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.     

Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa

Sex steroids play a crucial role in the gonad differentiation in various species of vertebrates. However, little is known regarding the localization and biological activity of steroid-metabolizing enzymes during gonadal sex differentiation in amphibians. In the present study, we showed by real-time RT-PCR analysis that the expression of CYP17, one of the key steroidogenic enzymes, was higher in the indifferent gonad during sex differentiation in male than in female tadpoles of Rana rugosa but that there was no difference detected in the 3betaHSD mRNA level between the male and female gonads. We next examined the localization of CYP17, 3betaHSD and 17betaHSD in the indifferent and differentiating gonads by using three kinds of antibodies specific for CYP17, 3betaHSD and 17betaHSD, respectively. Positive signals for CYP17, 3betaHSD and 17betaHSD were observed in somatic cells of the indifferent gonad of males and in the interstitial cell of the testis. The enzymatic activity of CYP17 was also examined in the gonad during sex differentiation in this species. [(3)H]Progesterone (Prog) was converted to [(3)H]androstenedione (AE) in the indifferent gonad in males and females, but the rate of its conversion was higher in males than in females. Moreover, fluorescence in situ hybridization (FISH) analysis revealed that the CYP17 gene was located on the q arm of chromosome 9, indicating that CYP17 was autosomal in R. rugosa. Taken together, the results demonstrate that the CYP17 protein is synthesized in somatic cells of the indifferent gonad during gonadal sex differentiation in R. rugosa and that it is more active in converting Prog to AE in males than in females. The data suggest that CYP17 may be involved in testicular formation during sex differentiation in this species.     

Ultrastructural aspects of gonadal morphogenesis in Bufo bufo (Amphibia Anura) 1. Sex differentiation

The morphogenesis of gonads in Bufo bufo tadpoles was studied, and ultrastructural differences between sexes were identified. All specimens analyzed initially developed gonads made up of a peripheral fertile layer (cortex) surrounding a small primary cavity. Subsequently a central layer of somatic cells (medulla) developed. Both layers were separated by two uninterrupted basal laminae between which a vestige of the primary cavity persisted. During female differentiation, the peripheral layer continued to be the fertile layer. In males, the central layer blended into the peripheral layer and the basal laminae disappeared. The somatic cells of the central layer came into direct contact with the germ cells; this did not occur in females. Testicular differentiation continued with the migration of germ cells towards the center of the gonad. The somatic elements surrounding the germ cells appeared to play an active role in their transfer to the center of the gonad. The peripheral layer shrank and became sterile. Two basal laminae then re-formed to separate the fertile central layer from the peripheral sterile one. Germ cells have always been thought to perform a passive role in sex differentiation in amphibians. Following the generally accepted "symmetric model", the mechanism of gonad development is symmetrical, with cortical somatic cells determining ovarian differentiation and medullary somatic cells determining testicular differentiation. In contrast, we found that sex differentiation follows an "asymmetric" pattern in which germ cells tend primarily toward a female differentiation and male differentiation depends on a secondary interaction between germ cells and medullary somatic cells.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

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

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

Rspo1对雌性脊椎动物性别分化的功能

长期以来雌性脊椎动物的性别分化被认为是一个“默认”的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.     

Gonadal development and sex differentiation in the cichlid fish Cichlasoma 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.     

The reproductive cycle of yellowfin bream, Acanthopagms australis (Günther), with particular reference to protandrous sex inversion

Yellowfin bream, Acanthopagrus australis , of all age classes were collected from Moreton Bay, Australia. The species possessed typical sparid ovotestes in which the testis and ovary occur in separate zones. During the spawning period (June-August) juveniles, functional males and functional females could be distinguished by the macroscopic appearance of the gonad. The sex ratio of males to females decreases with age, indicating protandrous sex inversion.
Histological and structural study of the ovotestis showed all fish have previtellogenic cells in the ovarian zone but only juvenile and male fish have developing spermatogenic cells in the testis. Most juveniles become functional males by the age of two years but a small proportion of juveniles develop directly into functional females (primary females). Protandrous sex inversion commences after the spawning period when male fish appear with spermatozoa and no other spermatogenic cells in the testis. During the period November-January male fish with no spermatogenic cells are common and a reduction in size of the testis occurs so that by March-April the ovotestis becomes structurally and histologically similar to the female ovotestis. Some fish remain functional males during their whole life-history (primary males). In functional females vitellogenic cells are present in the ovary only during the spawning period and the testis remains very small in size.     

