Avian vacuolar myelinopathy (AVM) is an often-lethal neurologic disease that affects waterbirds and their avian predators (i.e., bald eagles Haliaeetus leucocephalus) in the southern United States. Feeding trials and field surveys provided evidence that AVM is caused by a toxin-producing, undescribed cyanobacterium (UCB), which grows as an epiphyte on the leaves of submerged aquatic vegetation (SAV). Reservoirs with documented AVM epornitics support dense growth of nonnative SAV. Waterbirds ingest the toxin when feeding on aquatic plants with the epiphytic UCB, and secondary intoxication occurs when raptors consume these birds. Vegetation management has been proposed as a means to reduce waterbird exposure to the putative toxin. We fed aquatic vegetation with and without the UCB to triploid Grass Carp Ctenopharyngodon idella in laboratory and field trials. Only Grass Carp that ingested aquatic vegetation with the UCB developed lesions in the central nervous system. The lesions (viewed using light microscopy) appeared similar to those in birds diagnosed with AVM. Grass Carp that received aquatic vegetation without the UCB were unaffected. Grass Carp tissues from each treatment were fed to domestic chickens Gallus domesticus (an appropriate laboratory model for AVM) in a laboratory trial; the chickens displayed no neurologic signs, and histology revealed a lack of the diagnostic lesions in brain tissues. Results from our trials suggest that (1) triploid Grass Carp are susceptible to the AVM toxin, although no fish mortalities were documented; and (2) the toxin was not accumulated in Grass Carp tissues, and the risk to piscivorous avifauna is likely low. However, a longer exposure time and analysis of sublethal effects may be prudent to further evaluate the efficacy and risk of using triploid Grass Carp to manage aquatic vegetation in a system with frequent AVM outbreaks.

The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses (ICMs) and fetal neural stem (FNS) cells. In result, three rat chimeras were produced by day 5 (D5) Sprague-Dawley (SD) blastocysts injected with ICMs derived from day 6 (D6) and D5 Dark Agouti (DA) blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs are able to contribute to chimeras, but their potential decreases significantly after culture in vitro (P<0.05), and rFNS cells only have the potential to contribute to early fetal development.

The circulating and tissue-bound forms of follistatin (FST315 and FST288, respectively) modulate the actions of activins. FST knockout (KO/null) mice, lacking both isoforms, die perinatally with defects in lung, skin, and the musculoskeletal system. Using constructs of the human FST gene engineered to enable expression of each isoform under the control of natural regulatory elements, transgenic mouse lines were created and crossed with FST null mice to attempt to rescue the neonatal lethality. FST288 expression alone did not rescue the neonatal lethality, but mice expressing FST315 on the KO background survived to adulthood with normal lung and skin morphology and partial reversal of the musculoskeletal defects noted in FST KO mice. The FST315 rescue mice displayed a short period of neonatal growth retardation, impaired tail growth, and female infertility. The latter may be due to failure of corpus luteum formation, a decline in the ovarian follicular population, and an augmented uterine inflammatory response to mating. Failure of corpus luteum formation and impaired tail growth indicate abnormal vascularization and suggest that FST288 is required for the promotion of angiogenesis. The augmented uterine inflammatory response may result from the failure of FST315 to modulate the proinflammatory actions of activin A in the uterus or may result from the altered steroid milieu associated with the ovarian abnormalities. Although we cannot definitively conclude that the remaining defects are due to the absence of a particular isoform or due to variable expression of each, these models have demonstrated novel physiological processes that are influenced by FST.

