Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.     

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.     

Gonadal morphology of female diploid gynogenetic and triploid rainbow trout

Chromosome sets of fishes can be manipulated; this practice includes the production of triploid and gynogenetic salmonids. Such chromosomal modifications often result in abnormal ovarian development. In rainbow trout (RBT), triploid females have string-like gonads lacking significant developing oocytes and are suggested to be sterile due to the odd set of chromosomes disrupting oogenesis. Aberrant ovarian development is reported to occur in about 30% of gynogenetic females. It has been suggested that gynogenetic fish are more prone to expressing developmental abnormalities due to either increased homozygosity or to incomplete inactivation of the paternal chromatin. This investigation was done to compare the ovarian morphology of female triploid and induced gynogenetic diploid RBT. The objective was to determine whether the presence of supernumerary chromosomal fragments, potentially generated during the process of sperm genome inactivation, would result in abnormal gonadal development in gynogens comparable to that observed in triploid females. Gonadal morphology was observed and karyotypical analysis was completed on 21 gynogenetic fish. In 90% of the fish examined, the presence of chromosomal fragments was positively correlated with irregular ovarian development. The atypical gonadal morphology observed in the gynogens resembled triploid RBT ovarian morphology. The results of this investigation support the hypothesis that disruption of the normal diploid chromosomal complement alters germ cell development in gynogenetic female RBT due to the unbalanced nature of the genome. J. Exp. Zool. 286:505-512, 2000.     

Sex reversal and aromatase in chicken.

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens. To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor, in gonadal aromatase activity and in morphology and structure of the female genital system. Fadrozole was injected into eggs on day four of incubation, and its effects were examined during the embryonic development and for eight months after hatching. In control females, aromatase activity in the right and the left gonad was high in the middle third of embryonic development, and then decreased up to hatching. After hatching, aromatase activity increased in the left ovary, in particular during folliculogenesis, whereas in the right regressing gonad, it continued to decrease to reach testicular levels at one month. In treated females, masculinization of the genital system was characterized by the maintenance of the right gonad and its differentiation into a testis, and by the differentiation of the left gonad into an ovotestis or a testis; however, in all individuals, the left Müllerian duct and the posterior part of the right Müllerian duct were maintained. In testes and ovotestes, aromatase activity was lower than in gonads of control females (except in the right gonad as of one month after hatching) but remained higher than in testes of control and treated males. Moreover, in ovotestes, aromatase activity was higher in parts displaying follicles than in parts devoid of follicles. The main structural changes in the gonads during sex reversal were partial (in ovotestes) or complete (in testes) degeneration of the cortex in the left gonad, and formation of an albuginea and differentiation of testicular cords/tubes in the two gonads. Testicular cords/tubes transdifferentiated from ovarian medullary cords and lacunae whose epithelium thickened and became Sertolian. Transdifferentiation occurred all along embryonic and postnatal development; thus, new testicular cords/tubes were continuously formed while others degenerated. The sex reversed gonads were also characterized by an abundant fibrous interstitial tissue and abnormal medullary condensations of lymphoid-like cells; in the persisting testicular cords/tubes, spermatogenesis was delayed and impaired. Related to aromatase activity, persistence of too high levels of estrogens can explain the presence of oviducts, gonadal abnormalities and infertility in sex reversed females.     

Bi-directional sex change and gonad structure in the gobiid fish <Emphasis Type="Italic">Trimma yanagitai</Emphasis>

Bi-directional sex change in the deep-water gobiid fish Trimma yanagitai was examined. The gonads of all individuals consisted of ovarian and testicular elements, and an accessory gonadal structure. In no gonads were both testicular and ovarian parts simultaneously active. Bi-directional sex changes occurred during the rearing experiments in aquaria under conditions of which there was co-existence of two males or plural females. The sex of individuals could be determined by their relative body size or social dominance: the largest individuals acting as male and the remainder as female.     

Clinical,genetic, and pathological features of male pseudohermaphroditism in dog

Male pseudohermaphroditism is a sex differentiation disorder in which the gonads are testes and the genital ducts are incompletely masculinized. An 8 years old dog with normal male karyotype was referred for examination of external genitalia abnormalities. Adjacent to the vulva subcutaneous undescended testes were observed. The histology of the gonads revealed a Leydig and Sertoli cell neoplasia. The contemporaneous presence of testicular tissue, vulva, male karyotype were compatible with a male pseudohermaphrodite (MPH) condition.     

