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.     

Testes of XX in equilibrium with XY chimeric mice develop from fetal ovotestes

The majority of XX in equilibrium with XY chimeric mice develop into fertile males. The sexual differentiation of the gonads in these animals has been examined on days 12-14 postocoitum to determine if their development parallels that of normal testes. It was found that 50% of chimeric fetuses, the proportion predicted to be XX in equilibrium with XY, had neither normal testes nor ovaries. Instead, ovotestes were present, with varying proportions of presumptive ovarian and testicular tissue. On day 12 the ovotestes were organized with testicular tissue in the central region and ovarian tissue at the craniad and/or caudad poles. In the more advanced fetuses there was evidence of regression of the ovarian portion, which would account for the testes found in adults. These results are discussed in light of current theories of sex determination and differentiation and what was previously known about gonads of sex mosaics.     

Testes of XX ↔ XY chimeric mice develop from fetal ovotestes

The majority of XX ? XY chimeric mice develop into fertile males. The sexual differentiation of the gonads in these animals has been examined on days 12–14 postcoitum to determine if their development parallels that of normal testes. It was found that 50% of chimeric fetuses, the proportion predicted to be XX ? XY, had neither normal testes nor ovaries. Instead, ovotestes were present, with varying proportions of presumptive ovarian and testicular tissue. On day 12 the ovotestes were organized with testicular tissue in the central region and ovarian tissue at the craniad and/or caudad poles. In the more advanced fetuses there was evidence of regression of the ovarian portion, which would account for the testes found in adults. These results are discussed in light of current theories of sex determination and differentiation and what was previously known about gonads of sex mosaics.     

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.     

Juvenile bisexuality in the red sea bream,Pagrus major

The histology of the gonad of the red sea bream,Pagrus major, was examined in order to study the early gonadal development, sexual maturation and sex ratio in a natural population. A total of 1,117 fish between the ages of 4 months and 8 years were examined. Gonads of 4-month-old fish were either sexually undifferentiated with a central cavity, or ovarian in form. Gonads of 12- and 18-month-old fish were ovaries or bisexual gonads, while those of 2-year-old fish were ovaries, bisexual gonads or testes. Fish aged between 3 and 8 years had ovaries or testes, except for a few bisexual gonads found in 3- and 4-year-old fish. The chronological appearance of females, hermaphrodites and males in that order, and histological evidence, suggested that the testis originates from the ovary via a bisexual gonad in the juvenile stage. The sex ratio of females to males at the age of 2 years and over was about 1:1, suggesting that hermaphroditic red sea bream appear in about 50% of the juvenile population. The sexual pattern in this species, therefore, is concluded to be gonochorism with a bisexual juvenile stage.     

The problem of sexual inversion in the minnow, Phoxinus phoxinus (L.)

The incidence of sexual inversion in Phoxinus phoxinus has been studied by examining histologically (a) the gonads of 406 adult specimens caught from the wild during a period of 16 months, (b) the gonads of 168 adults kept in captivity in groups with an experimentally altered sex-ratio, and (c) the differentiation of the gonads in the fry.
All the adults had either testes or ovaries and no case of intersexuality was observed. For both testis and ovary, we recognized two phases, depending on the seasonal cycle: an active spring-summer phase, corresponding with the period of reproduction, and a quiescent autumn-winter phase. The ovaries showed an asynchrony in oocyte development, which is typical of species that spawn many times during the breeding season.
In P. phoxinus the ovaries are easily recognizable just one month after hatching and testes are well differentiated in 2-month-old fingerlings. Differentiation is, therefore, precocious and follows a pattern typical of gonochoristic species in which hermaphroditism never occurs.     

Gonadal morphology of normal and sex-reversed triploid and gynogenetic diploid coho salmon (Oncorhynchus kisutch)

In this study, the gonadal morphology of untreated and sex-reversed juvenile triploid and gynogenetic diploid coho salmon was compared with that of diploids. Testes of triploids were of the same size as those of diploids. Spermatogonia, however, were significantly bigger than those of diptoids in both diameter (P<0·001) and volume (P<0·01), suggesting that this characteristic can be a useful indicator of ploidy in the early stages of gonadal development. In females, induction of triploidy did not affect the lamellar structure of the ovaries but reduced their size considerably. Further, these ovaries had no oocytes. Treatment of triploids with oestrogen resulted in the feminization of genotypic males, which had ovaries similar to those found in tripioid females. However, gonads of triploid males partially sex-re versed into females were identified by their enlargement, the presence of remnants of the male vascular system, and by the appearance of ovarian lacunae and germinal and somatic cells typical of triploid females, Induction of gynogenesis resulted in 100% females, of which 34% had ovaries of reduced size with areas devoid of oocytes. However, and contrary to what has been found in cyprinids, no male germ cells were observed in these ovaries. This discrepancy may reflect differences, in the mechanisms of sex determination between salmonids and cyprinids. Treatment of gynogenetics with androgen increased the number of fish with abnormal ovaries but also resulted in the production of phenotypic-male gynogenetic diploids, of which 11% had testes indistinguishable from those of untreated control diploids.     

