Presumptive Ovaries Research Articles

Chronology of Gonadal Development in the Malayan Snail-eating Turtle Malayemys macrocephala.

The snail-eating turtle, Malayemys macrocephala, is a common freshwater turtle that can be used as an animal model for developmental biology. However, a thorough investigation of its development is needed before this species can be used as a model. Thus, this study aimed to examine the gonadal development of M. macrocephala. Turtle eggs were collected from rice fields in Phra Nakhon Si Ayutthaya Province, Thailand, and transported to the laboratory. Eggs were incubated in microprocessor-controlled incubators and randomly dissected on a weekly basis to reveal the developing embryos, then their developmental stage was identified according to Yntema (1968). Primordial germ cells and gonad structure were processed through the paraffin method. Moreover, the dynamics of germ cell proliferation and apoptosis were examined by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Examination of the gonad revealed four main stages of gonadal development: (i) germ cell migration, (ii) genital ridge appearance, (iii) testicular formation, and (iv) ovarian formation. In the male turtle (incubated at 26°C), gonad developed into the testis with medullary sex cords starting at Yntema stage 17. In the female turtle (incubated at 32°C), these sex cords then degenerated, followed by cortical development into an ovarian structure starting at Yntema stage 19. Subsequently, testicular and ovarian development occurred independently, and distinct sex organs were apparent at Yntema stage 25. In addition, the presumptive testis showed germ cell proliferation in the medulla at Yntema stages 17, 19, and 25 and germ cell apoptosis in the cortex at Yntema stages 19 and 25. The presumptive ovary showed germ cell proliferation in the cortex at Yntema stages 19 and 25, and germ cell apoptosis in the medulla at Yntema stages 19 and 25.

Gonadogenesis analysis and sex differentiation in cultured turbot (Scophthalmus maximus).

As a flatfish, the turbot (Scophthalmus maximus) is one of the most important farmed fish species with great commercial value, which has a strong sexual dimorphism on growth rate and sexual maturity. In this study, using histology, the basic information on proliferation and migration of germ cells and early gonadal development during sex differentiation in turbot were described in detail. There were six to nine individual primordial germ cells (PGCs) with large nuclei until 15days post-hatching (dph). The PGCs located under the mesonephric ducts undergo migration along the dorsal mesentery toward the region of the genital ridge from 0 to 15dph. During migration, the number of PGCs was constant, and the expression of vasa had no significant changes. At 20dph, the aggregation of somatic cells at genital ridge indicated the formation of primary gonad. Furthermore, the number of PGCs was increased to 60 and the expression of vasa was upregulated for the first time. The undifferentiated gonads with no morphological indications of sex differentiation grew larger with the increase in germ cells and somatic cells number/size from 20 to 35dph. During 36-52dph, cytological gonadal differentiation was observed. In presumptive testes of type I gonadal tissue (with a lance shape), the number of germ cells increased steadily and the germ cells had the same characteristics as before. Meanwhile, in presumptive ovaries of type II gonadal tissue (with a club-like shape), the germ cells proliferated and induced in two different populations of germ cells. One type had the morphological characteristics as undifferentiated germ cells, while the other type of germ cells underwent mitosis exhibiting smaller size and mottled nuclei. At 60dph, ovarian cavity was present in the gonad of type II, which would develop into ovaries. However, spermatogonial cysts were not noticed in the gonad of type I until 90dph, which indicated the formation of the testes.

Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressing cells in Lepidochelys olivacea

Bipotential gonads represent the structural framework from which alternative molecular sex determination networks have evolved. Maintenance of Sox9 expression in Sertoli cells is required for the structural and functional integrity of male gonads in mammals and probably in most amniote vertebrates. However, spatial and temporal patterns of Sox9 expression have diversified along evolution. Species with temperature sex determination are an interesting predictive model since one of two alternative developmental outcomes, either ovary or testis occurs under controlled laboratory conditions. In the sea turtle Lepidochelys olivacea, Sox9 is expressed in the medullary cords of bipotential gonads when incubated at both female- or male-promoting temperature (FT or MT). Sox9 is then turned off in presumptive ovaries, while it remains turned on in testes. In the current study, Sox9 was used as a marker of the medullary cell lineage to investigate if the medullary cords originate from mesothelial cells at the genital ridges where Sox9 is upregulated, or, if they derive from a cell population specified at an earlier developmental stage, which maintains Sox9 expression. Using immunofluorescence and in situ hybridization, embryos were analyzed prior to, during and after gonadal sex determination. A T-shaped domain (T-Dom) formed by cytokeratin (CK), N-cadherin (Ncad) and SOX9-expressing cells was found at the upper part of the hindgut dorsal mesentery. The arms of the T-Dom were extended to both sides towards the ventromedial mesonephric ridge before the thickening of the genital ridges, indicating that they contained gonadal epithelial cell precursors. Thereafter, expression of Sox9 was maintained in medullary cords while it was downregulated at the surface epithelium of bipotential gonads in both FT and MT. This result contrasts with observations in mammals and birds, in which Sox9 upregulation starts at a later stage in the inner cells underlying the Sox9-negative surface epithelium, suggesting that the establishment of a self-regulatory Sox9 loop required for Sertoli cell determination has evolved. The T-shaped domain at the upper part of the hindgut dorsal mesentery found in the current study may represent the earliest precursor of the genital ridges, previously unnoticed in amniote vertebrates.

