branches of the follicular cell cords, thus transforming to ovigerous cords. During the downward movement within these cords the germ cells transform to oöcytes which for their part proceed through first meiotic prophase and reach the dictyotene stage. The maturation of the germ cells seems to be controlled by the follicular cells and may even temporarily get out of control until an adequate number of follicular cells is found in vicinity of individual oöcytes to form primordial follicles.The alkaline phosphatase reaction reveals the presence of numerous persisting remnants of follicular cell cords in the developing and even adult ovary.It is suggested that the findings in the cattle gonads can be applied also to other mammals, mainly to those with longer gestation periods like man.Contribution No 58-66, Department of Biology, City of Hope Medical Center. This work was supported in part by a grant (CA 05138) from the National Cancer Institute, U.S. Public Health Service. The project was undertaken during a five-month visit to Dr. Ohno's laboratory by the senior author whose expenses were covered by the Deutsche Forschungsgemeinschaft.     

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.