New Eukaryotic Semaphorins with Close Homology to Semaphorins of DNA Viruses

Semaphorins were initially described as a family of repulsive guidance molecules in embryonal development. Their basic structure consists of an N-terminal signal sequence, the defining semaphorin domain ofapproximately 500 amino acids, an Ig-like domain,and a variable carboxy-terminus. We recently described a viral semaphorin homologue encoded by the alcelaphine herpesvirus type 1. Less conserved, truncated homologues were also identified in poxviruses. Here we describe new human and murine semaphorin homologues. The respective genes were cloned and sequenced, and they were termed H-Sema-L and M-Sema-L (HGMW-approved symbols SEMAL and Semal, respectively). A multiply spliced mRNA of 3.2 kb is expressed in human placenta, spleen, thymus, and gonadal tissue. H-Sema-L maps to chromosome 15q22.3–q23 and M-Sema-L to the homologous locus 9A3.3-B in the mouse genome. The expression patterns and the presence of related genes in large DNA viruses suggest that this new semaphorin has a relevant function in the immune system.     

Gonadal hormones during puberty organize environment-related social interaction in the male rat

This study examined the role of gonadal androgens during puberty on the development of environment-related social interaction (SI) in male rats. SI in an unfamiliar environment versus SI in a familiar environment was evaluated in young adult rats as a function of sex and gonadal status. Intact male rats at 60 days of age exhibited a differential response to the two environments, whereas SI in intact female rats at 60 days was equivalent in the two environments. Furthermore, male rats castrated as juveniles and tested for SI at 60 days displayed a pattern of environment-related SI similar to SI in intact adult female rats. This effect of juvenile castration on SI in male rats was prevented by chronic exposure to testosterone propionate (TP) over Days 30 through 60. SI in male rats castrated in adulthood, on the other hand, was not altered either 2 or 4 weeks postcastration. The results from this study indicate that pubertal secretions of gonadal androgen(s) are necessary for the development of environment-related SI in male rats. In contrast, secretions of gonadal androgens in adulthood do not appear to be critical for the continued expression of environment-related SI, as suggested by the observation that environment-related SI in male rats remains unchanged by castration in adulthood.     

Endocrine disruptors: present issues, future directions

A variety of natural products and synthetic chemicals, known collectively as endocrine-disrupting compounds (EDCs), mimic or interfere with the mechanisms that govern vertebrate reproductive development and function. At present, research has focused on (i) the morphological and functional consequences of EDCs; (ii) identifying and determining the relative potencies of synthetic and steroidal compounds that have endocrine-disrupting effects; (iii) the mechanism of action of EDCs at the molecular level; and (iv) the recognition that in "real life," contamination usually reflects mixtures of EDCs. Future research must examine (i) the interactive nature of EDCs, particularly whether the threshold concept as developed in traditional toxicological research applies to these chemicals; (ii) when and how EDCs act at the physiological level, particularly how they may organize the neural substrates of reproductive physiology and behavior; (iii) the various effects these compounds have on different species, individuals, and even tissues; and (iv) how adaptations may evolve in natural populations with continued exposure to EDCs. Several predictions are offered that reflect these new perspectives. Specifically, (i) the threshold assumption will be found not to apply to EDCs because they mimic the actions of endogenous molecules (e.g., estrogen) critical to development; hence, the threshold is automatically exceeded with exposure. (ii) Behavior can compound and magnify the effects of EDCs over successive generations; that is, bioaccumulated EDCs inherited from the mother not only influence the morphological and physiological development of the offspring but also the offsprings' reproductive behavior as adults. This adult behavior, in turn, can have further consequences on the sexual development of their own young. (iii) The sensitivity of a species or an individual to a compound is related to species (individual)-typical concentrations of circulating gonadal steroid hormones. Related to this is the recent finding that alternate forms of the putative receptors are differentially distributed, thereby contributing to the different effects that have been observed. (iv) Except in extraordinary situations, populations often continue to exist in contaminated sites. One possible explanation for this observation that needs to be considered is that animals can rapidly adapt to the nature and level of contamination in their environment. It is unlikely that successive generations coincidentally become insensitive to gonadal steroid hormones fundamentally important as biological regulators of development and reproduction. Rather, adaptive alterations in the genes that encode steroid receptors may occur with chronic exposure to EDCs, allowing the sex hormone receptor to discriminate natural steroids from EDCs.     