Androgen signaling is critical for normal fetal development but is not thought to regulate events in early embryogenesis. Given the interest in factors controlling the differentiation of embryonic stem (ES) cells, we have explored the possibility that androgens may play a role. This study demonstrates expression of androgen receptor (AR) RNA and protein in four independent mouse ES (mES) cell lines, and shows that the AR is functional and can interact with transfected androgen response elements to promote green fluorescent protein expression. AR mRNA was detected throughout 10-d differentiation in embryoid bodies (EBs). Exposure of EBs to testosterone (T) or dihydrotestosterone, at doses of 1 and 0.1 mum, respectively, promoted formation of beating cardiomyocytes. Flow cytometric analyses demonstrated a significant increase in the number of alpha-actinin and tropomyosin (cardiac markers) positive cells after these treatments. Addition of flutamide (1 microM) to T-treated EBs inhibited the T-induced proliferation of cardiomyocytes, confirming that, in this instance, androgens act via the classical AR-mediated genomic pathway. We also report that mES cells express key steroidogenic enzymes, as detected by RT-PCR, and during 24-h incubations secrete T at concentrations of 1.38 +/- 0.22 nM, levels comparable to those secreted by cultured Leydig cells. These novel data demonstrate the capacity of androgens to stimulate increased differentiation of mouse ES cells to cardiomyocytes, and are in keeping with recent observations that AR-deficient mice exhibit cardiac impairment in adulthood.

It has been proposed that follistatin can modulate the actions of activins and/or other members of the transforming growth factor-beta superfamily of proteins on testicular function, since mice overexpressing follistatin showed spermatogenic disruption. However, since mice with targeted disruption of the follistatin gene die soon after birth, it is not feasible to determine the effect of the absence of follistatin on testicular function using this model. To further understand the role of follistatin on the development and maintenance of spermatogenesis, fetal testes, collected by Caesarean section at day 18 of gestation from follistatin null mice, were transplanted to the external ear of castrated recombination activating gene 1 immunocompromised male mice. The testicular grafts were then analysed 7-8 weeks after transplantation and showed that full spermatogenesis developed in both the testes of wild-type and follistatin null mice. This study indicates that, if follistatin is required to modulate spermatogenic development, it is not supplied by local testicular production but by circulating follistatin from the host mouse.

Embryonic stem (ES) cells are pluripotent cells derived from inner cell masses (ICMs) of blastocysts. The capacity of pluripotency in differentiation is assumed to contribute to embryonic development to form a chimeric individual when these cells are reintroduced into embryos. Chimeric mice can be routinely generated by aggregation of ES cells with morulae or injection into blastocysts, which are then implanted in pseudopregnant foster mothers. Furthermore, recent studies have demonstrated that bone marrow-derived stem cells and neural stem cells can integrate into the embryonic development in mouse (Geiger et al. 1998 Cell 93, 1055–1065; Clarke et al. 2000 Science 288, 1660–1663). We therefore tried to assess the ability of rat ICMs and neural stem cells to form chimeras by injecting these cells into rat blastocysts. Forty-two rat ICMs from Day 6 blastocysts of Dark Agouti (DA) inbred rat were injected into Day 5 blastocysts of Sprague-Dawleyd (SD) outbred rats; 14 pups were born following embryo transfer of these blastocysts injected into Hooded Wistar (HW) recipients. One male of the 14 pups was coat color-patched and displayed germline transmission. Following embryo transfer of 22 SD blastocysts injected by Day 5 DA ICMs, 7 pups were born and 2 of them were coat color-patched. Nine pups were obtained from 23 DA blastocysts injected by Day 5 SD ICMs; 4 of them were coat color-patched. The ICM cells were isolated and cultured for 6 days. No chimeras were generated by injection of the cultured ICM cells, as assessed by coat color patching. These results suggest that rat embryonic ICMs have potential to develop into chimeras, but the chimeric potential of ICMs was rapidly lost in our culture system. Investigation of potential chimeric development of rat fetal neural stem (rFNS) cells transfected with Lac Z was carried out. Staining was observed in tissues from 2 of 41 E14 fetuses. These results demonstrated that rFNS cells can integrate into the early embryonic environment although the ability of these cells to contribute to chimeric formation was marginal. No coat color chimerism was observed in any of the 88 pups generated from the LacZ-rFNS cell experiments.

