Sex-reversed Males Research Articles

Estradiol‐17β content in developing eggs and induced sex reversal of the medaka (Oryzias latipes)

To clarify the effect of exogenous estradiol-17beta (E2) on sex differentiation, the E2 content of developing eggs of Oryzias latipes was measured by radioimmunoassay. Endogenous E2 was present in lower concentrations in ovulated, mature eggs in the ovarian cavity than in intrafollicular oocytes. The E2 content of eggs precipitously declined to a minimum level by 2 days post-fertilization. The E2 content of eggs was affected by 24 hr of incubation in medium containing exogenous E2 at concentrations above 10 ng/ml. Short (24 hr) exposure of fertilized eggs in the early developmental stage to exogenous E2 at concentrations of 10 ng/ml induced sex reversal of some genotypic males to functional females. However, endogenous E2 levels in fertilized eggs might not influence sexual differentiation in embryogenesis. The present results suggest the possibility that concentrations of exogenous E2 higher than that of endogenous E2 triggers a priming step in the cascade of sex differentiation toward the female, and this effect is maintained.

The XX-XY Sex-determination System in Oryzias luzonensis and O. mekongensis Revealed by the Sex Ratio of the Progeny of Sex-reversed Fish

The sex-determining gene in Oryzias latipes and O. curvinotus has been proved to be DMY. Although O. curvinotus has the DMY gene on the Y chromosome which is homologous to the Y chromosome of O. latipes, the sex-determining mechanism of other Oryzias fishes has not been identified. In order to uncover the sex-determining mechanism of O. luzonensis and O. mekongensis, which are most closely related species to O. latipes and O. curvinotus, we analyzed the sex ratio of the progeny of sex-reversed fish. We were able to obtain sex-reversed males by the administration of methyltestosterone, and found that these yielded all-female offspring in both species. These results indicate that O. luzonensis and O. mekongensis have the XX-XY sex-determination system.

Historical Perspectives

Fifty years ago, Professor Paul Polani and colleagues described young women who had Turner syndrome and were found to have apparently male “chromosomal sex.” (1) At that time, the generally accepted number of chromosomes in humans was 48. Five years earlier, Barr and Bertram described the presence of a prominent mass of chromatin lying close to the nucleolar membrane in neurons of some female cats. (2) This “sex-chromatin” was not found in the males. These “Barr bodies” later were detected in cells grown from skin biopsies. (3) Then, in 1956, Tjio and Levan reported that the number of diploid chromosomes was 46, (4) which rapidly was confirmed by others. (5) From this ripple, a wave soon developed. Three years later, the first association of a clinical syndrome with an abnormal number of chromosomes was reported from France (6) and quickly confirmed in Scotland. (7) This was the association of Down syndrome (mongolism) with an extra chromosome number 21 (trisomy 21), although this was called chromosome number 22 at the time. Not long afterward, translocation was noted in a girl who had Down syndrome born to a 21-year-old mother, (8) and translocation was described as a cause of familial mongolism. (9) Other trisomies soon were described. (10)(11) Based on the realization that the number of sex-chromatin bodies was one less than the number of X chromosomes present, the chromosomes of women who had Turner syndrome and appeared to be chromosomally “male” were investigated. They were found to have 45 chromosomes, with the sex chromosomes being XO. (12) Many of these exciting chromosomal discoveries were made by Professor Polani and his colleagues. Elsewhere, Professor Polani (now 90 years old) has described in detail the origins and evolution of clinical cytogenetics. (13) In this issue of NeoReviews , …

Long-range activation of Sox9 in Odd Sex (Ods) mice.

The Odd Sex mouse mutation arose in a transgenic line of mice carrying a tyrosinase minigene driven by the dopachrome tautomerase (Dct) promoter region. The minigene integrated 0.98 Mb upstream of Sox9 and was accompanied by a deletion of 134 kb. This mutation causes female to male sex reversal in XX Ods/+ mice, and a characteristic eye phenotype of microphthalmia with cataracts in all mice carrying the transgene. Ods causes sex reversal in the absence of Sry by upregulating Sox9 expression and maintaining a male pattern of Sox9 expression in XX Ods/+ embryonic gonads. This expression, which begins at E11.5, triggers downstream events leading to the formation of a testis. We report here that the 134 kb deletion, in itself, is insufficient to cause sex reversal. We demonstrate that in Ods, the Dct promoter is capable of acting over a distance of 1 Mb to induce inappropriate expression of Sox9 in the retinal pigmented epithelium of the eye, causing the observed microphthalmia. In addition, it induces Sox9 expression in the melanocytes where it causes pigmentation defects. We propose that Ods sex reversal is due to the Dct promoter element interacting with gonad-specific enhancer elements to produce the observed male pattern expression of Sox9 in the embryonic gonads.