METHYLTESTOSTERONE-INDUCED DIFFERENTIATION OF A SEMINAL VESICLE HOMOLOGUE IN JUVENILE GOLDFISH, CARASSIUS AURATUS L.

In male goldfish, a structure homologous to the seminal vesicles of other fishes develops from the posterior sterile region of testicular ridges coincidentally with the initiation of spermatogenesis in the anterior region. Sexually differentiated juvenile goldfish were administered with methyltestosterone (50 μg/g diet) for a period of 20 days from 40 (experiment I) and 60 days (experiment II) after hatching. In both experiments, androgen administration exerted inhibitory influences on germ cell development in both sexes, but rather promoted the formation of the ovarian lumen, and of its residues in male gonads as well, accompanied with columnar hypertrophy and ciliation of the epithelium. The same treatment brought about a prominent development of the seminal vesicle homologues in the caudalmost gonadal ridges of females as well as in those of males in experiment II but not in experiment I. The cause of difference in the effects between the two experiments seemed to be ascribable to an incomplete prolongation of the gonadal ridges up to the presumptive area of the organ at the start of experiment I. Thus the capacity of the seminal vesicle primordia, common to both sexes, to make their due differentiation in response to exogenous androgen is confirmed in this study.     

Increased fat deposition in injured skeletal muscle is regulated by sex-specific hormones

Sex differences in skeletal muscle regeneration are controversial; comparisons of regenerative events between sexes have not been rigorously defined in severe injury models. We comprehensively quantified inflammation and muscle regeneration between sexes and manipulated sex-specific hormones to determine effects on regeneration. Cardiotoxin injury was induced in intact, castrated and ovariectomized female and male mice; ovariectomized mice were replaced with low- or high-dose 17-β estradiol (E(2)) or progesterone (P4). Extent of injury was comparable between intact mice, but females were more efficient in removal of necrotic debris, despite similar tissue levels of inflammatory cells and chemokines. Myofiber size during regeneration was equivalent between intact mice and after castration or ovariectomy (OVX) but was decreased (P < 0.001) in ovariectomized mice with high-dose E(2) replacement. Intermuscular adipocytes were absent in uninjured muscle, whereas adipocyte area was increased among regenerated myofibers in all groups. Interestingly, intermuscular fat was greater (P = 0.03) in intact females at day 14 compared with intact males. Furthermore, castration increased (P = 0.01) and OVX decreased adipocyte accumulation. After OVX, E(2), but not P4, replacement decreased (P ≤ 0.03) fat accumulation. In conclusion, sex-dependent differences in regeneration consisted of more efficient removal of necrosis and increased fat deposition in females with similar injury, inflammation, and regenerated myofiber size; high-dose E(2) decreased myofiber size and fat deposition. Adipocyte accumulation in regenerating muscle was influenced by sex-specific hormones. Recovery following muscle injury was different between males and females, and sex-specific hormones contributed to these differences, suggesting that sex-specific treatments could be beneficial after injury.     

Gonadal stage-dependent effects of gonadal steroids on gonadotropin II secretion in the Atlantic croaker (Micropogonias undulatus).

Involvement of gonadal steroids in the control of gonadotropin II (GTH II) (homologous to LH) secretion was investigated in the Atlantic croaker (Micropogonias undulatus) using gonadectomy (Gx) and steroid replacement paradigms. Gonadectomy in males and females during the late gonadal recrudescence phase elicited significant increases in the gonadotropin response to stimulation by an LHRH analog (LHRHa), without altering basal GTH II secretion. Slow-release silicone elastomer implants of testosterone or estradiol significantly inhibited LHRHa-induced GTH II secretion in gonad-intact and Gx males, and in Gx females, whereas 5alpha-dihydrotestosterone, a nonaromatizable androgen, was ineffective. Pretreatment of fish with an aromatase inhibitor, 1,4, 6-androstatrien-3,17-dione, 2 days before the administration of testosterone implants, completely blocked the negative effect of testosterone on LHRHa-induced GTH II secretion in males, but only partially restored it in females. This suggests that the negative feedback of testosterone in males is primarily mediated by its conversion to estradiol at the level of the hypothalamus and/or pituitary gland, while in females the androgen may also exert a direct inhibitory effect on GTH II secretion, probably mediated via an androgen receptor. In addition, estradiol and testosterone exerted positive effects on basal and LHRHa-induced GTH II secretion during the early-recrudescence phase of the gonadal cycle. The steroids switched to a negative effect on LHRHa-induced GTH II secretion once the fish had fully developed gonads, possibly as a mechanism that prevents a precocious surge in GTH II secretion and final gamete maturation until gametogenesis is complete and the environmental conditions are appropriate for spawning.     