Gonadal sex differentiation in the bluegill sunfish Lepomis macrochirus and its relation to fish size and age

Bluegill (Lepomis macrochirus) monosex culture holds considerable potential as a method to improve bluegill aquaculture production as males grow much faster than females. A detailed understanding of the time of gonadal development and differentiation is critical to control sex and optimize culture. In the present study, we systematically studied gonadal sex differentiation of the bluegill sunfish and its relation to fish size and age from hatching to 90days post hatching (dph) using a slow-growing batch (SGB) and a fast-growing batch (FGB) of fish. The results indicated that the gonadal differentiation in bluegill was more related to body size than to age. In presumptive ovaries, germinal and somatic differentiation began between 13.2 and 16.0mm (60dph in SGB and 30dph in FGB) in total length (TL). The outgrowths from the proximal and distal portions of the gonads and the fusion of the outgrowths to form the ovarian cavity occurred between 16.0 and 21.0mm TL (80dph in SGB and 50dph in FGB) with germ cells undergoing meiosis. The gonads in the females larger than 25.5mm TL always had peri-nucleolus oocytes. In presumptive testes, the efferent duct formed in the fish ranging from 25.4 to 28.0mm TL (90dph in SGB and 70dph in FGB) with the onset of meiosis and testes contained spermatocytes exhibiting active meiosis in males larger than 33.0mm TL. These findings indicate that bluegill is a differentiated gonochorist and sex differentiation occurs earlier in females than males. Based on our results, we suggest that the critical period of sex differentiation in bluegill occurs between 13.2 and 16.0mm TL and histological sex differentiation is distinguishable in most fish larger than 21.0mm TL.

Gonadal development and sex differentiation in the cichlid fishCichlasoma dimerus (Teleostei, perciformes): A light- and electron-microscopic study

Although the overall pattern and timing of gonadal sex differentiation have been established in a considerable number of teleosts, the ultrastructure of early stages of gonadal development is not well documented. In this study, gonads from larval and juvenile stages of laboratory-reared Cichlasoma dimerus were examined at the light-microscopic and ultrastructural levels. This freshwater species adapts easily to captivity and spawns with high frequency during 8 months of the year, providing an appropriate model for developmental studies. Larvae and juveniles were kept at a water temperature of 26.5 +/- 1 degrees C and a 12:12 hour photoperiod. Gonadal development was documented from 14-100 days postfertilization, covering the period of histologically discernible sex differentiation. Gonadal tissue was processed according to standard techniques for light and electron microscopy. C. dimerus, a perciform teleost, is classified as a differentiated gonochorist, in which an indifferent gonad develops directly into a testis or ovary. On day 14, the gonadal primordium consists of a few germ cells surrounded by enveloping somatic cells. Ovarian differentiation precedes testicular differentiation, as usual in teleost fishes. The earliest signs of differentiation, detected from day 42 onward, include the onset of meiotic activity in newly formed oocytes, which is soon accompanied by increased oogonial mitotic proliferation and the somatic reorganization of the presumptive ovary. The ovarian cavity is completely formed by day 65. Numerous follicles containing perinucleolar oocytes are observed by day 100. In contrast, signs of morphological differentiation in the presumptive testis are not observed until day 72. By day 100, the unrestricted lobular organization of the testis is evident. The latest stage of spermatogenesis observed by this time of testicular development is spermatocyte II.

Morphological development of the gonads in zebrafish

Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine‐active compounds. At age 2 weeks post‐fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week‐old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst‐like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.