Histological study of the development and sex differentiation of the gonad in the European eel

The histological structure of the gonads was studied in yellow eels sampled from a coastal lagoon and from stocks reared in an aquaculture plant showing different sex ratios. Gonad development related to body size rather than to age and underwent an intermediate stage characterized by a structure of an early testis but containing oogonia and oocytes. This gonad was called the Syrski organ and the stage juvenile ambisexual. Ovaries were found in eels from 22–30 cm in length, possibly derived from undifferentiated gonads or from Syrski organs. Fully differentiated testes were found in eels >35 cm, derived from Syrski organs. These observations support the results of previous research. From elvers and in eels up to 15–16 cm in length, growth of the gonadal primordium is due to primordial germ cell migration. In eels > 15 cm multiplication of primordial cells begins. Oogonial clones were found in eels > 18 cm in length, whilespermatogonium B clones were observed in eels >30 cm in length. The dynamics of sex differentiation was different among stocks with different ultimate sex ratios: ovaries were found in shorter eels in stocks with a prevalence of females, in longer eels in stocks with a prevalence of males. This result supports the hypothesis of a metagametic (environmental) sex determination. The somatic cells in contact with germ cells and those in the interstitium appeared early during gonad development and preceded germ cell differentiation. This suggests that somatic cells are the targets of the environmental factors influencing sex differentiation.     

Sex Determination in Reptiles: An Update

Sex determination and sex differentiation are two separate butrelated phenomena. Sex differentiation is a programmed cascadeof events in which the indifferent gonad develops as a testisor an ovary with the appropriate urogenital and secondary sexcharacters. Sex determination is the event that sets this cascadein motion. In placental mammals, there is good evidence thatsex is determined by a gene on the Y chromosome (SRY) that initiatestestis formation. In the absence of SRY an ovary develops. Thereare, however, examples of placental mammal that develop as normalmales with no detectable SRY. In reptiles, sex differentiationappears to be similar to mammals (i.e., the same genes and hormonesact ina similar manner), but sex determination is clearly verydifferent. Ovarian differentiation in placental mammals canoccur in the absence of estrogen or an estrogen receptor. Ovariandifferentiation in reptiles requires the presence of estrogen.In the absence of estrogen a testis develops. In TSD reptiles,embryos will develop as females when treated with estrogen evenif eggs are incubated at male-inducing temperatures, and conversely,will develop as males when estrogen synthesis is blocked ineggs incubated at female-inducing temperatures. A number ofother genes have also been shown to be important in mammaliansex determination. One of these genes, Sox9, which is expressedin differentiating mouse testis, has recently been found tobe expressed in embryonic reptile testis. Other genes that appearto be common to both mammals and reptiles in the sex determiningcascade are SF- 1, MIH, and possibly DAX-1. Current researchis now focused on how the gene that produces the enzyme necessaryfor estrogen synthesis (aromatase) is regulated in the embryosof reptiles with genetic or environmental sex determination.Controversial issues in reptilian sex determination are 1) therole of the brain in gonadal sex determination, and 2) the roleof steroid hormones in the yolk prior to sex determination     

Sex determination and sex reversal

Sex determination in mammals is based on a genetic cascade that controls the fate of the gonads. Gonads will then direct the establishment of phenotypic sex through the production of hormones. Different types of sex reversal are expected to occur if mutations disrupt one of the three steps of gonadal differentiation: formation of the gonadal primordia, sex determination, and testis or ovary development.     

Gonadal morphogenesis and sex differentiation in cultured Ussuri catfish Tachysurus ussuriensis

The objective of this study was to investigate the optimal developmental time to perform sex reversal in Ussuri catfish Tachysurus ussuriensis, to develop monosex breeding in aquaculture. Systematic observations of gonadal sex differentiation of P. ussiriensis were conducted. The genital ridge formed at 9 days post fertilization (dpf) and germ cells begin to proliferate at 17 dpf. The ovarian cavity began forming on 21 dpf and completed by 25 dpf while presumptive testis remained quiescent. The primary oocytes were at the chromatin nucleolus stage by 30 dpf, the peri‐nucleolus stage by 44 dpf and the cortical alveoli stage by 64 dpf. The germinal vesicle migrated towards the animal pole (polarization) at 120 dpf. In presumptive testis, germ cells entered into mitosis and blood vessels appeared in the proximal gonad on 30 dpf. The efferent duct anlage appeared on 36 dpf and formation of seminal lobules with spermatogonia and lobules interstitium occurred at 120 dpf. Therefore, gonadal sex differentiation occurred earlier in females than in males, with the histological differentiation preceding cytologic differentiation in T. ussuriensis. This indicates that undifferentiated gonads directly differentiate into ovary or testis between 17 and 21 dpf and artificial induction of sexual reversal by oral steroid administration must be conducted before 17 dpf.     

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.     

Trichinella spiralis: Morphological characteristics of male and female intestine-infecting larvae

The sex of encysted and excysted intestine-infecting T. spiralis larvae can be distinguished by the following morphological characteristics: the male larva has a long (approx 50 μm) rectum, and the anterior part of the testis is curved posteriorly. The female larva has a shorter rectum (approx 25 μm), a telogonic ovary, coiled uterine and seminal receptacle primordia, and a vaginal primordium. In paraffin sections males can be recognized by the spermatocytes which are of the same size. The oocytes vary in size: the smallest are located in the ventral portion, the largest on the dorsal portion of the ovary. Sex of the larvae can be differentiated by the length of the rectum as early as the tenth day, by the curvature of the anterior part of the testis and by the uterine primordium by the eleventh day, and by the presence of the vaginal primordium by the thirteenth day of intramuscular development. Farre's Organ is believed to be the primordium of the seminal receptacle.     

