Neurogenesis in the neonatal rat hippocampus is regulated by sexually dimorphic epigenetic modifiers.

BACKGROUND: Neurogenesis in the hippocampus endures across the lifespan but is particularly prolific during the first postnatal week in the developing rodent brain. The majority of new born neurons are in the dentate gyrus (DG). The number of new neurons born during the first postnatal week in the DG of male rat pups is about double the number in females. In other systems, the rate of cell proliferation is controlled by epigenetic modifications in stem cells. We, therefore, explored the potential impact of DNA methylation and histone acetylation on cell genesis in the developing DG of male and female rats. METHODS: Cell genesis was assessed by quantification of BrdU + cells in the DG of neonatal rats following injections on multiple days. Methylation and acetylation were manipulated pharmacologically by injection of well vetted drugs. DNA methylation, histone acetylation and associated enzyme activity were measured using commercially available colorimetric assays. mRNA was quantified by PCR. Multiple group comparisons were made by one- or two-way ANOVA followed by post-hoc tests controlling for multiple comparisons. Two groups were compared by t test. RESULTS: We found higher levels of DNA methylation in male DG and treatment with the DNA methylating enzyme inhibitor zebularine reduced the methylation and correspondingly reduced cell genesis. The same treatment had no impact on either measure in females. By contrast, treatment with a histone deacetylase inhibitor, trichostatin-A, increased histone acetylation in the DG of both sexes but increased cell genesis only in females. Females had higher baseline histone deacetylase activity and greater inhibition in response to trichostatin-A treatment. The mRNA levels of the proproliferative gene brain-derived neurotrophic factor were greater in males and reduced by inhibiting both DNA methylation and histone deacetylation only in males. CONCLUSIONS: These data reveal a sexually dimorphic epigenetically based regulation of neurogenesis in the DG but the mechanisms establishing the distinct regulation involving DNA methylation in males and histone acetylation in females is unknown.

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks.

The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHbeta, LHbeta and GPalpha, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHbeta and GPalpha mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2>GnRH1>GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.

Relationship between expression of multiple drug resistance proteins and p53 tumor suppressor gene proteins in human brain astrocytes.

Multiple drug resistance occurs when cells fail to respond to chemotherapy. Although it has been established that the drug efflux protein P-glycoprotein protects the brain from xenobiotics, the mechanisms involved in the regulation of expression of multiple drug resistance genes and proteins are not fully understood. Re-entry into the cell cycle and integrity of the p53 signaling pathway have been proposed as triggers of multiple drug resistance expression in tumor cells. Whether this regulation occurs in non-tumor CNS tissue is not known. Since multiple drug resistance overexpression has been reported in glia and blood vessels from epileptic brain, we investigated the level of expression of multidrug resistance protein, multidrug resistance-associated proteins and lung resistance protein in endothelial cells and astrocytes isolated from epileptic patients or studied in situ in surgical tissue samples by double label immunocytochemistry. Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that multiple drug resistance, multidrug resistance protein, and lung resistance protein are expressed in these cells. Given that lung resistance proteins have been reported to be preferentially expressed by tumors, we investigated expression of tumor suppressor genes in epileptic cortices. The pro-apoptotic proteins p53 and p21 could not be detected in "epileptic" astrocytes, while endothelial cells from the same samples readily expressed these proteins, as did normal brain astroglia and normal endothelial cells. Other apoptotic markers were also absent in epileptic glia. Our results suggest a possible link between loss of p53 function and expression of multiple drug resistance in non-tumor CNS cells.

Cloning and functional expression of a thyrotropin receptor from the gonads of a vertebrate (bony fish): potential thyroid-independent role for thyrotropin in reproduction.