Cloning of the fibroblast growth factor receptor (FGFR) adaptor Snt-2 cDNA and the identification of FGFR-1 protein in association with sperm tails, suggested that FGFR-1 signaling was involved in either sperm tail development or function. This hypothesis was tested by the creation of transgenic mice that specifically expressed a dominant-negative variant of FGFR-1 in male haploid germ cells. Mating of transgenic mice showed a significant reduction in pups per litter compared with wild-type littermates. Further analysis demonstrated that this subfertility was driven by a combination of reduced daily sperm output and a severely compromised ability of those sperm that were produced to undergo capacitation prior to fertilization. An analysis of key signal transduction proteins indicated that FGFR-1 is functional on wild-type sperm and probably signals via the phosphatidylinositol 3-kinase pathway. FGFR-1 activation also resulted in the downstream suppression of mitogen activated protein kinase signaling. These data demonstrate the FGFR-1 is required for quantitatively and qualitatively normal spermatogenesis and has a key role in the regulation of the global tyrosine phosphorylation events associated with sperm capacitation.

Expansion of "low complex" repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP-involved in important housekeeping processes-retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asparagines, and alanine. Interestingly, while poly-Q repeats are found in vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling.

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.

Rat fetal neural stem cells(rFNSC),removed from E14.5-16.5 rat fetal brain tissues, were cultured in serum-free medium DMEM/F12 contained basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF).The inf1uences of bFGF and EGF on the proliferation of rat fetal neural stem cells were screened by ~3[H] thymidine incorporation. The results from BrdU incorporation show that these cells have mitotic ability during culture, and above 90% of cells in culture are nestin positive.They can differentiate into neurons,oligodendrocytes and astrocytes.It is proved that the obtained cells are rat neural stem cells.

The role of the inhibins, activins and follistatins in testicular function are being more clearly defined following studies describing the cellular localisation of these proteins to the testis and the availability of specific assay systems enabling measurement of these proteins. Taken together with the results of targetted gene inactivation experiments, several concepts emerge. Inhibin B is predominantly produced by the Sertoli cell in many adult male mammals whereas there is a perinatal peak of inhibin A in the rat. In contrast, activin A has its highest concentrations in the immediate post-natal period during which it is involved in the developmental regulation of both germ cells and Sertoli cells being modulated by follistatin. Activin A levels are considerably lower in the adult testis but Sertoli cell production is stimulated by interleukin-1 and inhibited by FSH. Little is known about the production of activin B due to the absence of a suitable assay but the βB subunit mRNA is expressed in germ cells and Sertoli cells and is stage-dependent. This pattern of expression suggest that it may be involved in autocrine or paracrine actions within the seminiferous epithelium.

This study describes the testicular levels of inhibin/activin subunits by Northern analysis and in situ hybridization and serum and testicular levels of inhibins A and B and activin A by enzyme linked immunosorbent assays (ELISA) during postnatal development in the rat. We show that serum inhibin A levels are less than 4 pg/ml throughout postnatal life. Serum inhibin B levels peak at 572 +/- 119 pg/ml (mean +/- se) at d 40 post partum (pp) before falling to 182 +/- 35 pg/ml in mature males. Serum activin A decreases from 294 +/- 29 pg/ml at d 6 to 132 +/- 27 pg/ml at maturity. Within the testis, inhibin A levels fall from 0.330 +/- 0.108 ng/g at d 15 to less than 0.004 ng/g at maturity. Inhibin B levels peak at 43.9 +/- 4.2 ng/g at d 6 before falling to 1.6 +/- 0.13 ng/g at maturity. Testicular activin A levels fall from 18.6 +/- 2.2 ng/g at d 6 to 0.094 +/- 0.013 ng/g at maturity. Northern profiles of testicular inhibin/activin subunits correlate with immunoreactive levels demonstrated by ELISA. In situ hybridization suggests that beta(A) and beta(B) subunit expression is largely restricted to the seminiferous tubule, particularly Sertoli cells, spermatogonia, and primary spermatocytes. These data support the view that inhibin B is the major inhibin in the male rat and that levels relate to Sertoli cell number and activity. Furthermore, the demonstration of high local concentrations of activin A during the period of Sertoli cell proliferation and the onset of spermatogenesis support its proposed role because a modulator of testicular development and function.