Gonadal differentiation and effects of temperature on sex determination in southern flounder ( Paralichthys lethostigma)

Southern flounder ( Paralichthys lethostigma) support valuable North American fisheries and show great promise for aquaculture. Because females grow faster and reach larger adult sizes than males, monosex culture of females is desirable for commercial operations. A detailed understanding of sexual development and its timing is critical to control sex and optimize culture. Structural and cellular sex-distinguishing markers were identified histologically, and then used to describe ovarian development in female and testicular development in male flounder. In presumptive ovaries of southern flounder, development of an ovarian cavity first occurs in fish ranging from 75 to 100 mm total length (TL). This is considerably delayed relative to that observed in the Japanese congener, Paralichthys olivaceus, where an ovarian cavity is seen in fish as small as 40 mm TL. The smallest southern flounder that possessed primary oocytes in the early perinucleolus stage was 115 mm TL. In presumptive testes, the formation of seminiferous tubules first occurs in fish of approximately 100 mm TL. Spermatogonia remained quiescent until most fish were over 100 mm TL. Overall, gonads from southern flounder greater than 120 mm TL commonly possess gonial cells undergoing meiosis, clearly differentiating sex. The effect of temperature on sex determination in southern flounder was addressed in a separate experiment. Juvenile southern flounder were grown at 18, 23, or 28°C for 245 days. High and low temperatures induced phenotypic sex reversal in juvenile southern flounder, producing a higher proportion of males (96% males at high temperature, P<0.001, 78% males at low temperature, P<0.01). Raising southern flounder at the midrange temperature held sex ratios close to 1:1. Sex ratios from these trials suggest that southern flounder possess a temperature-sensitive mechanism of sex determination similar to that shown for P. olivaceus, but possibly shifted towards warmer temperatures. These findings indicate that sex differentiation in southern flounder is distinguishable in most fish by 100–120 mm TL and that sex determination is sensitive to temperature. This information is critical to the development of strategies to maximize the number of faster-growing females for commercial flounder culture.

The P450 Aromatase (P450 arom) Gene Is Asymmetrically Expressed in a Critical Period for Gonadal Sexual Differentiation in the Chick

Steroid hormones appear to play an important role in gonadal sex differentiation of birds. Here we studied the steady-state level of the P450 arom mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) in the left and the right presumptive ovary and testis of developing chicken embryos. The gonads were evaluated every hour during the undifferentiated period, at 144–156 h of incubation (h/i), and every 24 h after sexual differentiation at 168 and 192 h/i. Activity of P450 arom was determined by estrone production from [3H]androstenedione at 144–192 h/i. Moreover, morphological development of the gonad was also examined by light microscopy. Results show that onset of P450 arom mRNA and its protein activity were simultaneously detected in the left and the right ovaries at 147 h/i. Asymmetric function of P450 arom gene expression was observed at 156 h/i when morphological gonadal differentiation is first recognized. Biotransformation of [3H]androstenedione to estrone was also asymmetrically detected between the left and right gonad at 156 h/i and asymmetry was maintained throughout the analyzed stages. It is proposed that there is a gene in birds that is asymmetrically expressed in the undifferentiated stage of the female and the male gonad. In the female this gene could promote P450 arom gene expression, increasing estrogen production, which in turn could induce ovarian cortex proliferation and expression of other structural estrogen-regulated genes involved in ovarian sexual determination.

Psychophysiological and neuropsychological distinction between familial and sporadic schizophrenia

Bipotential gonads represent the structural framework from which alternative molecular sex determination networks have evolved. Maintenance of Sox9 expression in Sertoli cells is required for the structural and functional integrity of male gonads in mammals and probably in most amniote vertebrates. However, spatial and temporal patterns of Sox9 expression have diversified along evolution. Species with temperature sex determination are an interesting predictive model since one of two alternative developmental outcomes, either ovary or testis occurs under controlled laboratory conditions. In the sea turtle Lepidochelys olivacea, Sox9 is expressed in the medullary cords of bipotential gonads when incubated at both female- or male-promoting temperature (FT or MT). Sox9 is then turned off in presumptive ovaries, while it remains turned on in testes. In the current study, Sox9 was used as a marker of the medullary cell lineage to investigate if the medullary cords originate from mesothelial cells at the genital ridges where Sox9 is upregulated, or, if they derive from a cell population specified at an earlier developmental stage, which maintains Sox9 expression. Using immunofluorescence and in situ hybridization, embryos were analyzed prior to, during and after gonadal sex determination. A T-shaped domain (T-Dom) formed by cytokeratin (CK), N-cadherin (Ncad) and SOX9-expressing cells was found at the upper part of the hindgut dorsal mesentery. The arms of the T-Dom were extended to both sides towards the ventromedial mesonephric ridge before the thickening of the genital ridges, indicating that they contained gonadal epithelial cell precursors. Thereafter, expression of Sox9 was maintained in medullary cords while it was downregulated at the surface epithelium of bipotential gonads in both FT and MT. This result contrasts with observations in mammals and birds, in which Sox9 upregulation starts at a later stage in the inner cells underlying the Sox9-negative surface epithelium, suggesting that the establishment of a self-regulatory Sox9 loop required for Sertoli cell determination has evolved. The T-shaped domain at the upper part of the hindgut dorsal mesentery found in the current study may represent the earliest precursor of the genital ridges, previously unnoticed in amniote vertebrates.