The thyroid stimulating hormone receptor (TSHR) mediates the pituitary control of the development, growth and function of the thyroid. The expression of the gene encoding this receptor is known only in the thyroid, lymphocytes, fibroblasts, retro-orbital tissue and fat cells. We have cloned a TSHR from the gonads of a non-mammalian vertebrate, a bony fish (striped bass, Morone saxatilis) in the course of our search for gonadotropin receptors (follicle stimulating hormone receptor, FSHR and luteinizing hormone receptor, LHR). RT-PCR analysis demonstrated that the striped bass TSHR (stbTSHR) transcripts were abundant in both the thyroid and gonads and detectable in skeletal muscle, heart and brain tissues. The stbTSHR cDNA encoded a 779-amino acid glycoprotein hormone receptor with much higher homology (57-59%) to the mammalian TSH receptors than the mammalian LH receptors (47-49%) and FSH receptors (47%), and salmon and catfish gonadotropin receptors (42-45%). There was a TSHR-specific insertion in the extracellular domain as seen in mammalian receptors. Moreover, PCR analysis of genomic DNA indicated the absence of the LHR-specific intron in the striped bass TSHR gene. Recombinant stbTSHR expressed in COS1 cells activated reporter genes (luciferase) driven by either a cAMP response element or the c-fos promoter in response to bovine TSH, stbLH or hCG, but not human FSH. In situ hybridization studies revealed the presence of stbTSHR transcripts in the gametes but not in the follicular cells. This pattern of expression is unique and suggests a direct, albeit unknown, role for TSH in gamete physiology.

Multiple GnRHs present in a teleost species are encoded by separate genes: analysis of the sbGnRH and cGnRH-II genes from the striped bass, Morone saxatilis.

GnRH is a neuropeptide which plays an essential role in the control of reproductive fitness for all vertebrates. Increasing evidence suggests that multiple forms of GnRH may exist in most vertebrate brains. Southern blot analysis of the three GnRHs known to be present in perciform fish, the seabream (sb)GnRH, the salmon(s) GnRH and the chicken (c) GnRH-II, demonstrates that each is present as a single gene copy in the genome of the striped bass, Morone saxatilis. In order to investigate the physiological consequences of multiple GnRHs in a single vertebrate species, we have isolated and characterized two of the GnRH genes, those for sbGnRH and cGnRH-II. Computer analysis of 3.5 kb of sequence upstream of the sbGnRH gene reveals a number of consensus DNA binding sites which implicate steroids, such as estrogen and glucocorticoids, and the steroidogenic transcription factor, SF-1, as being involved in the regulation of sbGnRH gene expression. Sequence analysis of the cGnRH-II gene reveals evidence of multiple promoters. Expression studies using (1) solution hybridization-RNAse protection mapping with several RNA probes directed at various regions of the proGnRH gene, (2) primer extension assays using two specific oligonucleotide primers, and (3) reverse transcription PCR with several oligonucleotide primers on cGnRH-II transcripts demonstrate that the cGnRH-II gene initiates transcription at numerous sites using a TATA-less promoter within the brains of sexually mature striped bass. This study is the first to characterize and compare the promoter structures of two GnRH genes present in a single vertebrate species.

Expression and inheritance of RSVLTR-rtGH1 complementary DNA in the transgenic common carp, Cyprinus carpio.

Transgenic common carp, Cyprinus carpio, possessing the long terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) fused to the rainbow trout (rt) growth hormone (GH1) complementary DNA (cDNA) were produced by microinjection. Initial studies showed that the transgenic common carp transmitted the foreign DNA to a significant fraction of their progeny in three of four crosses of transgenic males with control females. These progeny grew 20 to 40% faster than their nontransgenic full siblings. In this study, additional experiments were conducted to evaluate inheritance and expression of the foreign GH gene in transgenic common carp, and the growth performance of these transgenic fish. Four P1 (parental generation produced by microinjection) x nontransgenic controls, four P1 x P1, and one P1 x F1 matings of transgenic carp containing RSVLTR-rtGH1 cDNA were made. The percentages of transgenic progeny resulting from these matings were: 0, 32, 42, 100 (4 progeny only), 21, 21, 31, 30, and 23%, respectively. All crosses except 1 siblot (control x P1) exhibited progeny ratios below the expected 50 or 75% transgenic. These results indicate that most of these transgenic P1 had the foreign gene in their germ line but were mosaics, and at least one transgenic individual did not have the RSVLTR-rtGH1 cDNA in the gonadal tissue. Both P1 and F1 transgenic fish produce trout growth hormone mRNA and polypeptide as determined by reverse transcription polymerase chain reaction amplification, RNA dot-blot hybridization, and radio-immunobinding assay. Growth response by families of F1 transgenic fish to the addition of rtGH1 cDNA varied widely.(ABSTRACT TRUNCATED AT 250 WORDS)

Electroporation: a method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio).