Intersexuality in the mysid Neomysis integer

Intersexuality is described in Neomysis integer (Leach), apparently the first occurrence among the Mysidacea. Seven out of 13,700 specimens examined were masculinized females possessing elongated fourth pleopods, a male secondary sexual characteristic. Each masculinized female appeared to have reproduced as a normal female; there was no evidence of testicular tissue.     

Pathology of 46, XY pure gonadal dysgenesis: absence of testis differentiation associated with mutations in the testis-determining factor

Abstract. Individuals with 46, XY pure gonadal dysgenesis present with a completely female phenotype. These individuals develop bilateral streak gonads and have normal Müllerian structures. The apparent absence of testicular tissue in these individuals suggests a mutation in the initial steps of the male sex-determining pathway. A candidate gene for the primary signal in this pathway was recently cloned ( SRY ) which encodes a protein with a DNA-binding capacity. In a study of 14 XY females with pure gonadal dysgenesis harbouring SRY , we analysed the histology of the gonads and compared it to the presence or absence of mutations in the SRY open reading frame ( SRY -orf). The histological analysis revealed two distinct groups of streak gonads. In the first group, the gonad was composed of exclusively ovarian-like stroma, with sclero-hyaline nodules in some areas. No tubules were observed. The gonads in the second group were composed of undifferentiated stroma harbouring either tubules or a rete structure. This suggests that in the latter group some differentiation (towards testis formation) has occurred, whereas in the first group ovarian differentiation has been interrupted. Individuals with mutations in the SRY -orf were found to have streak gonads of the first group, whereas most of the remaining XY females without detectable mutation in the SRY -orf had streak gonads belonging to the second group.
On the basis of histology, it may be possible to distinguish between mutations in the sex-determining or sex-differentiation pathways. We suggest that SRY may play a role in rete testis formation. We also present arguments favouring the mesonephros as the origin of testicular somatic cells in humans.     

Frequent and unexplained gonadal abnormalities in whitefish (central alpine Coregonus sp.) from an alpine oligotrophic lake in Switzerland

The occurrence of a large number of whitefish with morphologically altered gonads in Lake Thun (Switzerland) has been reported by commercial fishermen since the year 2000. Macroscopic and microscopic examination of gonads revealed that 35% (281 out of 808) of whitefish from Lake Thun were affected. Frequency of gonadal abnormalities varied significantly with sex, age and ecotype of whitefish. Apparent females (26% [106/408]) showed significantly less gonadal alterations than apparent males (40% [151/225]). Fish with deformed gonads were observed in all age classes from 1 to 6 yr, but were most frequent in the year classes 3+ to 5+. The gonadal alterations showed different frequencies among the 3 ecotypes of whitefish living in the lake: 1 ecotype was less affected (26%) than the other 2 forms (41% and 32%, respectively). The gonadal alterations included adhesions/fusions to the peritoneal wall and the lateral trunk musculature (overall: 5%; in females: 5%; in males: 5%), asymmetry (4%; 6%; 4%), atrophy (4%; 6%; 1%), compartmentations (11%; 4%; 18%), constrictions (3%; 1%; 7%) and hermaphroditism (1.1% of sampled fish, and 10 additional specimens collected by fishermen during the filleting process). In some cases, more than one alteration was observed in the same fish. Hermaphroditism manifested as 3 different types: (1) the lobular type, in which a consecutive sequence of testicular and ovarian lobes are found on the same gonad strand; (2) discrete gonads within one fish, with one gonad strand being ovarian tissue and the other being testicular tissue; and (3) the mosaic type, in which oocytes lie in tissue with normal testicular morphology, or spermatids or sperm are present in ovarian tissue. Several features of the gonadal alterations described in this study of whitefish from Lake Thun have also been reported for whitefish from other areas. However, the Lake Thun situation appears to be unique because of the high prevalence of fish affected and the broad variation of abnormal features. The causes of the frequent appearance of these gonadal alterations in whitefish from Lake Thun remain unclear.     

