P450 aromatase expression in the temperature-sensitive sexual differentiation of salamander (Hynobius retardatus) gonads

Sex differentiation of gonads in amphibians is believed to be controlled genetically, but altered epigenetically or environmentally. When larvae of the salamander Hynobius retardatus were reared at defined temperatures from hatching to metamorphic stages, a high temperature (28 degrees C) induced exclusively female gonads (ovaries), whereas intermediate (20 and 23 degrees C) or lower (16 degrees C) temperatures produced a 1:1 sex ratio of the morphological gonads. The thermosensitive period was determined to be restricted from 15 to 30 days after hatching, just before or when sexual differentiation occurred. Hynobius P450 aromatase (P450arom) cDNA was isolated from adult gonads and the partial nucleotide or deduced amino acid sequences were determined, showing a high level of identity with various vertebrate species. The P450arom gene was expressed predominantly in the adult ovary and brain, weakly in testis, but not in other somatic organs. A typical sexual dimorphism in P450arom expression was detected in normally developing larvae by a quantitative competitive RT-PCR; strong expression in the female gonads but very weak in male gonads. The dimorphism was detected much earlier than the morphological sexual differentiation of the gonads. When larvae were reared at the female-producing temperature (28 degrees C), strong expression was detected in all the temperature-treated larvae, suggesting that P450arom was up-regulated, even in genetic males. Our results confirm the importance of the P450arom regulation in the sexual differentiation of gonads and demonstrate that an up-regulation of P450arom is involved in the process of temperature-sensitive sex reversal in this species.     

Biology of the amphioxus,Branchiostoma belcheri in the Ariake Sea,Japan II. Reproduction

We investigated the reproduction of the amphioxus Branchiostoma belcheri in the southern Ariake Sea, Japan, from 1999 to 2002. Gonads were very small or almost absent from September to December and began to develop in January. Changes in the gonad length index indicated that spawning began in mid June, after the gonads had attained maximum size. Although most gametes were extruded during the first spawning, some remained in the gonads. Shrunken gonads were much smaller but contained gametes, and the second spawning occurred around 10 July. It was unclear whether all adults spawned twice, but a large proportion of individuals did. After the first spawning, both males and females lost 30% of their body weight. Most one-year-old individuals did not spawn, and the minimum size at maturity was ca. 20 mm. We found no termination of reproduction among very large individuals. The average number of gonads was significantly larger on the right side of the body (26.2 in males and 26.1 in females) than on the left side (24.0 in males and 23.5 in females). We found two hermaphrodites in a total of 11,184 specimens examined. Each had four or three ovaries among a total of 45 or 54 gonads.     

Tamoxifen and sex differentiation of the gonads in chick embryo

Tamoxifen or 4-hydroxytamoxifen were injected either alone or in combination with oestradiol into 4-5 day-old chick embryos in order to study their action on the sex differentiation of the gonads. The results of the histological study of the gonads performed at the stage of 16-19 days warrant the following conclusions: None of both anti-oestrogens exerts an effect on the testes. None of both compounds modifies the sex differentiation of the female gonads. Tamoxifen exerts an antagonistic action on the feminization of the testes by oestradiol. These conclusions do not lend support to the hypothesis according to which oestrogens play a role in normal sex differentiation of the female gonads.     

Nutrient allocation in the gonads of the sea urchin Arbacia dufresnii in different stages of gonadal development