Recombinant plasmids containing the Rous sarcoma virus long-terminal repeat (RSVLTR) promoter linked to either rainbow trout (Oncorhyncus mykiss) growth hormone 1 (rtGH1) or growth hormone 2 (rtGH2) cDNA were linearized and introduced into the fertilized eggs of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus), and common carp (Cyprinus carpio) by both electroporation and microinjection. The latter two species had these rainbow trout constructs (RSVLTR-rtGH1cDNA or RSVLTR-rtGH2) electroporated into both gametes (i.e., sperm and unfertilized eggs) prior to fertilization, into eggs shortly after fertilization, and at the first cell division stage. Survival was determined just after hatching and again between 3 and 5 months after hatching. Polymerase chain reactions and Southern blot analyses were used to detect those individuals carrying the introduced foreign genes 3 to 5 months after hatching, respectively. Individuals analyzed by both methods yielded identical results in a double-blind study. The electroporation results were compared with groups that were microinjected. Although survival was similar, electroporation tended to produce a greater number of transgenic individuals than the microinjection procedure, and many more eggs could be treated per unit time by electroporation than microinjection. Survival was better for common carp when electroporation was performed shortly after fertilization, whereas channel catfish fared better at the first cell division stage. Electroporation prior to and shortly after fertilization, and at the first cell stage appeared to generate a large fraction of transgenic fish. We cautiously conclude that electroporation is an efficient method for introducing foreign DNA into fish gametes and embryos and may be an ideal method for treating large numbers of gametes in a modest period.

Transfer, expression, and inheritance of salmonid growth hormone genes in channel catfish, Ictalurus punctatus, and effects on performance traits.

We examined expression and inheritance of salmonid growth hormone genes RSVLTR-rtGH1 cDNA and RSVLTR-csGH cDNA, transferred to channel catfish (Ictalurus punctatus) by microinjection. One to 9 copies of the foreign DNA were inserted in either head-to-tail tandem array at single insertion sites or single copies at multiple insertion sites. All P1 transgenic catfish evaluated produced salmonid growth hormone regardless of the construct. Five P1 x P1 matings were accomplished. The spawning rate and fertility of these P1 transgenics in artificial spawning conditions were comparable to those of normal channel catfish. In two of three years, 100% spawning and 100% hatch were obtained. Percent transgenic progeny observed in the five matings were 20, 52, 7, 47, and 0%, which was lower (P < 0.001, chi 2) than the 75% inheritance expected assuming the P1 brood stock had at least one copy of the foreign gene integrated and were not mosaics in the germ line. At least 7 of 10 P1 were mosaics, and a minimum of 2 of 10 P1 did not possess the salmonid growth hormone genes in their germ line. P1 transgenics grew at the same rate as their nontransgenic full siblings, which is not surprising because the P1 were mosaics. F1 transgenic progeny in two families possessing RSVLTR-csGH cDNA grew 26% faster, to 40 to 50 gm, than their nontransgenic full siblings when evaluated communally. One F1 progeny group produced by RSVLTR-rtGH1 cDNA x RSVLTR-csGH cDNA mating and one F1 progeny group (parents either RSVLTR-rtGH1 cDNA or RSVLTR-csGH cDNA) grew at the same rate as normal full siblings when grown communally to 25 gm and 60 mg, respectively. In families where F1 progeny grew faster than controls, the range in body weight and coefficient of variation for the transgenic full siblings were less than those for controls. In families where F1 progeny grew at the same rate as controls, range in body weight and coefficient of variation were similar for transgenic and normal individuals. The percent deformities observed in P1 transgenics (13.6%) was higher (P < 0.05) than in microinjected P1 nontransgenics (5.1%). Percent deformities in transgenics and control F1 channel catfish was not different (p > 0.05; 0.5 and 2.8%, respectively).