Gonadal development and non-functional protogyny in a coral-reef damselfish, Dascyllus albisella Gill

All gonads of the Hawaiian dascyllus Dascyllus albisella , irrespective of the final sex of individuals, developed an ovarian lumen and primary-growth-stage oocytes after an initially undifferentiated state. From this ovarian state or from more differentiated ovaries, some gonads redifferentiated into testes. None of 117 individuals examined had a gonad containing degenerating vitellogenic oocytes and proliferating spermatogenic tissue. Eleven individuals had gonads containing degenerating cortical-alveolus-stage oocytes and developing spermatogenic tissue. The size of these individuals overlapped with the female size range in which the majority of the females were still in the middle of the maturation process. They were absent from the larger size range where the majority of females had vitellogenic oocytes. This indicated that the transition toward maleness is likely to have occurred after the onset of cortical-alveolus stage, but before final oocyte maturation and spawning as females. Therefore the protogynous pattern of gonadal development was non-functional. There was no dimorphism in the sperm duct configuration, and all the testes were secondary testes reported for diandric, protogynous species with undelimited gonads. Very early development of an ovarian lumen appeared to have resulted in a secondary-male configuration in all testes, although redifferentiation into males appeared to have occurred before sexual maturity and spawning as females.     

Immunolocalization of cytochrome P450 17alpha-hydroxylase/c17-20 lyase in the ovary of pregnant pigs and fetal gonads

The study was designed to localize P450 17alpha-hydroxylase/c17-20 lyase (P450c17) in the ovaries of pregnant pigs and fetal gonads. Immunoexpression of P450c17 was investigated in the porcine ovaries (follicles and corpora lutea; CL) collected on days 10, 18, 32, 50, 70 and 90 post coitum (p.c.), and fetal gonads (testes and ovaries) on days 50, 70 and 90 p.c. The presence of P450c17 in ovarian follicles was demonstrated on all examined days of pregnancy but was restricted to theca interna cells. In CL, P450c17 was detected on all examined days of pregnancy but only in small luteal cells. In the female porcine fetuses, P450c17 immunostaining was found in oocyte nests and granulosa cells of primary ovarian follicles, while in the male fetuses in fetal Leydig cells. In conclusion, the immunolocalization of P450c17, detected in the ovaries of pregnant pigs and fetal porcine gonads, indicates the potential sites of androgen synthesis. We suggest that androgens may play a role in the maintenance of pregnancy and in the development of prenatal gonads in pigs.     

Induction of gynogenesis and gonad development in the muskellunge

Muskellunge Esox masquinongy eggs activated with UV-irradiated yellow perch Perca flavescens sperm were subjected to heat shock at 31 ± 1° C for 6 min, 20 min after fertilization in three experiments. Survival at eyed stage was 1·7 ±1·6, 6·8 ± 4·8 and 2·3 ±0·5% in experiments I, II and III, respectively. After rearing the gynogenetic muskellunge in troughs and then in ponds, the sex ratio of gynogens in experiment I did not significantly (P>0·05) differ from the expected ratio 0:1 (male: female), however, one male and one intersex were observed. In experiments II and III, the sex ratio of gynogens differed significantly from expected (0 : 1). Three months after hatching, the growth of females did not significantly (P>0·05) differ among control and gynogenetic groups, whereas male growth was significantly (P<0·01) higher in the control v. gynogenetic group. The histological structures of the gynogenetic fish gonads in both sexes were similar to those described in the gonads of control muskellunge. After the overwintering period, signs of active spermatogenesis were observed in the testis of 1 + year old gynogenetic fish, whereas ovaries contained only oocytes at the perinucleolar stage. At this stage, plasma sex steroids testosterone (T) and oestradiol-17β (E2) cannot be used to discriminate the sex of gynogenetic muskellunge, as intersex, male and female fish had similar levels of T and E2.     

Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature-dependent sex determination

Cellular infiltrations forming lymphoid-like aggregates were previously observed in gonads of two turtle species exhibiting temperature-dependent sex determination (TSD): at hatching in Chelydra serpentina; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid-like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female-producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids.     

Morphological and histological features of the gonad structure in hermaphroditic walleye pollock <Emphasis Type="Italic">Theragra chalcogramma</Emphasis> (Gadidae)

Morphology of hermaphroditic gonads is studied in the individuals of walleye pollock Theragra chalcogramma collected in different regions of the seas of the North Pacific from 1987 to 2014. Based on the external morphological characteristics of sexual glands of 17 fishes, the most frequent types of the structure of bisexual gonads are described. Histological analysis shows the absence of differences in the composition and condition of oocytes in the left and right-paired ovaries, and the testes and ovaries of each individual are at the same maturity stage. In the ovigerous lamellae of ovaries in the areas adjacent to testes the zones of oocyte resorption are observed. These zones are located exclusively near the joining of the female and male parts of the gonads. The development of ovaries and testes of bisexual gonads is similar to that in the majority of normally maturing males and females collected during the same period. The occurrence of hermaphrodites is 0.0003–0.0016% from the total number of investigated exemplars.     

Gonadogenesis in early developmental stages of Acipenser naccarii and influence of estrogen immersion on feminization

Gonad development processes and the effects of a single 8‐hour immersion treatment with 17β‐estradiol (E2, 400 μg L⁻¹) on sex differentation in the Adriatic sturgeon, Acipenser naccarii, were investigated. After migration of germ cells, gonadal ridges appeared in 16‐ to 18‐day old larvae and undifferentiated gonads in 55‐ to 60‐day old larvae. Putative ovaries with notches in the germinal epithelium and presumed testes with smooth germinal epithelium appeared in 180–185‐day old juveniles. Ovaries with proliferating oogonia and early meiotic oocytes clusters were observed in 292‐day old juveniles. Testes did not exhibit germ cell mitosis until 430 days of age. Developmental stages in E₂‐treated animals closely followed those of controls up to 430 days. The treatment significantly increased the percentage of ovaries when administered to embryos about 1.5 day before hatching, while did not significantly altered the normal 1/1 sex ratio when administered to 1.5‐day old pre‐larvae and 10‐day old larvae. It is likely that in A. naccarii exogenous E₂ administration may act through a feedback mechanism of self‐supporting steroid production and that steroids are the physiological inducers of sex differentiation, as in most teleosts. The E₂‐immersion treatment, easier than time‐consuming administration through food, could be a good approach to control sex differentiation and caviar production.     

Gender differences in transcriptional signature of developing rat testes and ovaries following embryonic exposure to 2,3,7,8-TCDD

Dioxins are persistent organic pollutants interfering with endocrine systems and causing reproductive and developmental disorders. The objective of our project was to determine the impact of an in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on reproductive function of male and female offspring in the rat with a special emphasis on the immature period. We used a low dose of TCDD (unique exposure by oral gavage of 200 ng/kg at 15.5 days of gestation) in order to mirror a response to an environmental dose of TCDD not altering fertility of the progeny. We choose a global gene expression approach using Affymetrix microarray analysis, and testes of 5 days and ovaries of 14 days of age. Less than 1% of the expressed genes in gonads were altered following embryonic TCDD exposure; specifically, 113 genes in ovaries and 56 in testes with 7 genes common to both sex gonads. It included the repressor of the aryl hydrocarbon receptor (Ahrr), the chemokines Ccl5 and Cxcl4 previously shown to be regulated by dioxin in testis, Pgds2/Hpgds and 3 others uncharacterized. To validate and extend the microarray data we realized real-time PCR on gonads at various developmental periods of interest (from 3 to 25 days for ovaries, from 5 to the adult age for testes). Overall, our results evidenced that both sex gonads responded differently to TCDD exposure. For example, we observed induction of the canonic battery of TCDD-induced genes coding enzymes of the detoxifying machinery in ovaries aged of 3-14 days of age (except Cyp1a1 induced at 3-10 days) but not in testes of 5 days (except Ahrr). We also illustrated that inflammatory pathway is one pathway activated by TCDD in gonads. Finally, we identified several new genes targeted by TCDD including Fgf13 in testis and one gene, Ptgds2/Hpgds regulated in the two sex gonads.     