The 3.0 kb 5' arm (long arm, LA) of rat HPRT gene knock-out vector was cut by Sal I from rat HPRT gene genomic bacterial artificial chromosome (BAC) , and the 1.7 kb 3' arm (short arm, SA) was proliferated by PCR. Neo', 5' arm, 3' arm were sequentially cloned into pBS vector's relative restriction enzyme sites. For acquirement of tk gene, the 5' arm -Neo' -3' arm fragment inserted into pKO vector to construct pKO-HPRT. The pKO-HPRT was linearized by Not I, extracted by ethidium bromide, butanol and phenol/chloroform, and dialyzed by 0.025 microlmol/L Millipore. At the same time, rat neural stem cells cultured from E14.5-16.5 rat fetal brain. Passage 2 rFNSCs was tranfected by linearized pKO-HPRT with Fugene-6t transfection reagents. After 80 microg/ml G418 and 0.2 micromol/L ganciclovir selection, the survived cells was cultured in suspension to form neural spheres. The spheres can be picked up under the microscopy, and proliferated in 96- ,48- and 24-well plates sequentially. When the cell number reached 4 x 10(3)/well, half cells was lysed by lysis buffer to extract DNA, the other half was kept on growing to freeze and extract RNA. The knock-out cell colonies first detected by PCR, then confirmed by Southern blot and RT-PCR. All the results show that we have knocked out HPRT gene in three rat fetal neural stem cell colonies from 32 colonies.

This study uses a high purity cell culture system to extend previous observations of factors controlling the end of the Sertoli cell proliferative phase. Thyroid hormone, retinoic acid, and testosterone were assessed for their ability to halt the proliferative phase and regulate the expression of markers associated with maturation of the Sertoli cell. We show that these hormones share similar suppressive effects on the rate of Sertoli cell division without any apparent additive effects. We demonstrate that these hormones induce the progressive accumulation of cell cycle inhibitors p27Kip1 and p21Cip1 in Sertoli cells, a likely regulatory mechanism controlling the suppression of proliferation. We used real-time RT-PCR to examine the effects of these factors on the expression of mRNA encoding the Id proteins, demonstrating an increase in Id2 and Id3 expression in Sertoli cells treated with thyroid hormone, retinoic acid, or testosterone. Finally, we examined the expression of a number of genes that have been implicated in the Sertoli cell differentiation process. Our results suggest that these hormones can induce aspects of Sertoli cell differentiation in vitro, providing a valuable in vitro model for studying Sertoli cell function.

The present study was performed to determine suitable methods for parthenogenetic activation and subsequent development of rat oocytes in vitro. In the first series of experiments, the ability of electrical pulses, strontium, ethanol and ionomycin to activate Sprague-Dawley (SD) rat oocytes was examined. The synergistic effect of strontium and cycloheximide or puromycin was also examined in the second series of experiments. In the third series of experiments, the development of F1 hybrid ( SD x Dark Agouti) parthenotes activated with different concentrations of strontium ( 10 - 0.08 mM) was compared with that of SD parthenotes. The effect of the timing of activation ( 10 min and 2, 4 and 6 h after cervical dislocation) was also assessed in a fourth series of experiments. The oocytes activated by strontium showed higher pronuclear formation and cleavage rates than those in the other groups ( P < 0.05). Higher blastocyst development was obtained from parthenotes activated by strontium and strontium - cycloheximide compared with the strontium - puromycin group ( P < 0.01). However, the total cell number of blastocysts from the strontium - cycloheximide activation group was higher than that of other groups ( P < 0.05). With strontium ( 2.5 - 10 mM) treatment, 40.9% of blastocysts were obtained from F1 hybrid oocytes, whereas 22.9% were obtained from SD ( P < 0.01). The oocytes activated 10 min or 2 h following cervical dislocation showed higher blastocyst development than those of the 4 and 6 h groups ( P < 0.01). These results suggest that strontium - cycloheximide produces the highest parthenogenetic activation rate in the rat and that oocytes must be activated by 2 h after cervical dislocation.