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

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

miR-202-5p在牙鲆性腺中的表达分析及其与cbx2靶向关系验证

为研究miR-202-5p在鱼类中作用的靶基因及功能,采用荧光定量PCR和原位杂交技术构建了miR-202-5p在牙鲆(Paralichthys olivaceus)不同组织和雌雄性腺中的表达谱。定量结果发现, miR-202-5p在牙鲆性腺中具有特异性的高表达,且在精巢中的表达水平高于卵巢。原位杂交结果显示, miR-202-5p主要在精巢的精原细胞和精母细胞中表达,而仅在卵巢的Ⅳ和Ⅴ期卵母细胞中有较强烈表达。研究发现色素框同源蛋白2(Chromobox homolog 2, CBX2)在性腺发育中具有重要调节作用,为进一步研究miR-202-5p在牙鲆性腺发育中的功能,采用生物信息学方法预测和双荧光素酶报告基因技术鉴定了二者的靶向关系,结果证实, cbx2为miR-202-5p直接调节的靶基因,为深入阐述miR-202-5p在牙鲆性腺发育中的作用机制提供了基础。     

Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes

In germ cells, the function of which is to form the next generation, apoptotic cell death occurs during development, as in the case of somatic cells. In this study, we show that Bcl-x knockout heterozygous (Bcl-x(+/-)) mice exhibit severe defects in male germ cells during development. A substantial increase in apoptosis of male germ cells occurs at around embryonic day 13.5 (E13.5) in Bcl-x(+/-) embryos, leading to hypoplasia of postnatal testes and reduced fertility. On the other hand, female germ cells at the same stages do not show discernible differences between wild-type and Bcl-x(+/-) embryos. This phenotype of Bcl-x haploinsufficiency shows that regulation of apoptosis becomes different between the sexes at around the onset of sex differentiation. Through this study, we found that, in wild-type embryos, (1) apoptosis is much more frequent (approximately 10 times) in the male than in female germ cells, and (2) expression of Bcl-xL, but not that of Bax, is higher in female than in male germ cells, at around E13.5. Male fetal germ cells, cultured with gonadal somatic cells in vitro, showed higher frequencies of apoptosis than those cultured without gonadal somatic cells. On the other hand, in the absence of gonadal somatic cells, both male and female fetal germ cells in vitro showed similar frequencies of apoptosis to female fetal germ cells in vivo. Therefore, male germ cell apoptosis, of which the default pathway is similar to that of the female, is likely to be influenced by male gonadal environments.     

The role of plasma-binding proteins in the cellular uptake of lipophilic vitamins and steroids.

Steroid hormones and many other lipophilic compounds are believed to enter cells solely by free diffusion through the plasma membrane. However, recent work using a megalin-deficient mouse model has identified a new endocytic pathway responsible for the delivery of steroids to renal and gonadal tissues. This review describes these new pathways for uptake of 25-hydroxy-vitamin-D3 and the gonadal sex-steroids (17beta-estradiol and testosterone) bound to vitamin D-binding protein and sex hormone-binding globulin respectively. Furthermore examples of other lipophilic molecules that enter cells by receptor-mediated pathways will be presented and the receptors responsible for their uptake described.     

The mechanism for primordial germ-cell migration is conserved between Japanese eel and zebrafish

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).     

Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.     

Alterations in gonadal steroidogenesis in individuals expressing a common genetic variant of luteinizing hormone

Some pathologies of the pituitary-gonadal function have recently been found to be due to mutations of the gonadotropin or gonadotropin receptor genes. Although these conditions are extremely rare, they are very informative, by elucidating some less well characterized facets of normal gonadotropin function and the molecular pathogenesis of disturbances in sexual differentiation and fertility. In contrast, there is a common polymorphism in the Luteinizing Hormone (LH) β-subunit gene, where two point mutations cause two alterations in the amino acid sequence (Trp⁸→Arg and Ile¹⁵→Thr) and introduce an extra glycosylation signal to Asn¹³. The carriers of this variant gene are largely healthy, but certain mild differences in their gonadal function have been found, as reflected by alterations in gonadal steroidogenesis, pubertal development and predisposition to diseases such as infertility, polycystic ovarian syndrome, and breast and prostatic cancer. The purpose of this chapter is to review the current knowledge of the occurrence, special functional features and clinical correlates of this LH variant.     

Prodomain-dependent tissue targeting of an ADAMTS protease controls cell migration in Caenorhabditis elegans

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted proteins play important roles in animal development and pathogenesis. However, the lack of in vivo models has hampered elucidation of the mechanisms by which these enzymes are recruited to specific target tissues and the timing of their activation during development. Using transgenic worms and primary cell cultures, here we show that MIG-17, an ADAMTS family protein required for gonadal leader cell migration in Caenorhabditis elegans, is recruited to the gonadal basement membrane in a prodomain-dependent manner. The activation of MIG-17 to control leader cell migration requires prodomain removal, which is suggested to occur autocatalytically in vitro. Although the prodomains of ADAMTS proteases have been implicated in maintaining enzymatic latency, polypeptide folding and secretion, our findings demonstrate that the prodomain has an unexpected function in tissue-specific targeting of MIG-17; this prodomain targeting function may be shared by other ADAMTSs including those in vertebrates.     