The overlapping effects of thyrotropin and gonadotropins on chick embryo gonads in vitro

Pieces of 12- and 15-day-old chick embryo testes and ovaries were cultured in vitro in the presence of thyrotropin (TSH), gonadotropins (FSH + LH) and adrenocorticotropin (ACTH) for different periods. All the explants of treated gonads differentiated into typical testes or ovaries according to their genetic sex. The gonads of 12-and 15-day-old chick embryos showed a good response to both thyrotropic and gonadotropic stimulation. On the other hand, they did not respond to adrenocorticotropic stimulation. Fifteen-day-old chick embryo testes were grown in tissue culture in the presence of the said hormones. Gonadotropins and TSH enhanced the growth and migration of testicular cells as compared with the control or ACTH treated group. In addition, they maintained the germ cells on the upper surface of epithelial cells. These results have confirmed our previous results in vivo in that gonadotropins and thyrotropin hormones accelerated the development of 12- or 15-day-old chick embryo gonads.     

Observations on mass atresia and skipped spawning in northern Atlantic cod, from Smith Sound, Newfoundland

Atlantic cod Gadus morhua were collected from Smith Sound, Newfoundland in January 1999. Visual examination of females (n=150) and males (n=126) revealed that some large fish (42–79 cm) had underdeveloped gonads. Histological examination of underdeveloped ovaries indicated that the majority of these females were undergoing mass resorption of oocytes and would not have spawned in 1999. Fish in this condition included females that were aborting their first attempt at maturation and females that had spawned the previous year but were failing to re-ripen. Somatic and liver condition were significantly lower (P<0·05) for fish undergoing mass oocyte resorption than ripening females, suggesting that the interruption in the maturation cycle may have been related to insufficient nutrient storage. In males, testes of some adult fish were considered to be non-reproductive as they showed no signs of ripening and probably would not have spawned in 1999. Liver condition was significantly higher (P<0·05) for non-reproductive males than those that were ripening. Disruptions in male and female reproductive cycles may also have been related to water temperatures that were too cold (0–0·5° C) for successful gamete development. Immature gametes (perinucleolar oocytes in females; spermatogonia in males) showed no signs of breakdown in non-reproductive individuals, suggesting that they retained the potential to develop and spawn gametes in 2000.     

Immunohistochemical Localization of Laminin and Cytokeratin in Embryonic Alligator Gonads

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.     

Sex-related differences in blood and gonad levels of testosterone in silver fox fetuses

The mass of silver fox fetuses of both sexes, their gonads, and adrenals, and the levels of testosterone in blood serum and in gonads and adrenals were determined from day 31 of gestation and every five days thereafter until its termination. Marked sex-related differences were revealed: the blood and gonad levels of testosterone in male fetuses were much higher than those in female fetuses. The fetal adrenals contained significantly less testosterone than the gonads. No sex-related differences in the content of testosterone in the fetal adrenals were found. No differences were found in the body and adrenal mass in female and male fetuses at all the developmental stages studied, while the mass of ovaries exceeded that of testes from day 45 of gestation. The data obtained suggest sex dimorphism in the production of testosterone by gonads in silver foxes appears after day 35 and appears to correspond to the period of morphological differentiation of gonads.     

Gonadal differentiation and hormonal sex reversal in Arctic charr (Salvelinus alpinus)

The purpose of this study was to develop a practical protocol for the production of female populations of Arctic charr (Salvelinus alpinus). Achieving this required knowledge of the timing of gonadal differentiation. Undifferentiated gonads were observed microscopically to be present by 194 degrees C-days post-hatch and definitive germ cells by 346 degrees C-days post-hatch, where " degrees C-days" denote acquired thermal units calculated as the product of temperature and days. Some of the gonads had developed a lumen by 510 degrees C-days post-hatch, and by 681 degrees C-days post-hatch anatomical divergence into two types of gonads was clear. Two protocols (immersion and feeding) were tested for hormonal sex reversal of genotypic females using the synthetic androgen 17alpha-methyldihydrotestosterone (MDHT). Six-hour MDHT immersions (0.5, 1, 3, 5 and 10 mg/L) were carried out weekly from hatch to first feeding (140 degrees C-days post-hatch), whereas daily feeding treatments (0.5 mg/kg) went from 140 to 600 degrees C-days post-hatch. The sex ratios of all immersion experimental groups were significantly different from the control, with the proportion of presumptive males increasing as MDHT concentration increased. The highest immersion treatment, 10 mg/L, yielded a population of 90% presumptive males and 10% with atypical gonads. However, the most effective treatment, yielding a population of 90% presumptive males and no fish with atypical gonads, was the feeding treatment. Given that female salmonid fishes are homogametic, sex-reversed (masculinized) genotypic females produced in this way can serve as broodstock for the creation of all-female charr populations for aquaculture.