Psychobiology of sexual behavior in a whiptail lizard, Cnemidophorus inornatus

This study investigated the effects of social environment on gonadal recrudescence and sexual behavior in male and female Little Striped Whiptail lizards (Cnemidophorus inornatus). The presence of sexually active males facilitates ovarian recrudescence in conspecific females. Similarly, the presence of reproductively active females facilitates testicular recrudescence in conspecific males. Males housed with females, however, had lower average circulating concentrations of testosterone and dihydrotestosterone, and higher average concentrations of corticosterone compared to intact males housed in isolation. In other studies, the presence of reproductively active females partially restored courtship behavior in castrated males compared to castrated males housed in isolation. Despite the stimulatory effects of females on castrates, exogenous androgens are required for complete restoration of all components of sexual behavior in male C. inornatus. Females are receptive to male courtship and copulatory behavior only during the vitellogenic stages; females in previtellogenic or postovulatory ovarian stages aggressively reject male courtship advances. These findings demonstrate reciprocal effects of sexual behaviors of males and females upon each other's reproductive behavior and physiology.     

Sex reversal and aromatase in the European pond turtle: treatment with letrozole after the thermosensitive period for sex determination

In the European pond turtle (Emys orbicularis), gonadal sex differentiation is temperature-dependent. The temperature sensitive period (TSP) of gonadogenesis lies between stages 16 and 22 of embryonic development. Previous studies have shown that embryos incubated at 30 degrees C, a temperature yielding 100% phenotypic females, can be sex reversed by treatments with an aromatase inhibitor administered during TSP or even somewhat after TSP (as of stage 22+). The goal of the present study was to determine whether the ovary still retains male potential at later stages of embryonic development and whether the induced male characters persist after hatching. For this purpose, eggs of E. orbicularis were treated with letrozole, a nonsteroidal aromatase inhibitor, at or as of stages 23, 24 or 25, then gonadal aromatase activity in each individual and the related gonadal structure were studied at hatching (stage 26) and for one year after hatching. Two kinds of treatments were carried out: 1) repeated applications of 10 microg of letrozole in ethanolic solution onto the eggshell; and 2) a single injection of 10 microg of letrozole in olive oil. Similar results were obtained with either application or injection of the aromatase inhibitor. In treatments as of or at stage 23, individuals with gonadal aromatase activity lower than 20 fmoles/hour/gonad had ovotestes, i.e., 22% of the treated individuals. At hatching, the inner part of these ovotestes contained testicular cords and also mixed lacunae presenting various degrees of transdifferentiation of the epithelium into a Sertolian epithelium. The cortex was maintained, although some germ cells degenerated within it. These processes continued after hatching. However, at 12 months, gonads were still ovotestes displaying some follicles with a growing oocyte in the remaining parts of the cortex. In treatments as of or at stages 24 or 25, only a few individuals were masculinized. One had ovotestes; in others, the cortex was absent in some parts and when it was present oocytes were degenerating. These results show that in the European pond turtle, differentiation of ovotestes from ovaries can be induced by treatment with an aromatase inhibitor starting at late stages of embryonic development (between the end of TSP and hatching), although such differentiation is less frequent as embryonic development proceeds. Sex reversal persists for at least one year after hatching. J. Exp. Zool. 290:490-497, 2001.     

Testosterone propionate treatment of an XY gonadal dysgenetic chacma baboon

Behavioral studies of an XY gonadal dysgenetic chacma baboon prior to and during testosterone propionate treatment were carried out. The orchidectomized dysgenetic individual, two intact males, a castrate male, and two ovariectomized females were pair-tested with a group of eight ovariectomized stimulus females prior to and during their treatment with estradiol benzoate. Three test series were carried out. One series occurred prior to any treatment of the agonadal focal subject animals. During this series it was only the intact males who showed behavior change during their testing with the estrogen treated females. A second test series occurred after a month of daily testosterone propionate injections (1 mg/kg/day) had been given to the four agonadal subjects. During this test series the castrate male ejaculated once with one of the estrogen-treated females. All of the treated subjects showed increases in their frequency of yawning. Upon completion of this test series the androgen dosage was increased (2 mg/kg/day) and 2 weeks later a third test series was carried out. During this series the castrate male ejaculated with five of his eight estrogen-treated partners. The yawning of all the treated subjects continued. As had been the case in the second series the XY gonadal dysgenetic individual continued to behave as did the ovariectomized females. None of these animals showed any increase in any measure of male sexual behavior. This study establishes the fact that a genetic male primate deprived of in utero exposure to testicular hormones will go on to develop as a normal genetic female and will fail to exhibit increased levels of male sexual behavior during androgen treatment.     

