Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila

The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to

A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats

A nerve growth factor (NGF) was isolated from the venom of Chinese cobra (Naja naja ntr a) by ion exchange chromatography, gel filtration and fast protein liquid chromatography (FPLC). The N-terminal sequence of 22 amino acid residues was identical with other NGFs previously purified from the venom of the same genus. The NGF monomer molecular weight was estimated to be 13 500 by reducing SDS-PAGE and the isoelectric point was determined to be 7.2 by isoelectric focusing electrophoresis. NGF improved the epididymal sperm motility of male rats and increased the pregnancy rate and fetus number of mated female rats. The serum levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male rats administrated NGF + gossypol was lower than that of male rats administrated gossypol. Histological sections of testes and epididymides showed that NGF reduced the destructive effects of gossypol on rat testes. (C) 1999 Elsevier Science Inc. All rights reserved.

HORMONAL-REGULATION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR AND PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 IN CULTURED MONKEY SERTOLI CELLS

Sertoli cells play a central role in the control and maintenance of spermatogenesis. Isolated Sertoli cells of mouse and rat testes have been shown to secrete plasminogen activator (PA) and a plasminogen activator inhibitor type-1 (PAI-1) in culture. In this study, we have investigated the hormonal regulation of PA and PAI-1 activities in cultured monkey Sertoli cells. Sertoli cells (5x10(5) cells/well) isolated from infant rhesus monkey testes were preincubated at 35 degrees C for 16 h in 24-well plates precoated with poly(D-lysine) (5 mu g/cm(2)) in 0.5 mi McCoy's 5a medium containing 5% of fetal calf serum and further incubated for 48 h in 0.5 mi serum-free medium with or without various hormones or other compounds, PA as well as PAI-1 activities in the conditioned media were assayed by fibrin overlay and reverse fibrin autography techniques respectively. The Sertoli cells in vitro secreted only tissue-type PA (tPA), no detectable amount of urokinase-type PA (uPA) could be observed, Monkey Sertoli cells were also capable of secreting PAI-1, Immunocytochemical studies indicated that both tPA and PAI-1 positive staining localized in the Sertoli cells, spermatids and residual bodies of the seminiferous epithelium; Northern blot analysis further confirmed the presence of both tPA and PAI-1 mRNA in monkey Sertoli cells. Addition of follicle-stimulating hormone (FSH) or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generating agents and gonadotrophin-releasing hormone (GnRH) agonist or phorbol ester (PMA) to the cell culture significantly increased tPA activity. PAI-1 activity in the culture was also enhanced by these reagents except 8-bromo-dibutyryl-cAMP, forskolin and 3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPA activity, whereas decreased PAI-1 activity, implying that neutralization of PAI-1 activity by tile high level of tPA in the conditioned media may occur. These data suggest that increased intracellular signals which activate protein kinase A (PKA), or protein kinase C (PKC) can modulate Sertoli cell tPA and PAI-1 activities, The concomitant induction of PA and PAI-1 by the same reagents in the Sertoli cells may reflect a finely tuned regulatory mechanism in which PAI-1 could limit the excession of the proteolysis.

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3'-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

KHAWIA SAUROGOBII N. SP (CESTODA: CARYOPHYLLIDEA) FROM FRESHWATER FISH SAUROGOBIO SPP. (CYPRINIDAE) IN CHINA

Khawia saurogobii n. sp. is described from the intestine of the cyprinid fishes Saurogobio dabryi and Saurogobio dumerili in China. The new species belongs to the Lytocestidae and is placed in Khawia because of its afossate scolex, vitelline follicles in the cortical parenchyma, uterus not looping anterior to the cirrus sac, gonopores separate but close together in distinct genital atrium, external seminal vesicle absent, and postovarian follicles present. The new species differs from other congeneric species by the shape of the body markedly tapering posteriorly from the end of the anterior third, the shape of the scolex that is very short and markedly wider than the neck, spatulate without incisions, but shallow superficial grooves, vitelline follicles and testes starting immediately posterior to the scolex, and an ovary with long, posterior arms bent medially, thus giving the shape of an inverted A.

Discovery of a male-biased mutant family and identification of a male-specific SCAR marker in gynogenetic gibel carp Carassius auratus gibelio

Gibel carp ( Carassius auratus gibelio) is a uniquely gynogenetic species with a minor ratio of males in natural habitats, but its male origin and sex determination mechanisms have been unknown. In this study, a male-biased mutant family was discovered from the gynogenetic gibel carp, and a male-specific SCAR marker was identified from the mutant family. Normal spermatogenesis was observed in the male testes by immuno. fluorescence histochemistry. Nearly identical AFLP profiles were observed between males and females, but a male-specific 86 bp AFLP fragment was screened by sex-pool bulked segregant analysis and individual screening. Based on the male-specific AFLP fragment, a total of 579 bp sequences were cloned by genome walking. Subsequently, a male-specific SCAR marker was designed, and the male-specific DNA fragment was confirmed to be steadily transmitted to the next generation and consistently detected only in males. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.

