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.     

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.     

Involvement of oestrogens in sexual differentiation of gonads as a function of temperature in turtles

Abstract. To investigate the possible involvement of oestrogens in the phenomenon of temperature sex-reversal in the turtle Emys orbicularis , the effects of oestrone, oestradiol and an antioestrogen. tamoxifen, on sexual differentiation of gonads were examined at a male-producing temperature of 25° C and at a female-producing temperature of 30° C. When oestrone or oestradiol were injected into eggs incubated at 25° C just before or at the beginning of the thermosensitive period, the gonads differentiated into ovaries instead of testes. Conversely, when tamoxifen was injected, at the same stages, into eggs incubated at 30° C, epithelial cords or tubes, similar to potential seminiferous cords, differentiated in the interior part of the gonads. However, an ovarian-like cortex persisted at their surface. At 25° C, treatment with tamoxifen or with both tamoxifen and oestradiol also resulted in differentiation of ovotestes. These experiments show that tamoxifen binding to oestrogen receptors prevented the inhibitory action of oestrogens on testicular cord development. Maintenance (at 30° C) or development (at 25° C) of ovarian cortex in the presence of tamoxifen can be expected from an agonistic action of this drug, as already described. Preliminary data on steroid content in the gonads indicate that, during the early stages of the thermosensitive period, the level of estrogens is higher at 30° C than at 25° C. It is proposed that in species displaying temperature sensitivity for the sexual differentiation of gonads, temperature acts on the processes regulating the synthesis or the activity of cyto-chrome-P450 aromatase.     

Time required for temperature-induced changes in gonadal aromatase activity and related gonadal structure in turtle embryos

Abstract. In the turtle Emys orbicularis , sexual differentiation of gonads is temperature-dependent. Oestrogens have been shown to be involved in this phenomenon and temperature has been expected to act, directly or indirectly, on regulation of synthesis or activity of cytochrome P-450 aromatase (P-450 arom). We have studied the effects of temperature shifts and of exposure at female- or male-producing temperatures for different times on gonadal aromatase activity and gonadal structure. In a first series of experiments, eggs were incubated at 25°C (masculinizing temperature) up to stage 18 and then exposed for 1 to 8 days at 35°C, a highly feminizing temperature. The response was exponential: aromatase activity increased clearly only after 4 day exposure at 35°C, then it was considerably enhanced. After 1 and 2 days at 35°C, the structure of gonads was not modified. With longer exposures at 35°C, gonads were progressively feminized: medullary epithelial cords disappeared, whereas an ovarian cortex was forming. In another type of experiment, eggs incubated at 30°C (feminizing temperature) until stage 19 were transferred at 25°C for 6 days. In embryos of these shifted eggs, gonadal aromatase activity was about ninefold lower than that in control embryos (maintained at 30°C). However, this activity did not fall to the level measured in embryos of the same stage incubated at 25°C from egg-laying and was about twofold higher than that measured at the time of transfer. Gonads exhibited a cortex anlage but the medulla was more voluminous than that of controls and epithelial cords were beginning to form within. Together these results show that changes in gonadal aromatase activity and in gonadal structure are correlated, and that temperature acts on regulation of P-450 arom synthesis. Amplification of this synthesis during the thermosensitive period at higher temperatures could reflect amplification of expression of the P-450 arom gene.     

Sexual differentiation of the zebra finch song system parallels genetic, not gonadal, sex.

Mechanisms regulating sexual differentiation of the zebra finch song system present an intriguing puzzle. Masculine development of brain regions and behavior can be induced in genetic females by posthatching estradiol treatment. That result is consistent with the hypothesis that estradiol, converted within the brain from testicular androgen via the aromatase enzyme, masculinizes neural structure and function. In contrast, treatment during specific stages of development with the aromatase inhibitor Fadrozole has not prevented masculine development, and the presence of testicular tissue in genetic females did not induce masculine organization of neuroanatomy or singing behavior. Fadrozole treatments in those previous studies were limited, however, and most genetic females had both ovarian and testicular tissue. The present experiments were designed to provide increased aromatase inhibition and to reliably produce genetic females with only testicular tissue. Eggs received a single injection at a later age or with higher doses of Fadrozole than had been used previously. Some embryos were exposed to Fadrozole more frequently by either injecting eggs on 2 days of development or dipping them for 10-12 days in Fadrozole. Finally, in some individuals from Fadrozole-treated eggs, the left gonad was removed, leaving each genetic male and female with a single right testis. None of these treatments significantly affected development of the song system compared to appropriate control groups. These results suggest that sexual differentiation of the zebra finch song system is not regulated by embryonic aromatase activity or by gonadal secretions and instead involves events that need not be mediated by steroid hormones.     

Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of the European pond turtle, sexual differentiation of gonads is temperature-dependent. Production of oestrogens appears to play a key role in this phenomenon. Gonadal aromatase activity was measured in embryos incubated at 25°C (masculinizing temperature) and at 30°C (feminizing temperature). At the beginning of the thermosensitive period, the aromatase activity was low at both temperatures but was somewhat higher at 30 than at 25°C. Afterwards, it remained low in differentiating testes at 25°C, whereas it increased in differentiating ovaries at 30°C to form a marked peak when germ cells underwent meiotic prophase. Eggs were shifted either from 25 to 30°C (highly feminizing) or from 30 to 35°C for 6 days at different stages of embryonic development. The 25–35°C shifts performed during the thermosensitive period strongly increased the aromatase activity but were ineffective after this period. The 30–35°C shifts increased the aromatase activity at all stages. Altogether, results indicate that, in differentiating gonads of turtle embryos, temperature acts on the regulation of synthesis (and therefore activity) of cytochrome P-450 aromatase (P-450-aro). The expression of the P-450-aro gene itself could be temperature-dependent. However, temperature could also act upon the expression of another gene involved in P-450-aro regulation.     

Expression of P450(arom) in Malaclemys terrapin and Chelydra serpentina: a tale of two sites.

The formation of estrogens from androgens in all vertebrates is catalyzed by the "aromatase" complex, which consists of a membrane bound P(450) enzyme, P(450) aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH-cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determination in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terrapin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incubated at male temperatures (26.5 degrees C). The level of expression is similar to that of a normal embryonic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5 degrees C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5 degrees C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5 degrees C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5 degrees ), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important.     

南方鲶性腺分化的组织学观察

用芳香化酶抑制剂(Fadrozole)、雌激素受体拮抗剂(Tamoxifen)对人工孵化的南方鲶(Silurus meridionalis)幼鱼进行雄性化诱导处理(口服),获得雄鱼。对孵化后第5—130d的南方鲶幼鱼性腺进行组织学观察,结果表明,在实验条件下,南方鲶性腺分化发生在孵化后7d左右,雌雄性分化过程差异明显。雌鱼卵巢腔在孵化后12d左右形成,生殖细胞在孵化后35d左右快速增殖,成熟分裂最早发生在孵化后55d左右;雄鱼生殖细胞在孵化后130d左右快速增殖,成熟分裂最早发生在孵化后130d左右。雌性性腺分化早于雄性。     

An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal

Dietary administration of a cytochrome P450 aromatase (P450arom) inhibitor (fadrozole) in genetic female juveniles of zebrafish (Danio rerio) was performed at 15-40 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, treated with 0, 10, 100 and 1000 microg fadrozole g(-1) diet(-1) were 0, 62.5, 100 and 100%, respectively. Rearing at high water temperature in genetic all-females was performed at 15-25 days post-hatching. The percentage of gonadal masculinization in the genetic all-females at 40 days post-hatching, at water temperatures of 28.5, 35 and 37 degrees C were 0, 68.8 and 100%, respectively. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive oocytes of early diplotene and perinucleolar stages in fadrozole-treated genetic females (1000 microg g(-1) diet(-1)) were observed at 15-40 days post-hatching during sex-reversal. In contrast, apoptotic oocytes of early diplotene stage in high temperature-treated genetic females (at 35 and 37 degrees C) during sex-reversal and presumptive males of wild-type fish during sex differentiation were found at 15-27 days post-hatching. Our findings indicate that oocyte apoptosis, depletion of P450arom activity and differentiation of spermatogonia during gonadal sex-reversal are caused by treatments of aromatase inhibitor or high water temperature.     

Expression and characterization of the cyp19a gene and its responses to estradiol/letrozole exposure in Chinese soft‐shelled turtle (Pelodiscus sinensis)

