Aggression in male mice lacking functional estrogen receptor α.

Estrogen receptor alpha knockout (ERαKO) male mice fail to display sexual behavior. The authors hypothesized that ERαKOs require higher testosterone (T) concentrations than wild-type (WT) males to exhibit copulatory behavior. Increasing T stimulated sexual behavior and preference for females in WT males but failed to do so in ERαKOs. However, T did induce female-directed aggression in ERαKOs. In aggression tests, WT residents selectively attacked T-treated male intruders. ERαKO residents attacked female, T-treated male, and estrogen-treated male intruders equally. Increased access to olfactory cues prior to direct contact reduced overall aggression in ERαKO versus WT males but did not cause ERαKOs to differentially attack male and female opponents. Results suggest that ERα is essential for normal social behavior, perhaps via processing of chemoinvestigatory cues, which are required to discriminate males from females. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Regional distribution of 5alpha-reductase type 1 and type 2 mRNA along the human epididymis

BACKGROUND AND PURPOSE: In women, symptoms of coronary artery disease are delayed by 10 to 15 years in comparison with men, most likely because of the protective effect of ovarian hormones. This report compares the prevalence and degree of carotid atherosclerosis between 292 premenopausal women and 294 women at 5 to 8 years after menopause. METHODS: Scans were performed in the same laboratory over the same time period for both groups. Intima-media thickness (IMT) was averaged across the common, bulb, and internal carotids. The plaque index summarized degree of focal plaque based on the size and number of plaques throughout both carotid systems. RESULTS: Mean IMT was 0.69 mm for premenopausal women and 0.77 mm for postmenopausal women (P < 0.001). Prevalence of plaque was 25% among premenopausal women and 54% among postmenopausal women (P < 0.001). In both premenopausal and postmenopausal women, risk factors measured before menopause were associated with carotid atherosclerosis. Premenopausal risk factors independently associated with IMT were higher pulse pressure (P < 0.001), triglycerides (P = 0.002), body mass index (P < 0.001), and study group (a surrogate for both age and menopausal status; P < 0.001). Premenopausal risk factors independently associated with focal plaque were ever smoking (P = 0.002), higher pulse pressure (P = 0.028), higher LDL (P = 0.003), age at baseline (P = 0.050), and study group (P < 0.001). CONCLUSIONS: Subclinical carotid atherosclerosis can be observed in middle-aged women. Risk factors measured before menopause are clearly associated with subclinical disease measured both concurrently and at 5 to 8 years after menopause.     

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.     

Generation and reproductive phenotypes of mice lacking estrogen receptor beta

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERalpha and ERbeta. We previously generated and studied knockout mice lacking estrogen receptor alpha and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor beta (ERbeta -/-) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERbeta -/- mice lack normal ERbeta RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERbeta is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERbeta in bone and cardiovascular homeostasis.     

Identification of type 1 5alpha-reductase in myelin membranes of male and female rat brain

Following its release from cells during infection and inflammation, calreticulin (CRT) can act as an autoantigen in diseases such as SLE. Why CRT is a target of protective immunity and whether it may interfere with innate immunity once released from cells during inflammation is unclear. In the present study, we found that CRT was detected more frequently in SLE sera and in higher amounts than found in control sera. Approximately 40% of SLE sera tested contained autoantibodies against CRT as detected by ELISA and immunoblotting. CRT was found to be predominantly in the sera of SLE patients associated with immune complexes and C1q, and only bound to the surfaces of neutrophils in the presence of low levels of calcium and magnesium. In order to further investigate the C1q-CRT interaction, recombinant CRT and its discrete domains (N-, P-, and C-domains) were produced in Escherichia coli. CRT binds to globular head region of C1q primarily via its N- and P-domains. The N-domain was shown to be the most autoantigenic region of CRT, as the anti-CRT autoantibodies from most patients reacted against this region. CRT also altered C1q-mediated immune functions. The P-domain of CRT bound to C1q and reduced the binding of immune complexes in SLE sera to immobilized C1q. Full length CRT and its N- and P-domains were able to reduce the C1q-dependent binding of immune complexes to neutrophils and solid-phase bound C1q. We conclude that CRT, once released from leucocytes during inflammation, may not only induce an antigenic reaction, but also interfere with C1q-mediated inflammatory processes.     