U19/Eaf2 Binds to and Stabilizes von Hippel-Lindau Protein

Studies have firmly established a key regulatory role for the tumor suppressor pVHL in the regulation of the vascular system and normal spermatogenesis. Here, we report that knockout of the newly identified tumor suppressor U19/Eaf2 also caused vascular system abnormalities and aspermatogenesis, suggesting a potential link between U19/Eaf2 and pVHL. Coimmunoprecipitation and in vitro binding assays showed an association between U19/Eaf2 and pVHL, whereas deletion mutagenesis revealed the requirement of the NH2 terminus of U19/Eaf2 and both the alpha and beta domains of pVHL for this binding. U19/Eaf2 stabilizes pVHL, as shown by protein stability and pulse-chase studies. Testes and mouse embryonic fibroblasts (MEF) derived from U19/Eaf2 knockout mice expressed reduced levels of pVHL, indicating that full in vivo expression of pVHL indeed requires U19/Eaf2. As expected, U19/Eaf2 knockout MEF cells exhibited an increased level and activity of hypoxia-inducible factor 1 alpha (HIF1 alpha), a protein typically regulated via a pVHL-mediated degradation pathway. Furthermore, angiogenesis in a Matrigel plug assay was significantly increased in U19/Eaf2 knockout mice. The above observations argue that U19/Eaf2 can modulate HIF1 alpha and angiogenesis, possibly via direct binding and stabilization of pVHL. [Cancer Res 2009;69(6):2599-606]

Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

Hir/Hira (histone regulation) genes were first identified in yeast as negative regulators of histone gene expression. It has been confirmed that HIRA is a conserved family of proteins present in various animals and plants. In this paper, the cDNAs of the Hira homolog named CagHira and CaHira were isolated from gynogenetic gibel carp (gyno-carp) and gonochoristic color crucian carp (gono-carp) respectively. The full-length CagHira is 3,860 bp in length with an open reading frame (ORF) of 3,033 bp that encodes 1,011 amino acids, while the full-length CaHira is 3,748 bp in length and also has an ORF of 3,033 bp. The deduced amino acid sequences of both Hira homologs contain seven WD domains and show high identity with other HIRA family members. RT-PCR analyses revealed strong expression of Hira in the ovaries, whereas no expression was detected in the testes of either of the fishes. Hira transcription was not detected in the liver of gyno-carp, but a high level of Hira mRNA was observed in gono-carp. The temporal expression pattern showed that the Hira mRNA is consistently expressed during all embryonic development stages in gyno-carp. However, the abundance of CaHira mRNA significantly decreased (P < 0.05) shortly after fertilization and then increased again and remained stable from gastrula till hatching. The varying spatiotemporal expression patterns of Hira genes in gyno-carp and gono-carp may be associated with the differing reproductive modes used by these two closely related fishes. Our results suggest that Hira may play a role not only in the decondensation of sperm nucleus and the formation of pronucleus during fertilization, but also in gastrulation and the subsequent development of embryos.

A new species of Allocreadium (Trematoda : Allocreadiidae) from freshwater fishes in the Danjiangkou Reservoir in China

A new species of Allocreadium, Allocreadium danjiangensis n. sp., is described from the intestine of several species of freshwater fish, including Abbottina rivularis (Basilewsky, 1855), Sarcocheilichthys nigripinnis nigripinns (Gunther, 1873), Gnathopogon argentatus (Sauvage et Dabry 1874), Opsariichthys uncirostris bidens (Gunther, 1873), and Erythroculter mongolicus mongolicus (Basilewsky, 1855) (Cyprinidae) from the Danjiangkou Reservoir in central China. The main morphological characters of the new species are as follows: vitelline follicles numerous, extending from the level of acetabulum to posterior extremity, distributed over both sides around the ceca; cirrus sac relatively large, developed, lying obliquely anterior to the acetabulum, extending from the level of the intestinal bifurcation to the central level of acetabulum, and overlapping left or right cecal; and ovary much smaller than testes, generally close to or even overlapping the anterior border of anterior testis. Observation by scanning electron microscopy shows only 2 kinds of tegumental formations, i.e., papillae and tubercles, instead of 3 types of tegumental formations, i.e., papillae, bosses, and minute sensor receptors observed on other species of the Allocreadiidae. The tegumental striations of the present species vary on the different parts of the body. In addition, a new structure, identified as the "groove" with a tonguelike tubercle, was observed on the inner wall of acetabulum.