The consequences of altered somatotropic system on reproduction

Although the primary control of gonadotropin secretion is by the hypothalamic GnRH and the gonadal function is controlled by the pituitary gonadotropins and prolactin, the emerging evidence suggests a vital role of the somatotropic axis, growth hormone (GH), and insulin-like growth factor-I (IGF-I) in the control of the pituitary and gonadal functions. It has been shown that GH deficiency, GH resistance, and experimental alterations in IGF-I secretion modify folliculogenesis, ovarian maturation, ovulation, and pregnancy, and in the male, GH/IGF-I plays an important role in spermatogenesis and the Leydig cell function. The primary focus of this review is to examine the role of GH/ IGF-I on the onset of puberty, fertility, pituitary, and gonadal endocrine functions. A number of studies have revealed that fertility is affected in GH-deficient dwarf and in IGF-I gene-ablated mice, possibly due to subnormal function of either the pituitary gland or the gonads. In the female GH receptor gene knockout (GHR-KO) mice, there was impairment in follicular development, ovulation rate, sexual maturation, production of and responsiveness to pheromonal signals, and the corpus luteum function. In IGF-I-deficient male GHR-KO mice, puberty is delayed, spermatogenesis is affected, and neuroendocrine-gonadal function is attenuated. Similarly, in some of the human Laron syndrome patients, puberty is delayed due to GH resistance. These data suggest that, in addition to GnRH and gonadotropins, GH/IGF-I influences the pituitary and gonadal functions in animals and humans.     

A new era for small molecule screening: from new targets, such as JAK2 V617F, to complex cellular screens

Traditionally reserved to research and development in pharmaceutical companies, screening of small molecule libraries is rapidly becoming an approach undertaken by academic laboratories. Novel cellular assays, sensitive systems to probe function, emerging new molecular targets are just some of the reasons explaining this shift. Targets of small molecules identified in cellular screens begin to be amenable to identification by elegant genetic approaches, such as probing toxicity of candidate small molecules on libraries of genetically modified yeast strains. Several new targets, such as JAK2 V617F, an activated JAK2 (Janus Kinase 2) mutant genetically associated with the majority of human myeloproliferative neoplasms, are being actively pursued. In this Review Series, we will learn how libraries of small molecules are harnessed to identify novel molecules, that alone or in combination, have the ability to alter cell fate, cell signalling, gene expression or response to extracellular cues.     

Photorefractoriness and energy availability interact to permit facultative timing of spring breeding

In seasonally breeding mammals, vernal reproductive development is not directly triggered by increases in day length, rather, an endogenous program of photorefractoriness to short winter days initiates spontaneous development in advance of spring. The transition to the reproductive phenotype is energetically demanding. How food availability in late winter and early spring impacts the onset and expression of photorefractoriness is not known. In this study, male Siberian hamsters were born into a simulated natural photoperiod, and at the winter solstice, they were subjected to a restricted feeding protocol in which a daily food ration was provided in an amount equal to ad libitum (AL) intake during the weeks preceding the solstice. Over the next several months, AL-fed control hamsters exhibited spontaneous recrudescence or spontaneous development. In contrast, vernal reproductive development was abolished in most food-rationed hamsters. In food-rationed hamsters that did exhibit recrudescence, conspicuous delays in the onset of gonadal development and decreases in the magnitude of growth were evident. In all hamsters, the termination of food rationing triggered rapid gonadal development. The data indicate that late winter/early spring increases in environmental food availability are required for the normal manifestation of photorefractoriness-induced reproductive development and suggest that a function of photorefractoriness may be merely to disinhibit the reproductive axis from photoperiodic suppression. Vernal gonadal development or recrudescence appears to be strongly affected by proximate energy availability.     

SMAD3 regulates gonadal tumorigenesis

Inhibin is a secreted tumor suppressor and an activin antagonist. Inhibin alpha null mice develop gonadal sex cord-stromal tumors with 100% penetrance and die of a cachexia-like syndrome due to increased activin signaling. Because Sma and Mad-related protein (SMAD)2 and SMAD3 transduce activin signals in vitro, we attempted to define the role of SMAD3 in gonadal tumorigenesis and the wasting syndrome by generating inhibin alpha and Smad3 double mutant mice. Inhibin alpha and Smad3 double homozygous males were protected from early tumorigenesis and the usual weight loss and death. Approximately 90% of these males survived to 26 wk in contrast to 95% of inhibin-deficient males, which develop bilateral testicular tumors and die of the wasting syndrome by 12 wk. Testicular tumors were either absent or unilaterally slow growing and less hemorrhagic in the majority of double-knockout males. In contrast, development of the ovarian tumors and wasting syndrome was delayed, but still occurred, in the majority of the double-knockout females by 26 wk. In double mutant females, tumor development was accompanied by typical activin-induced pathological changes. In summary, we identify an important function of SMAD3 in gonadal tumorigenesis in both sexes. However, this effect is significantly more pronounced in the male, indicating that SMAD3 is the primary transducer of male gonadal tumorigenesis, whereas SMAD3 potentially overlaps with SMAD2 function in the ovary. Moreover, the activin-induced cachexia syndrome is potentially mediated through both SMAD2 and SMAD3 or only through SMAD2 in the liver and stomach. These studies identify sexually dimorphic functions of SMAD3 in gonadal tumorigenesis.     

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.     

A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.