Uptake of 3H‐estradiol by embryonic crocodile gonads during the period of sexual differentiation

AbstractEgg incubation temperature controls sexual differentiation of the gonads in the saltwater crocodile, Crocodylus porosus. Embryos incubated at 30°C develop ovaries and hatch as females while most embryos incubated at 32–32.5°C develop testes and hatch as males. Several lines of evidence suggest that estrogen is involved in gonadal sex differentiation in crocodilians and other reptiles with temperature‐dependent sex determination (TSD). In vivo autoradiography was used here to identify sites of 3H‐estradiol uptake in the urogenital tissues of C. porosus embryos incubated at 30°C (female‐producing) and at 32.5°C (predominantly male‐producing). During the period of gonadal differentiation (developmental stage 23), 3H‐estradiol was specifically concentrated in the gonads of presumptive females at 30°C and presumptive males at 32.5°C. Binding was localised primarily in the medulla of the gonad in both sexes, with little or no uptake in the cortex. No uptake was detected in the adjoining adrenal glands, at either temperature, although some uptake was observed in the mesonephros at the female temperature (30°C). No specific binding of estradiol by the gonads was found prior to (stage 19) or after (stage 28) the period of gonadal differentiation. These results are consistent with the hypothesis that estrogen binding sites are present in the gonads of both sexes during a specific period of embryogenesis, which coincides with the time of gonadal sex differentiation. Furthermore, these observations suggest that the role of estrogen in TSD is not mediated through differential uptake of the steroid by presumptive ovaries and testes. It is therefore hypothesised that estrogen synthesis is temperature‐sensitive, rather than estrogen binding by the gonad. © 1994 Wiley‐Liss, Inc.

Gonadal development and differentiation in Alligator mississippiensis at male and female producing incubation temperatures

The development and differentiation of the gonads of embryonic alligators incubated at 30 °C (100% female producing) and 33 °C (100% male producing) was investigated histologically. The stage of development of the gonad and differentiation into an ovary or a testis occurred at essentially the same time at both temperatures. This contrasts with the overall development of the embryos which was slower at the lower temperature. A few days prior to differentiation, gonads grew more quickly at 33 °C than they did at 30 °C. However, once differentiated into a presumptive testis, gonads reduced in volume so that at hatching presumptive testes were smaller than presumptive ovaries. It is hypothesized that synchrony/asynchrony of development of the gonad and the rest of the embryo may account for temperature‐dependent sex determination.

Differentiation and development of the reproductive system in the iguanid lizard, Sceloporus undulatus

Embryos of the lizard Sceloporus undulatus were sampled throughout incubation, and the differentiation and development of the reproductive system was documented histologically. The undifferentiated gonads possess both a cortex and medulla, both of which contain germ cells until embryonic stage 34. Beginning at stage 34, the cortex of the presumptive ovary thickens, and cortical germ cells are more abundant. By the time of hatching, the ovarian cortex is 6 to 10 cells thick and filled with oogonia and oocytes; primordial follicles, however, are not yet present. In males at embryonic stage 34, seminiferous tubules appear in the medulla of the testis, and Sertoli cells begin to differentiate. Seminiferous tubule formation is complete by hatching, and both Sertoli and Leydig cells are apparent. The mullerian ducts develop in both sexes but begin regressing in the male at embryonic stage 37. The wolffian ducts also develop in both sexes and are present in males and females at hatching.

Asymmetrical Development of the Gonads in the Embryos and Fry of the Fish, Oryzias celebensis: (asymmetry/gonad/primordial germ cell/migration/teleost (Oryzias celebensis)).

The development of the gonad in Oryzias celebensis was studied by light and electron microscopy. The path of PGC-migration, the time of the sex-differentiation, and the pattern of germ cell proliferation were almost identical to those in O. latipes. The most conspicuous difference was in the distribution of germ cells after migration. The gonadal anlage in O. celebensis developed only on the right side of the dorsal mesentery, although PGCs were stituated on the both sides of the embryos before migration. The testis retained its unilateral condition throughout development and acquired a unilobed shape. In the female, the right presumptive ovary developed over the mesentery, and the ovary became bilobed. Thereafter, right and left parts of the presumptive ovary fused to develop into a single ovary in the adult fish. The situation appears to be comparable to the gonadal asymmetry in birds, but the present observations suggest that the developmental processes of the asymmetrical gonads in this fish are quite different from those in birds.