Chromosome set manipulation and sex control in common carp: a review

The development of techniques for production of gynogenetic, androgenetic, polyploid, and monosex progenies in common carp ( Cyprinus carpio L.) is described from a chronological perspective. Gynogenetic progenies were obtained either by suppression of the second meiotic division in eggs (meiotic gynogenesis) or by suppression of the first mitotic division in haploid embryos (mitotic gynogenesis). As a rule, gynogenetic progenies of common carp were all-female, revealing female homogamety (females—XX, males—XY) in this species. Induced gynogenesis results in increased homozygosity; the rate of increase depends on the type of gynogenesis. Inbreeding coefficient ( F) for one generation of meiotic gynogenesis in common carp is about 0.6, while diploids obtained by mitotic gynogenesis are homozygous for all genes ( F = 1.0). Mitotic gynogenesis was used for production of clones in common carp. In androgenetic progenies of common carp, YY males were identified, that after crossing with normal females (XX) produced all-male progenies. Triploids of common carp are characterized by a significant reduction in gonad development (especially ovaries). However, the reduction in gonad development did not result in an increase of somatic growth rate of fish. The procedure for androgen treatment to induce phenotypic sex reversal in genotypic females (XX) was elaborated. All-female progenies of common carp were produced on a large scale by crossing normal females (XX) with hormonally sex-reversed males (XX). Rearing of all-female progenies in conditions when fish normally reach sexual maturity before reaching of market size increased production yield by 7–8%. In a few cases distant hybridization resulted in polyploidy of fish without application of any physical treatment. The ability of hybrid females between crucian carp ( Carassius auratus) and common carp to produce diploid (with unreduced chromosome number) gametes resulted in opportunities to produce triploid and tetraploid hybrid progenies.

WT1 gene mutation responsible for male sex reversal and renal failure: the Frasier syndrome

We report the case of a young woman with primary amenorrhea. In her childhood, she suffered from renal failure requesting kidney transplantation at the age of 11. The investigations for primary amenorrhea revealed a hypergonadotropic hypogonadism associated with 46 XY karyotype. The association of primary amenorrhea with renal failure suggested Frasier syndrome (FS) or Denys–Drash syndrome (DDS). Genetic analysis revealed a Wilms’ tumour (WT1) gene mutation characteristic of the Frasier syndrome. Dysgenetic ovaries were removed laparoscopically due to the risk of gonadal cancer.

Tributyltin causes masculinization in fish

We examined the effect of tributyltin (TBT) on the sex differentiation process in genetically female Japanese flounder (Paralichthys olivaceus). The fish were fed an artificial diet containing tributyltin oxide (TBTO) at concentrations of 0.1 and 1.0 microg/g diet from 35 to 100 d after hatching, which includes the sex differentiation period. The ratio of sex-reversed males significantly increased to 25.7% of the flounder fed the 0.1 microg/g diet and to 31.1% of those fed the 1.0 microg/g diet compared with the control (2.2%). From morphological and histological examination of the fish in the TBT-treated groups, normal females had typical ovaries and sex-reversed males had typical testes. These results clearly demonstrated the masculinization of flounder exposed to TBTO. This is the first report of TBT inducing sex reversal in vertebrates.

Effect on growth and reproduction of hormone immersed and masculinized fighting fish Betta splendens.

To produce all-male progenies in the fighting fish, Betta splendens, six groups of fry were subjected to discrete immersion treatment at different 17alpha-methyltestosterone (MT) doses (viz. 100, 200, 500, 700, 900, and 1,000 microg/l) for a constant duration (3 hr/day) and frequency (second, fifth, and eighth day after hatching). The treatment at 900 microg/l led to 98% masculinization and 71% survival at sexual maturity. Treated groups, which showed significant deviation from the 1:1 sex ratio, were classified into two different series: S1 and S2. The groups that showed nearly cent-percent masculinization were classified as S1, and the other groups were classified as S2. The S1 males showed remarkably slower growth and attained 3.5 cm total length compared to 6.0 cm attained by a normal male. The S2 males attained 5.4 cm total length. Apart from these morphological defects, both S1 and S2 males suffered functional (decreased sperm count and sperm motility) and behavioral defects (incomplete embracing during mating) in their reproductive ability, leading to approximately 50% and 30% reduction in fecundity per mating, respectively. The cumulative fecundity loss suffered by the S1 male during its active reproductive phase is discussed. When normal and sex-reversed males were presented, a female preferred the former. Progeny testing of the sex-reversed males showed the occurrence of 12.75% males, indicating the possible role of autosomal genes in the sex determination mechanism of this species. Discrete immersion treatment at optimal/super-optimal doses ensured not only a higher percentage of masculinization, but also a higher frequency of homogametic males (XX).