The social interactions of adult conspecifics with an adult XY gonadal dysgenetic chacma baboon (Papio ursinus)

Two separate behavioral studies on an XY gonadal dysgenetic chacma baboon were carried out. In the first experiment the focal subject was treated identically as the control females by her adult male test partners. In contrast to the controls in this experiment the dysgenetic animal reciprocated the agonistic behaviors directed by the males toward her. In the second study intact and castrated males as well as ovariectomized females served as controls for the dysgenetic subject in tests with ovariectomized adult females. The stimulus females presented more to both classes of males than either the subject females or the gonadal dysgenetic individual, suggesting that the focal subject was treated by other female conspecifics as a female.     

Treatment of pregnant rhesus macaques with testosterone propionate: observations on its fate in the fetus

Castrated male primates, unlike castrated male rodents, respond to exogenous estrogen by releasing gonadotropin. Although this disparity is unexplained, it may occur because the amount of testicular androgen secreted during a critical period for sexual differentiation is not sufficient to completely androgenize the anlagen of the central nervous system (CNS) in male primates. Therefore, male primates might be incompletely masculinized, and if fetal males were exposed to additional androgen during sexual development, they would no longer display the positive feedback to estrogen that usually characterizes females. Besides the development of mechanisms mediating the release of gonadotropins, questions about relationships between adult male sexual behaviors and the intensity of the androgen stimulus upon developing neural structures are of interest. We tested some of these possibilities by injecting androgen into 8 pregnant rhesus macaques from Days 40 through 50 of gestation, and we compared serum levels of testosterone (T), dihydrotestosterone (DHT), and androstenedione (delta 4) in the fetal circulation with that of 5 untreated control males. Fetal sera (both male and female) from treated pregnancies did not contain significantly greater quantities of T and DHT than sera of intact control males from untreated mothers. The maternal sera, however, contained large amounts of T (125.05 +/- 27.40 [SEM] ng/ml, n = 8) and significant elevations of DHT and delta 4 after treatment. The concentrations of delta 4 in the fetal circulation were significantly elevated (p less than 0.01) in treated fetuses compared to intact control males, probably due to the actions of the 17 beta-hydroxysteroid dehydrogenases in the placenta, the fetus, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

A quantitative study of testicular germ cell populations in masculinized neomale common carp (Cyprinus carpio L.)

The main objective of the present study was to investigate the effects of 17 alpha-methyltestosterone treatment upon the testicular germ cells of gynogenetic masculinized neomale common carp (Cyprinus carpio L.) in comparison with diploid carp. Gynogenetic common carp progeny (mean body weight, BW, 2.6+/-0.3g; mean total length, 10.4+/-0.5 cm) were treated for a period of 40 days with 17 alpha-methyltestosterone (MT) at a dose of 100mg kg(-1). The oral administration of MT resulted in 61.5-100% of fish exhibiting male gonads. The masculinized neomales exhibited reduced (P<0.05) body weight (BW=22.9+/-0.8) but significantly increased (P<0.05) mean testis weight (2.1+/-0.3) and mean gonadosomatic index (GSI=9.5+/-0.2%) in comparison with fish not treated with MT (BW 54.8+/-1.3; GSI=0.61%). Furthermore, treatment with MT also resulted in an increased number of fish exhibiting abnormal gonads. However, neomales did not exhibit abnormalities in the development of sperm ducts. MT treatment significantly increased germ cell volume, nuclear diameter, nuclear volume and cyst volume (P<0.01 in all cases) in MT-treated fish compared to untreated fish. The area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells were significantly (P<0.05) greater in fish treated with MT. The carp neomales exhibited approximately 20-60% more Sertoli cells per cyst (P<0.05). Leydig cell nuclear volume and Leydig cell individual volume were significantly reduced in MT-treated groups (P<0.05) compared with untreated groups. In conclusion, our study strongly suggests that the abnormal gonadal structure evident in masculinized neomales could be explained by a combination of MT-induced genetic (homozygosity) and anabolic effects (upon germ and somatic cells).