This study reports nutrient allocation in different stages of gonadal development for two populations of the sea urchin Arbacia dufresnii off the Patagonian coast of Argentina (Nuevo Gulf and San Jorge Gulf). The biochemical composition of gonads was used to assess nutrient allocation by measuring ash, soluble protein, lipid and trichloroacetic acid-soluble carbohydrate concentrations, and absolute contents over a 24-month period. Reproductive output in terms of energy was calculated for females. Results were correlated with histological stage of the gonads. Soluble proteins were the main component for the Nuevo Gulf population while unmeasured organic material (i.e. insoluble proteins and nucleic acids, especially in testes) was prevalent in gonads from San Jorge Gulf. Soluble protein and lipid concentrations followed the gonadal cycle, while carbohydrate concentration was almost negligible, especially in the Nuevo Gulf population. The different patterns in the gonadal cycle in the two populations were reflected in the biochemical composition of gonads. Concentrations and contents of the biochemical components and reproductive output were higher in the population from San Jorge Gulf owing to the larger size of gonads and gametes. These findings contribute to the better understanding of the plasticity of the reproductive biology of A. dufresnii in different environments.     

Temperature regulates SOX9 expression in cultured gonads of Lepidochelys olivacea, a species with temperature sex determination

Although sex determination starts in the gonads, this may not be the case for species with temperature sex determination (TSD). Since temperature affects the whole embryo, extragonadal thermosensitive cells may produce factors that induce gonadal sex determination as a secondary event. To establish if gonads of a species with TSD respond directly to temperature, pairs of gonads were cultured, one at female-promoting temperature (FPT) and the contralateral at male-promoting temperature (MPT). Histological and immunohistochemical detection of SOX9 revealed that the response to temperature of isolated gonads was similar to that of the gonads of whole embryos. While gonads cultured at MPT maintained SOX9 expression, it was downregulated in gonads at FPT. Downregulation of SOX9 took longer in gonads cultured at stage 23 than in gonads cultured at stage 24, suggesting that a developmental clock was already established at the onset of culture. To find out if sex commitment occurs in vitro, gonads were switched from FPT to MPT at different days. Results showed that the ovarian pathway was established after 4 days of culture. The present demonstration that gonads have an autonomous temperature detector that regulates SOX9 expression provides a useful starting point from which the molecular pathways underlying TSD can be elucidated.     

Germinal cell ectopism in the strepsirhine prosimian Galago crassicaudatus crassicaudatus

The presence of germinal cells outside of the embryonal and fetal gonads of the strepsirhine prosimian Galago crassicaudatus crassicaudatus is described. Forty-three embryos and fetuses from day 26 or 27 of gestational age to near term were studied: more than 90% possessed germinal cells in ectopic sites situated either far from (extragonadal ectopism) or close to the gonads (perigonadal ectopism). The first sites were the walls of the aorta and mesenteric artery, the stroma between the aorta and the cardinal vein, and the retroperitoneal neuroganglia. The second were the mesenchyme dorsal to the gonads and around the vestigia of the mesonephric glomeruli and tubules, and the rete ovarii and testis. The ectopic cells were generally present in conscpicuous numbers, in some animals being more numerous than in the gonads. Those situated far from the gonads underwent degeneration and decreased significantly in numbers during post-embryonal stages of development, while the others remained numerous and functionally active up to near term. While the differentiation of the extragonadal germinal cells after day 60 of gestational age could not be studied due to technical difficulties, the XX and XY cells in perigonadal sites appeared to follow patterns of differentiation identical to those of their entopic counterparts.     

Variations of lipid and fatty acid contents during the reproductive cycle of the anthozoan <Emphasis Type="Italic">Renilla koellikeri</Emphasis>

Lipid and fatty acid contents were monitored in the sea pansy Renilla koellikeri during its reproductive cycle. The fluctuations of lipids and fatty acids were closely related to sexual development and gonads represented the principal site of lipid storage in the colony. Between April and May, fatty acids and lipids greatly accumulated just prior to spawning. The subsequent decrease of lipid and fatty acid contents in June probably resulted from loss due to spawning. Fatty acid composition was assessed for the whole animal and was compared between male and female gonads, and between gonads and somatic tissues. Arachidonic acid (AA) was the principal fatty acid in the whole colony followed by palmitic acid and eicosapentaenoic acid (EPA). The examination of fatty acid variations in gonads showed that EPA and AA were highly incorporated in eggs during the month preceding spawning, whereas in the same period, EPA was the only fatty acid to accumulate in spermatophores. The differences of fatty acid levels observed in gonads reflected sex differences observed in colonies. The seasonal variations of the omega6/omega3 ratio of the colonies were largely influenced by EPA and AA, and appeared as a reliable indicator of the sexual maturity of the sea pansy.     