Incidence of Phenotypic Female Chinook Salmon Positive for the Male Y-Chromosome-Specific MarkerOtY1in the Central Valley, California

Abstract Fall-run chinook salmon Oncorhynchus tshawytscha collected during 1999 carcass surveys of seven locations within the Sacramento River basin and six locations within the San Joaquin River basin, California, were screened using the Y-chromosome-specific marker OtY1 to evaluate inconsistencies between sexual genotype and phenotype. Of 287 phenotypic females screened, 46 (16% overall) tested positive for the Y-chromosome marker. Stream populations had higher frequencies of sex-reversed males (American River, 20%; Battle Creek, 35%; Feather River, 20%; Merced River, 24%; Mokelumne River, 38%; and Yuba River, 25%) than hatchery populations (Feather River Hatchery, 0%; Nimbus Hatchery, 12%; Merced River Hatchery, 14%; and Mokelumne River Hatchery, 4%), and several other streams had intermediate frequencies. All 150 male fall-run chinook salmon from the sampling locations tested positive for the Y-chromosome marker. These results present evidence that some genetic males have been sex reversed and have th...

Influence of sexual genotype on the behaviour of females (genotype WZ) and pseudofemales (genotype ZZ) in the tilapia Oreochromis aureus

17α-ethynylestradiol sex-reversed males of Oreochromis aureus (pseudofemales, ΔF, genotype ZZ) are used in aquaculture to produce a male monosex population by crossing with ZZ homogametic normal males. When placed with males (M) and females (F) in the same spawning tank, the spawning rate of F is higher than for ΔF. In order to understand this phenomenon, comparisons were made between the behaviour of 18 F (446 ± 96 mm) and 18 ΔF (401 ± 59 mm). ΔF showed a more aggressive behaviour and were significantly more dominant than normal F in fighting pair experiments (F × ΔF) or in fighting group experiments in four different stocking densities (8, 12, 16 and 83 fish 10 –3 l with F/ΔF ratio = 1). ΔF were also more aggressive towards males than F were. The results support the idea that behavioural differences exist between F and ΔF. These differences are probably due to the effect of the sexual genotype on behaviour.

Hormonal Sex Reversal and Evidence of Female Homogamety in Black Crappie

Abstract This study reports the results on hormone-induced sex reversal in black crappie Pomoxis nigromaculatus and the sex ratios of progenies obtained from test crosses of androgen-treated males with normal females. The androgen 17α-methyltestosterone (MT) was orally administered to fish with an artificial diet (30 mg/kg) for 30 d beginning 37 d after hatching; mean fish lengths were 35 and 55 mm at the beginning and conclusion of the treatment, respectively. The MT-treated group consisted of 95% males and 5% intersex fish, while the sex ratio in the control group was not significantly different from 1:1. Four males from the androgen-treated group were individually test-crossed with normal females to identify sex-reversed males according to the sex ratios in progenies and to reveal the chromosomal mechanism of sex determination in black crappie. Two out of three analyzed progenies consisted of females only, while one progeny had a sex ratio not significantly different from 1:1. The appearance of all-fem...

Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan

The purpose of this review is to introduce recent advances in research, development and application of chromosome manipulation techniques in Japan. Triploids have been induced and utilized to improve growth. In most cases, triploids are produced as all-female populations by using spermatozoa of artificially sex-reversed males, so as to assure their complete sterility. In contrast, triploid males show better gonadal development and sometimes generate functional spermatozoa, which give rise to inviable aneuploids in most species. Although induced tetraploids can be useful for mass production of triploids by mating with normal diploids, tetraploid lines have only been produced in rainbow trout by a few institutes belonging to prefectural governments. In most cases, the techniques to inhibit the first cleavage often result in very low survival and mosaicism. In the loach (Cobitidae), polyploid lines such as hexaploids have been developed by using natural tetraploid individuals as an intermediate step. Cloned fish can be produced by the second cycle of gynogenesis in the eggs of completely homozygous diploids, which were produced by inhibiting first cleavage after induction of gynogenetic and androgenetic development. The second cycle of androgenesis, using spermatozoa of completely homozygous males, can also generate clonal lines. The most serious technical problem of cloning is the extremely low survival of homozygous gynogenetic and androgenetic diploids, probably due to the expression of deleterious recessive genes and side effect of treatments. However, cloned populations have been realized in commercially important ayu Plecoglossus altivelis, amago salmon Oncorhynchus masou ishikawae, coho salmon O. kisutch, hirame (Japanese flounder) Paralichthys olivaceus, fancy carp Cyprinus carpio, and red sea bream Pagrus major. In hirame, a practical method for mass production of clones was proposed and better performance has been reported in a heterozygous clone, produced by hybridization between two different homozygous clonal lines. Repeated meiotic (polar body) gynogenesis may be more practical than cloning from homozygous gynogens as a method to generate isogenic lines. In meiotic gynogenesis, the proximal region of chromosomes should be homozygous, whereas the distal region should be heterozygous due to high rates of gene–centromere recombination. Consequently, similar genotypes are predicted in the second and later generations of gynogenetic progeny. Their isogenic nature has been confirmed by minisatellite, microsatellite, and other DNA analyses. Gynogenesis, androgenesis and cloning can be used for elucidation of genetic sex determination. The involvement of environmental factors has been indicated from the sex ratios of chromosomally manipulated populations of several species. Finally, the regulation of chromosomally manipulated fish by the guidelines and the integration of such techniques with molecular genetics for further gene mapping and transgenics are discussed.

Permanent and functional male-to-female sex reversal in d-rR strain medaka (Oryzias latipes) following egg microinjection of o,p'-DDT.

Complete sex reversal of fish is accomplished routinely in aquaculture practices by exposing fish to exogenous sex steroids during gonadal differentiation. A variety of environmental chemicals are also active at sex steroid receptors and theoretically possess the potential to alter normal sexual differentiation in fish. However, in controlled environmental chemical exposures to date, only partial alterations of fish sexual phenotype have been observed. Here we report complete, permanent, and functional male-to-female sex reversal in the Japanese medaka (Oryzias latipes, d-rR strain) after a onetime embryonic exposure to the xenoestrogen o, p'-DDT. d-rR strain medaka are strict gonochorists that possesses both sex-linked pigmentation, which distinguishes genotypic sex, and sexually dimorphic external secondary sexual characteristics, which distinguish phenotypic sex. We directly microinjected the xenoestrogen o, p'-DDT into the egg yolks of medaka at fertilization to parallel the maternal transfer of lipophilic contaminants to the embryo. At 10 weeks of age, microinjected medaka were examined for mortality and sex reversal. A calculated embryonic dose of 511 +/- 22 ng/egg o, p'-DDT (mean +/- standard error) resulted in 50% mortality. An embryonic exposure of 227 +/- 22 ng/egg o, p'-DDT resulted in 86% (6 of 7) sex reversal of genetic males to a female phenotype (XY females). XY females were distinguished by sex-linked male pigmentation accompanying female secondary sexual characteristics. Histologic examination of the gonads confirmed active ovaries in 100% of the XY females. In 10-day breeding trials in which XY females were paired with normal XY males, 50% of the XY females produced fertilized embryos; this represents a comparable breeding success rate to normal XX females. Fertilized eggs produced from XY females hatched to viable larvae. These results clearly indicate that a weakly estrogenic pesticide, o, p'-DDT, when presented during the critical period of gonadal development, can profoundly alter sexual differentiation.

Notes on first attempt at artificial spawning and rearing of early stages with goldblotch grouper, Epinephelus costae (Steindachner, 1875).

Notes on first attempt at artificial spawning and rearing of early stages with goldblotch grouper, Epinephelus costae (Steindachner, 1875).

An in vivo model fish system to test chemical effects on sexual differentiation and development: exposure to ethinyl estradiol

A model system was characterized which may be used as an in vivo screen for effects of chemicals or environmental mixtures on sexual differentiation and development of reproductive organs and gametes. We evaluated the effects of a model environmental estrogen, ethinyl estradiol (EE 2), on the d-rR strain of medaka, Oryzias latipes, using a nano-injection exposure. Gonad histopathology indicated that a single injection of 0.5–2.5 ng EE 2/egg can cause phenotypic sex-reversal of genetic males to females. Sex-reversals could be detected as early as 7 days post-hatch. Sex-reversed males had female-typical duct development and the secondary sex characteristics we measured were generally consistent with phenotype, with the exception of a few EE 2-exposed XX and XY females which possessed ambiguous anal fins. Using discriminant analysis, we determined that the presence or absence of the secondary sex characteristic, a dorsal fin notch, was a very reliable indicator of gonadal sex. No instances of gonadal intersexes were observed. Ethinyl estradiol also appeared to reduce growth but not condition (weight-at-length) and exposed XX females appeared to have a higher incidence of atretic follicles relative to controls. Our results suggest that estrogenic chemicals may influence sexual differentiation and development and that the medaka model is well suited to assessing these effects.

Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.).

Fully inbred clonal lines of fish are likely to be of great value in research on immunology, sex determination, quantitative genetics, and toxicology. In this study on the Nile tilapia (Oreochromis niloticus), gynogenesis or androgenesis were used to produce a first generation of completely inbred fish, from which clonal lines were established using gynogenesis, androgenesis, hormonal sex reversal and intraline crosses. The clonal nature of these lines was verified by using multilocus DNA fingerprinting and the isozyme locus ADA*. Although these lines might be expected to be monosex in nature (all-female XX or all-male YY depending on the clone), one line did contain both sexes of fish. The presence of males in this gynogenetic clonal line and data from progeny testing of these males suggested that this line was homozygous for an allele or combination of alleles at an autosomal locus or loci which caused female to male sex reversal but with limited penetrance. Outbred clonal lines were also produced by crossing between different inbred clones. J. Exp. Zool. 284:675-685, 1999.

Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese flounder (Paralichthys olivaceus).

The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.

Evidence for the presence of sex chromosomes in the Nile tilapia ( Oreochromis niloticus) from synaptonemal complex analysis of XX, XY and YY genotypes

A cytogenetic analysis of chromosome synapsis was carried out during the first meiotic prophase of the Nile tilapia, Oreochromis niloticus. Three different genotypes were studied: XX sex-reversed males, `wild-type' (XY) males and YY `supermales'. TEM analysis of synaptonemal complex (SC) spreads revealed the presence of 22 fully paired bivalents during pachytene in both homogametic genotypes. In the heterogametic genotype, an incompletely paired segment was frequently observed during the process of meiotic synapsis in the terminal region of the longest bivalent. The presence of this unpaired segment, together with several features characteristic of sex-chromosome behaviour during meiosis, suggests the existence of a non-homologous region in this chromosomal pair in the heterogametic genotype, and provides cytological evidence for the chromosomal basis of sex determination in O. niloticus. The usefulness of SC analysis for the understanding of sex determination and its relevance in the management of species of aquacultural importance are discussed.

Convenient method for sex reversal in a freshwater teleost, the medaka

In order to analyze the mechanism of sex differentiation, fertilized eggs of Oryzias latipes with sex-linked R (orange-red) and r (white) phenotypes were exposed to estradiol-17β at various concentrations until hatching, and fry were reared on a normal diet up to sexual maturity. When embryos 0–6 days after fertilization were immersed in saline containing estradiol-17β at 1 μg/ml for only 4–10 days until hatching, the sex reversal of genotypic males to females was induced. The majority of the sex-reversed females (XrYR) were fertile, as indicated by the presence of vitellogenetic oocytes in their ovaries. These results show that steroid treatment during embryonic stages is a very convenient method for sex reversal in the medaka. J. Exp. Zool. 283:210–214, 1999. © 1999 Wiley-Liss, Inc.

Germ cell loss in the XXY male mouse: altered X-chromosome dosage affects prenatal development.

Male mammals with two X chromosomes are sterile due to the demise of virtually all germ cells; however, the underlying reasons for the germ cell loss remain unclear. The use of a breeding scheme for the production of XXY male mice has allowed us to experimentally address the question of when and why germ cells die in the XXY testis and whether the defect is due to the presence of an additional X chromosome in the soma, the germ cells themselves, or both. Our studies demonstrate that altered X-chromosome dosage acts to impair germ cell development in the testis at a much earlier stage than suggested by previous studies of XX sex-reversed males or XX/XY chimeras. Specifically, we noted significantly reduced germ cell numbers in the XXY testis during the period of germ cell proliferation in the early stages of testis differentiation. Although the somatic development of the XXY testis is morphologically and temporally normal, our studies indicate that germ cell demise reflects a defect in somatic/germ cell communication, since, in an in vitro system, the proliferative potential of fetal germ cells from XXY males is indistinguishable from that of normal males.