Sex reversal in the medaka Oryzias latipes by brief exposure of early embryos to estradiol-17beta

To induce sex reversal of male to female, freshly-fertilized eggs of the S-rR strain medaka (Oryzias latipes) were immersed in saline containing estradiol-17beta (E2) in different concentrations for various durations until hatching. Results of the present experiment showed that the immersion duration in 1 microg/ml E2 to induce 100% reversal of sex differentiation in the genotypic males was enough only for one day (24 hr) post-fertilization (dpf) and that treatment with E2 for 1 dpf resulted in a dose-dependent manner with the maximum sex reversal of 100% at 1 microg/ml. To ascertain early developmental periods efficacious for inducing sex reversal, additional brief immersion treatments of eggs with E2 were further performed individually for four different early developmental periods (Stages 4-9, 10-12, 13-15 and 16-18) within 1 dpf. As a result, induction of sex reversal was observed in all these short immersion periods without any restricted efficacy. Between both experimental and control groups treated with or without E2 for 1 dpf, differences in the number of germ cells in a gonad were compared in newly-hatched fry. It was found that gonads of the genotypic males (XY) treated with E2 revealed the female type which contained many germ cells with much dividing activity. These data suggest that a possible switch mechanism that exogenous E2 could trigger to change the genetic cascades involved in sex determination upon fertilization exists in early developmental stages.     

An electron microscopic study of early gonadogenesis in the hermaphroditic appendicularian Oikopleura gracilis (Tunicata,Oikopleuridae)

The postembryonic development of the gonad in the hermaphroditic appendicularian O. gracilis was studied using transmission electron microscopy. The primordial germ cells were detected first in 10-h-old larvae and represent migrating primordial germ syncytium (mPGS) localized in the hemocoel of the tail/trunk junction and several haemocoel areas of the digestive compartment. The mPGS consisted of primordial germ nuclei (PGN) 2 μm in diameter, and elongate somatic-line nuclei 1.8 μm in diameter. In 12.5-h-old juveniles the gonad primordium 40 × 90 μm in size, was separated by a narrow space of haemocoel between the gut and the epidermis of the reproductive compartment. The gonad primordium consisted of the central syncytial part of primordial germ nuclei (PGN), enclosing a single layer of somatic epithelium. In 3-day-old juveniles, the gonad was differentiated into testis and ovary. The testis, 400 × 550 μm in size, is a syncytium of spermatogonial nuclei, covered by a single layer of somatic epithelium. The ovaries, 350 × 850 μm in size, consist of a syncytium with nurse nuclei and meiotic nuclei. The hermaphroditic gonad originates from extragonadal mPGS. Early gonadogenesis in appendicularians has ultrastructural features in common with early gonadogenesis in ascidians.     

Embryonic gonadal and sexual organ development in a small viviparous skink, Niveoscincus ocellatus

The majority of research into the timing of gonad differentiation (and sex determination) in reptiles has focused on oviparous species. This is largely because: (1) most reptiles are oviparous; (2) it is easier to manipulate embryonic developmental conditions (e.g., temperature) of eggs than oviductal embryos and (3) modes of sex determination in oviparous taxa were thought to be more diverse since viviparity and environmental sex determination (ESD)/temperature-dependent sex determination (TSD) were considered incompatible. However, recent evidence suggests the two may well be compatible biological attributes, opening potential new lines of enquiry into the evolution and maintenance of sex determination. Unfortunately, the baseline information on embryonic development in viviparous species is lacking and information on gonad differentiation and sexual organ development is almost non-existent. Here we present an embryonic morphological development table (10 stages), the sequence of gonad differentiation and sexual organ development for the viviparous spotted snow skink (Niveoscincus ocellatus). Gonad differentiation in this species is similar to other reptilian species. Initially, the gonads are indifferent and both male and female accessory ducts are present. During stage 2, in the middle third of development, differentiation begins as the inner medulla regresses and the cortex thickens signaling ovary development, while the opposite occurs in testis formation. At this point, the Müllerian (female reproductive) duct regresses in males until it is lost (stage 6), while females retain both ducts until after birth. In the later stages of testis development, interstitial tissue forms in the medulla corresponding to maximum development of the hemipenes in males and the corresponding regression in the females.     

Sex differences in dog corpus callosum.

Human studies reported sex differences in size and shape of the corpus callosum. These observations have been contested. The purpose of the present study is to investigate possible sex differences in the corpus callosum of dogs. The entire brains including the medulla from 12 female and 9 male adult mongrel dogs were removed and weighed. Total and partial area measurements of the callosum were made from photographic tracings of its outline. The callosum was partitioned into 3 regions; anterior half, posterior half, posterior one-fifth. The total corpus callosum, anterior half, posterior half, and posterior fifth or splenium areas were measured. Sex differences were found. The anterior half, the posterior half, the posterior fifth, and the total callosum were significantly greater in absolute area in males than in females.