Metals in the tissues of the starry flounderPleuronectes stellatus from Nyiskii Bay (Southeastern Sakhalin)

We examined the concentrations of the heavy metals Zn, Cu, Cr, Pb, and Cd in the liver, gonads, and muscles of male and female starry flounderPleuronectes stellatus from Nyiskii Bay (off the northeastern coast of Sakhalin) in August 1996. It is shown that the accumulation of the metals was different in the organs and tissues of males and females. The greatest differences were recorded in the gonads. The concentrations of Zn and Cu were greater in the gonads of females, whereas the concentrations of Cd, Pb, and Cr were greater in male gonads. More Cd and Pb accumulated in the liver of females, whereas the concentrations of Zn and Cd were greater in the muscles of males. The results obtained seem to reflect the specific features of physiological processes in specimens of different sexes and the biochemical composition of the tissues of fish organs during the period of foraging and gonad development. The concentrations of metals in the muscles of starry flounder from Nyiskii Bay did not exceed the sanitary standards.     

Gonads of Sea Urchins as the Source of Medication Stimulating Sexual Behavior

In experiments on male rats it was established that native and lyophilized gonads of sea urchins have a stimulating effect on sexual behavior. An effective amount and optimal duration of a dose of the gonads of sea urchins were determined. It is suggested that sexual behavior is connected with the influence of the components of gonads on the adrenoreceptors of the reproductive system of animals.     

Morphometric characteristic of sex cells and seasonal changes of female gonads of Cucumaria japonica (Semper, 1868) (Echinodermata: Holothuroidea) from Peter the Great Bay, Sea of Japan

The oocytes at different stages of development were characterized morphometrically, and seasonal changes in female gonads of the Far-eastern edible holothurian Cucumaria japonica (Semper, 1868) from the Peter the Great Bay were described. Groups of oocytes and categories of sexual tubules corresponding to different stages of development of gonads were distinguished. Free laying oocytes were divided into several groups, i.e., oocytes 3, 4 and 5. The greatest size of C. japonica oocytes was 460 μm. In various seasons of the year there was greater growth of oocytes in the gonads, which reached its maximum volume during reproduction of the holothurians. The gonads developed asynchronously. All the stages of development were observable simultaneously in the gonads of each individual of this species during the reproductive cycle, and the stage of fast growth prevailed. C. japonica spawned twice a year, in May–June and from July to October. Unreleased oocytes were subjected to resorption.     

The histological picture of the hypothalamus (the nucleus preopticus) and hypophysis in male Anguilla anguilla L. treated with hormones

Studies were conducted on the effect of human chorionic gonadotropin (HCG), testosterone and desoxycorticosterone acetate (DOCA) on the histological picture of the nucleus preopticus (NPO), hypophysis and gonads of male European eels. It was ascertained that none of the injections used changed the content of the Gomori-positive material within the NPO cells. HCG and testosterone injections altered the histological picture of the hypophysis and injections of HCG influenced the gonads by accelerating their maturity. The injections of DOCA did not change the histological picture of the hypophysis and gonads in male eels.     

An in vitro model of gonad differentiation in the chicken. Estradiol-induced sex-inversion results in the occurrence of serological H-Y antigen

Dissociated cells from the gonads and mesonephros of 8-day-old chicken embryos were reorganized in rotation culture. The aggregates obtained from gonadal cells exhibited specific morphologic and histologic sex differences. In the presence of estradiol, aggregates from testicular cells showed characteristics similar to control ovarian aggregates, while in ovarian aggregates under estradiol treatment the female organization became more pronounced. Determination of serological H-Y antigen revealed that male aggregates of gonads and mesonephros were negative for H-Y and those of female embryos were positive for H-Y. Administration of estradiol did not change the H-Y findings in female aggregates. In contrast, in the male, gonadal cultures became H-Y positive while mesonephros cultures remained negative. It is assumed that estradiol induces the occurrence of H-Y antigen in the gonads.     