Cytochrome P450 aromatase (CYP19) catalyzes the conversion of androgens to estrogens and is critical in sex differentiation. CYP19 exists as the ovarian type and brain type. Herein, we cloned the full‐length ovarian cyp19a gene from the Chinese soft‐shelled turtle, Pelodiscus sinensis (pscyp19a). We determined the distribution of pscyp19a in adult tissue and evaluated its expression during embryonic development, following treatment with 17β‐estradiol (E2) or letrozole (LE). The pscyp19a complementary DNA is 2,285 bp in length and comprises a 1,512 bp open reading frame that encodes a protein of 503 AA. The nucleotide sequence and amino acid of pscyp19a shared significant identity with other vertebrate sequences. Expression of pscyp19a was high in the ovary (p < 0.01), and exhibited modest expression in the female brain and intestine. Expression of pscyp19a displayed significant differences between sexes during early embryo development stages; expression increased gradually during embryonic development in females, but the opposite trend was observed in males. Female embryos treated with different concentrations of E2 and LE displayed altered pscyp19a expression compared with untreated individuals, and E2 clearly induced pscyp19a expression. These results indicate that pscyp19a gene plays important roles in early developmental stages in Chinese soft‐shelled turtle, and may assist future studies on sex differentiation and sex control in this and similar species.     

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.     

Histochemical and immunohistochemical studies of the gonads and paramesonephric ducts of male and female hatchlings of loggerhead sea turtles (Caretta caretta)

The sex of neonatal sea turtles is difficult to determine, because neonates lack heteromorphic sex chromosomes and dimorphic external characteristics; internal dimorphic morphology is defined at hatching. We used histochemical staining and made measurements in the gonads and paramesonephric ducts (PD) of both sexes to determine structural differences in female and male loggerhead sea turtle (Caretta caretta) hatchlings. We detected differences in the gonads and PD between the sexes including the amounts of mucopolysaccharides, collagen and elastic fibers. We determined that the thickness of the gonadal cortex and the diameter of the PD lumen are reliable sex-specific characteristics. We also assessed immunolocalization of aromatase, an enzyme complex that converts androgens to estrogens, and found differences in the localization and intensity of aromatase immunostaining in the gonads and PD of female and male hatchlings. Comprehensive studies of the sexual differences of sea turtles are important for conservation programs.     

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent K_m 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (V_max, 895 fmol/h/mg protein) than the female (V_max, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5-reduced metabolite of testosterone, 5-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (K_i = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.     

Degree of sex reversal as related to plasma steroid levels in genetic female chickens (Gallus domesticus) treated with Fadrozole

The objectives of this work were to determine whether or not plasma levels of testosterone and estradiol reflect the various grades of sex reversal in genetic female chickens treated with Fadrozole (CGS 16949 A), a nonsteroidal aromatase inhibitor, and whether gonadal aromatase activity and plasma levels of testosterone and estradiol in treated females can or not be modified by post-hatch treatments with Fadrozole or Fadrozole + testosterone. Eggs were injected with 1 mg Fadrozole on day 4 of incubation. In females having developed sex-reversed gonads, endocrine parameters (estradiol and testosterone) at and after 13 weeks of age were indicative of the degree of sex reversal, with, for example, sex-reversed females with two testes having the highest levels of testosterone and the lowest levels of estradiol. Among these females, eight (from a total of 13) produced ejaculates with scarce and abnormal spermatozoa. Some motility was observable in the ejaculates from five of them. None of the post-hatch treatments had a significant effect on plasma levels of testosterone or estradiol (measured at 3-week intervals from week 4 to week 28 post-hatch) or on gonadal aromatase activity (measured at 12 and 28 weeks). In conclusion, these results indicate that plasma levels of testosterone and estradiol at and after 13 weeks of age are valuable indicators of the degree of sex reversal in female chickens treated with Fadrozole prior to gonadal sex differentiation. In pre-cited conditions, post-natal treatments with either Fadrozole or Fadrozole + testosterone had no apparent effect on the degree of sex reversal in these birds. Finally, the occurrence of ejaculates with motile although scarce and abnormal spermatozoa, revealed that epididymes and ducti deferens can develop and become functional in sex-reversed female chickens.     

Experimentally induced true hermaphroditism in genetically female fowl

Summary Two types of hermaphroditism were experimentally induced in genetically female fowls by grafting of embryonic testes in embryos. Of the 27 hermaphrodites observed during the 8 months after hatching, 20 possessed a right testis and a left ovary and 7 a right testis and a left ovotestis. The testes and ovotestes contained seminiferous tubules with a more or less developed germ cell complement, attaining in many cases the early spermatid stage. The interstitial tissue was poorly functional, as shown by the absence of male secondary sex characters. The ovary or ovarian part of the ovotestes possessed numerous small ovarian follicles. The female arrangement of the plumage and the absence of spurs demonstrated the secretion of oestrogens. A mechanism is proposed for explaining this partial masculinization of genetically female gonads, a phenomenon which occurs during the period of embryonic sex differentiation, and is responsible for this experimental true hermaphroditism.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.