Transplantations de gonades indifférenciées chez l’hermaphrodite simultané Eisenia foetida (Oligochète, Lumbricidae): Mise en évidence de facteurs locaux (inducteurs?) de la différenciation sexuelle

ABSTRACT Transplantations of undifferentiated gonads in the simultaneous hermaphrodite Eisenia foetida (Oligochaeta, Lumbricidae’) leading to evidence of local factors (inductors ?) of sexual differentiation The hypothesis of a ♀ sex capable of autodifferentiation and of a ♂ sex depending on an androgen hormone has been verified in several gonochoric or hermaphrodite invertebrates. In order to test the validity of this hypothesis in the hermaphrodite Eisenia foetida we have investigated the influence of the gonads’ environment on their differentiation by transplanting undifferentiated gonads, dorsally and ventrally, on different parts of the body. The gonads, taken with the adjacent wall of the body from new-born worms, are grafted on worms of the same age; some grafts do, others do not, retain their nervous system. The control transplantation in which a graft of a given presumptive sex is transplanted in a region of the same sex can lead to inversions. These inversions have a rare and short-lived character in the gonads coming from presumptive testes. They are more frequent and more or less durable in the gonads coming from presumptive ovaries. These results can only be explained by the existence, within the grafts of the two presumptive sexes, of a dominant relationship between one and the other sexual potentiality which can be momentarily inverted by trophic disturbances caused by the transplantation. The heterosexual transplantations, in which a graft of a given presumptive sex is transplanted in the region of the opposite sex, show that the frequency of the inversions is doubled in relation to the frequency obtained in the control transplantations. The inversions which occur in the presumptive testes can be lasting and even permanent. These results lead to the opinion that to the effects of transplantation is added an inductive action from the host, leading to masculinity in the 3 region and femininity in the ♀ region. The transplantations outside of the sexual ventral regions lead to inversions of which the character and frequency recall those obtained in the control transplantations. They suggest that these regions are devoid of inductive sexualizing power. The very slight variations of frequency of the inversions observed along the antero-posterior and dorso-ventral axes can be explained by the existence of physiological gradients acting on the balance of the graft’s own potentialities. The removal of the nervous system in the grafts shows that this system plays a part, probably trophic, in favour of the masculine potentialities of the graft. In conclusion, our experiments lead to the rejection of the hypothesis of the ♀sex capable of autodifferentiation in Eisenia, in order to adopt that of a sexual balance between the ♂ and ♀ potentialities determining, according to their dominant relationship, the synthesis of (♂or ♀ ‘inductors’ respectively in the ♂ and ♀ ventral regions. These inductors act in turn on the bipotential sexual cells.

Freemartins in cattle: the first steps of sexual organogenesis.

Summary. Multiple pregnancies were obtained in cows treated with gonadotrophic hormones and the fetuses were removed 39 to 62 days after mating. This covers the period of sexual organogenesis in males from the initial differentiation of the testes to the final establishment of most male structures. Twenty-nine pregnancies resulting in heterosexual fetuses with fused chorions and vascular anastomoses provided forty-eight presumptive freemartins. The genital tract was studied histologically and gonadal size was determined by the paper weight method. No difference was observed between control females and presumptive freemartins up to 48 days. Thereafter, growth of the presumptive ovaries practically stopped in freemartins and the diameter of the upper part of their Müllerian ducts decreased, as in males. Contrary to older views, the first freemartin effects occurred at the same stage (49 to 52 days) in all the presumptive freemartins. Ovarian and Müllerian inhibition begins simultaneously and a significant correlation between the degree of inhibition of both structures was obtained. The penis, scrotum and prostate were absent in all freemartins until Day 60. In only one case was the ano-genital distance slightly increased. A delayed appearance of seminal vesicles was found in three out of seven freemartins at 61 and 62 days. There was a notable absence of seminiferous tubules in the gonads of all these freemartins. It is suggested that if these tubules are present after birth it is because they have developed later. The factors responsible for the freemartin effect still remain to be discovered. Ovarian and Müllerian inhibitions occur during the period of time when the external genitalia of males become masculinized. This suggests that a factor of male origin which inhibits both the ovaries and the Müllerian ducts and which is distinct from the masculinizing hormone could be involved. Other possibilities are also discussed.