Gonadal development and spawning of female ocean whitefish, Caulolatilus princeps (Pisces: Branchiostegidae) in the Bay of La Paz, B.C.S., Mexico

The developmental stages of the gonads and the spawning season of the ocean whitefish, Caulolatilus princeps , were determined. Gonad maturation is annual, and the spawning season is prolonged, from late autumn to early spring. From November to March, around 80% of the females were in an advanced mode of gonadal development, and in April most gonads were spent. From May to October the gonads were at rest and preparing for the next spawning season.
The gonadosomatic index describes the reproductive activity of the species adequately.     

Effect of Fixed Daylengths on the Photoperiodic Regulation of Gametogenesis in the Sea Urchin Strongylocentrotus purpuratus

Summary

The gametogenic response of the sea urchin Strongylocentrotus purpuratus was compared under treatments of different photoperiod regimes of fixed and seasonal changing daylength. Quantification of gametogenic activity in en- zymatically dissagregated ovaries and in histological sections of testes have shown that the sea urchin is sensitive to both fixed and variable daylengths. After one year at fixed short day (8L:16D) or fixed neutral day (12L:12D) the gonads were ripe and in active gametogenesis, the same as were gonads of animals reared at changing photoperiod and sampled during the short-day phase of the cycle. Under fixed long day (16L:8D) the gonads lacked significant amount of gametes and had the cell constitution found in gonads of animals reared at changing photoperiod and sampled during the long-day phase of the cycle. Measurement of a critical daylength related to the autumnal equinox seems to be part of the mechanism in the photoperiodic regulation of vitellogenic oocyte growth and spermatogenesis in these animals.     

A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4+/+ and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.     

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.     

Genetic and developmental evidence for a repressed genital primordium in Drosophila melanogaster

Agametic, a maternal-effect mutation, causes the absence of germ cells in approximately 40% of the gonads of flies derived from homozygous females. The nature of the deficiency in the eggs produced by these flies was examined. Ultrastructural abnormalities were seen in the polar granules of some eggs shortly after fertilization. Although a normal number of pole cells form, some are abnormal with degenerating polar granules and nuclear bodies and they contain myeloid bodies. The pole cells reach the gonads and at 14 hr of development all the gonads contain germ cells. However, in 40% of the gonads the germ cells become necrotic and disappear. Thus, the source of agametic gonads in the adult is embryonic death of pole cells in some gonads. To test whether this gonadal death is an autonomous deficiency of the mutant pole cells, mosaic pole cell populations were produced by reciprocal pole cell transplantation. In both types of transplants, the mutant pole cells died autonomously. In eight instances gonads containing only donor pole cells were obtained. Since mutant pole cells die when wild-type pole cells normally begin dividing, we suggest that the lesion affects the ability of these mutant pole cells to reenter the cell cycle.     

Evidence for gonadotropin-releasing hormone peptides in the ovary and testis of rainbow trout.

GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA is present also in the ovary and testis. However, isolation of the peptide from testis has not been reported. We used HPLC and specific RIAs to determine whether the GnRH peptide can be detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH that are present in the ovary and testis. Extracts of immature and mature ovarian and testicular tissue were examined from 17- to 21-mo-old rainbow trout (Oncorhynchus mykiss). For the first time, GnRH peptides were isolated from testis and identified by HPLC-RIA with specific antisera and by elution position compared with synthetic standards. GnRH peptides were also present in the ovary. In addition, multiple forms of GnRH, including a form not normally detected in the brain of trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages in the gonads, which may explain the inability to detect and isolate the GnRH peptides from gonads in earlier studies.