EST-based identification of genes expressed in the hypothalamus of male orange-spotted grouper (Epinephelus coioides)

The orange-spotted grouper, Epinephelus coioides, is an important marine aquaculture fish, but its large-scale aquaculture has been hindered by the rarity of natural males because it is a protogynous hermaphroditic fish. Hypothalamus-pituitary-gonad is an important endocrine axis in regulating reproduction and sex differentiation. To reveal the molecular mechanism of hypothalamic physiological functions, we performed he studies on identification of genes expressed in the hypothalamus of male orange-spotted grouper using EST and RT-PCR strategy. A total of 1006 ESTs were sequenced, and 402 (39.96%) clones were identified as known genes and 604 (60.04%) as unknown genes. The 402 clones of known gene products represent transcripts of 18 1 genes. Moreover, the expression patterns of 26 unknown genes were analyzed in various tissues, such as liver, kidney, spleen, fat, heart, muscle, pituitary, hypothalamus, telencephalon, cerebellum, midbrain, medulla oblongata, ovary and testes. Five different categories of expression patterns were observed from them. Several unknown ESTs, such as DN551996, DN551998, DN552082, and DN552070, were detected to be hypothalamus-specific, brains-specific, or hypothalamus and gonad-specific genes. Interestingly, DN551996, not only exhibiting expression differences between ovary and testis, but also showing sex-dependent differences in hypothalamus of grouper, might play significant role in grouper reproduction or sex inversion. Further functional studies on these genes will provide more information on molecule regulation mechanism of sex inversion in groupers. (c) 2006 Elsevier B.V All fights reserved.

The Chinese rare minnow (Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full life-cycle test with diethylstilbestrol

Chinese rare minnow (Gobiocypris rarus), a freshwater teleost,. was exposed to diethylstilbestrol (DES) at 0.05, 0.5, 1 and 5 mug/L from fertilized eggs for up to mature period under flow-through condition. Several endpoints that related to development, reproductive fitness and transgenerational effects were evaluated. It was found that body length and body weight were significantly reduced and vitellogenin (Via) levels were significantly increased for fish exposed to DES. Histological examination showed that the sex ratios of F-0 fish skewed to female and about 2% of the fish exposed to 0.05 mug/L DES developed testes-ova. The reproductive success, as determined from data on egg production, was reduced in female fish exposed to 0.05, 0.5, 1 and 5 mug/L DES. The lowest-observed-effect concentrations (LOEC) for chances of sex ratios, reproductive success and histology alteration of F-0 are 0.05 mug/L. In the offspring, transgenerational effects on egg hatching rate. egg fertilization and Vtg levels of juvenile individuals were not observed. However. survival of F, generation fry significantly declined. The analysis of sex steroid levels revealed a significant decrease of testosterone (T) in the whole body homogenates (WBH) of male progeny and somewhat elevation of estradiol (E-T) in the WBH of female offspring. These findings indicate that exposure to DES causes a variety of developmental, reproductive and transgenerational effects. (C) 2004 Elsevier B.V. All rights reserved.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Observations on two species of phyllodistomes (Trematoda : Gorgoderidae) from fishes of the Hubei Province, central China, with an erection of Neophyllodistomum gen. n.

Two rare trematode species so far reported in the genus Phyllodistomum Braun, 1899, P. pawlovskii (Zmeev, 1936) and P. serrispatula Chin, 1963, were found in the urinary system the yellow catfish Pelteobagrus fulvidraco (Richardson) of Bao'an Lake (prevalence 8 %) and the swamp eel Monopterus albus (Zouiev) of Liangzi Lake (prevalence 6 9/6), respectively, from the Hubei Province, central China. In contrast to the original description, P. pawlovskii showed a considerable morphological variability particularly in the shape, size and topography of testes. A unique morphological feature of P. serrispatula is the presence of many conspicuous lateral outgrowths on the hindbody and, principally based on this character, a new genus Neophyl-lodistomum is erected (type species N. serrispatula (Chin, 1963) comb. n.)) to accommodate this species. Both trematode species are briefly redescribed. P. pawlovskii and N. serrispatula are reported for the first time from the Hubei Province and the former from the Yangtze River drainage system.

Induction of cytogenetic adaptive response in spermatogonia and spermatocytes by pre-exposure of mouse testis to low-dose C-12(6+) ions

To investigate the effects of pre-exposure of mouse testis to low-dose C-12(6+) ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation. the testes of outbred Kun-Ming strain mice were irradiated with 0.05 Gy of C-12(6+) ions as the pre-exposure dose, and then irradiated with 2 Gy as challenging dose at 4 h after per-exposure. Poly(ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4 h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8 h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2 Gy of C-12(6+) ions. However, pre-exposure of mouse testes to a low dose of C-12(6+) ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose C-12(6+) ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations. (C) 2008 Elsevier B.V. All rights reserved.