Biotransformation of tirilazad in human: 3. tirilazad A-ring reduction by human liver microsomal 5alpha-reductase type 1 and type 2

Connexin 43, a member of the highly conserved connexin family of gap junction proteins, is expressed in the pig ovary. In other species, ovarian connexin 43 expression and phosphorylation are hormonally regulated. We characterized connexin 43 expression and phosphorylation in the ovaries of mature pigs during the estrous cycle and in prepubertal gilts during follicular development induced by eCG (750 IU)/hCG (500 IU; 72 h later). Ovarian connexin 43 protein expression and phosphorylation were examined by immunoblot analysis. Connexin 43 was localized to specific follicular cell types during development by immunofluorescence. While no change in total connexin 43 protein expression was seen during the cycle, connexin 43 phosphorylation was significantly higher (p < 0.05) during the late follicular stage of the cycle than during the early luteal and early to mid-follicular stages. In ovaries of eCG/hCG-primed prepubertal pigs, connexin 43 protein levels remained steady, while phosphorylation of the protein increased significantly at 72 h and 84 h after eCG treatment (p < 0.05), then declined to pretreatment levels by 96 h (24 h post-hCG administration). Immunoreactive connexin 43 was localized predominantly to granulosa cells of cyclic pigs and eCG/hCG-primed prepubertal gilts. Follicular connexin 43 was highest between 60 h and 84 h after eCG and declined after hCG administration. Connexin 43 was not detected in morphologically atretic follicles, stroma, or vascular tissue of the ovary. This is the first evidence that porcine ovarian connexin 43 phosphorylation is differentially regulated during follicular development. The results suggest that hormonally induced changes in connexin 43 phosphorylation may play a coordinating role in porcine follicular development.     

Nonisotopic single strand conformation analysis of the 5 alpha-reductase type 2 gene for the diagnosis of 5 alpha-reductase deficiency

5 alpha-Reductase deficiency is a rare autosomal recessive disorder of defective virilization in karyotypic males due to reduced conversion of testosterone to dihydrotestosterone. The gene encoding the affected 5 alpha-reductase type 2 enzyme has recently been cloned, and mutations within the coding region have been discovered as the cause of this disease. We address the possibility of a rapid nonradioactive molecular genetic screening technique for initial diagnosis and report different point mutations in this gene in eight unrelated patients with clinical features of 5 alpha-reductase deficiency. For molecular genetic analysis, DNA from peripheral blood leukocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific PCR amplification, nonradioactive single strand conformation analysis, and direct sequencing. In seven patients, homozygous point mutations were identified (Leu55-Gln, delta Met157, Gly196-Ser, Arg227-Gln, Ala228-Thr, and His231-Arg). One individual was a compound heterozygote carrier of two mutations (Ile112-Asn and Gln126-Arg). We conclude that molecular genetic characterization of point mutations in the 5 alpha-reductase type 2 gene may be used as an additional valuable procedure for the diagnosis of this disorder.     

Anti-GBM glomerulonephritis in mice lacking nitric oxide synthase type 2

To determine the relationship between quantitative Doppler parameters of portal, hepatic, and splanchnic circulation and hepatic venous pressure gradient (HVPG), variceal size, and Child-Pugh class in patients with alcoholic cirrhosis, we studied forty patients with proved alcoholic cirrhosis who underwent Doppler ultrasonography, hepatic vein catheterization, and esophagoscopy. The following Doppler parameters were recorded: time-averaged mean blood velocity, volume flow of the main portal vein flow, and resistance index (RI) of the hepatic and of the superior mesenteric artery. Doppler findings were compared with HVPG, presence and size of esophageal varices, and Child-Pugh class. There was a significant inverse correlation between portal velocity and HVPG (r = -.69), as well as between portal vein flow and HVPG (r = -.58). No correlation was found between RI in the hepatic artery or superior mesenteric artery and HVPG. No correlation was found between portal vein measurements and presence and size of varices. Severe liver failure was associated with lower portal velocity and flow. In patients with alcoholic cirrhosis, only portal vein blood velocity and flow, but neither hepatic nor mesenteric artery RI, are correlated to the severity of portal hypertension and to the severity of liver failure.     

Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication.     

Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor

Signaling by tumor necrosis factor type 1 receptor (TNFR-1) is required for the initiation of liver regeneration after partial hepatectomy. Using knockout mice that lack either TNFR-1 or TNFR-2, we determined whether signaling through TNF receptors is important for liver injury and hepatocyte proliferation induced by carbon tetrachloride (CCl4). Lack of TNFR-1 inhibited hepatocyte DNA synthesis after CCl4 injection. At 44 hours after the injection, replication of hepatocytes in TNFR-1 was 50% to 90% lower than in wild-type (WT) animals, depending on the dose injected. In WT animals, hepatocyte replication was essentially completed by 4 days after CCl4 injection, but replication at a low level persisted in TNFR-1 mice for at least 2 weeks. TNFR-1 knockout mice had little detectable NF-kappa B and STAT3 binding during the first 5 hours after CCl4, high plasma TNF, and reduced levels of plasma interleukin (IL)-6 and liver IL-6 mRNA. Injection of IL-6 30 minutes before CCl4 administration corrected the deficiency of hepatocyte replication at 44 hours and restored STAT3 binding to normal levels. In contrast, mice lacking TNFR-2 did not differ significantly from WT mice in NF-kappa B and STAT3 binding, IL-6 and TNF levels, or hepatocyte replication. Although AP-1 binding was induced in WT TNFR-1 and TNFR-2 knockout mice, binding in TNFR-2 knockouts was lower than in WT mice. C/EBP binding was much lower in TNFR-1 and TNFR-2 knockout mice than in WT mice. As assessed by morphological analysis and alanine aminotransferase levels, the acute injury caused by CCl4 appeared to be similar in the three groups of animals, but subsequent regeneration was impaired in mice lacking TNFR-1. We conclude that a TNFR-1 signaling pathway involving NF-kappa B, IL-6, and STAT3 is an important component of the hepatocyte mitogenic response induced by CCl4 injury in mouse liver.     

Characterization of type I 5 alpha-reductase activity in DU145 human prostatic adenocarcinoma cells

Each amino acid is represented by a vector of numerical measurements for the attributes of volume, area, hydrophilicity, polarity, hydrogen bonding, shape, and charge. Inter-residue distances are then calculated according to common metrics, and we introduce a new clustering objective function derived from information-theoretic considerations. The arguments of the function are the inter-object distances of the things to be clustered: in this case the amino acids. By means of approximating the solution of an integer programming problem, then, the residues are partitioned into clusters. The clusters obtained are compared with groups obtained in substitution/mutation studies and found to be similar. Thus, probably the strongest and most objective evidence to date is supplied for believing that physico-chemical properties account for the viability of substitutions and that the important similarities/differences are explained by a relatively small and simple set of properties.     

Molecular characterization of 5 alpha-reductase type 2 deficiency and fertility in a Swedish family

The molecular background of 5 alpha-reductase type 2 deficiency was investigated in a Swedish family with no known consanguinity and in which the affected males were fertile. The three male siblings were born with ambiguous external genitalia, and the diagnosis of 5 alpha-reductase deficiency was established at the ages of 16, 14, and 10 yr, respectively. All three siblings underwent surgery for hypospadias repair. At least two of the brothers are demonstrably fertile. Molecular analysis showed the three brothers to be compound heterozygotes, carrying two different mutations in exon 4 of the 5 alpha-reductase type 2 gene. The two mutations (G196S and H231R) have been described previously and reported to give rise to partially functioning enzymes, which may explain the milder phenotype and perhaps the fertility in the preset three patients.     

PNU 157706, a novel dual type I and II 5alpha-reductase inhibitor

PNU 157706 is a novel dual inhibitor of 5alpha-reductase (5alpha-R), the enzyme responsible for the conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT). Tested on a crude preparation of human or rat prostatic 5alpha-R, PNU 157706 caused enzyme inhibition with IC50 values of 20 and 34 nM, respectively, compared to the values of 32 and 58 nM shown by finasteride. Furthermore, PNU 157706 was highly potent in inhibiting human recombinant 5alpha-R type I and II isozymes, showing IC50 values of 3.9 and 1.8 nM and, therefore, it was several folds more potent than finasteride (IC50 values of 313 and 11.3 nM), particularly on the type I isozyme. PNU 157706 was shown to have no binding affinity for the rat prostate androgen receptor (RBA 0.009% that of DHT). In adult male rats, a single oral dose of 10 mg/kg of PNU 157706 caused a marked and longer lasting reduction of prostatic DHT than did finasteride (at 24 h inhibition by 89 and 47%, respectively). In prepubertal, T- or DHT-implanted castrated rats, PNU 157706, given orally for 7 days at the dose of 10 mg/kg/day, markedly reduced ventral prostate weight in T- but not in DHT-implanted animals, thus showing to be devoid of any anti-androgen activity. In adult rats treated orally for 28 days, PNU 157706 resulted markedly more potent (16-fold) than finasteride in reducing prostate weight, the ED50 values being 0.12 and 1.9 mg/kg/day, respectively. These results indicate that PNU 157706 is a promising, potent inhibitor of both type II and I human 5alpha-R with a very marked antiprostatic effect in the rat.     

Abnormal astrocyte development and neuronal death in mice lacking the epidermal growth factor receptor

Stimulation of the epidermal growth factor receptor (EGF-R) produces numerous effects on central nervous system (CNS) cells in vitro including neuronal survival and differentiation, astrocyte proliferation and the proliferation of multipotent progenitors. However, the in vivo role of EGF-R is less well understood. In the present study, we demonstrate that EGF-R null mice generated on a 129Sv/J Swiss Black background undergo focal but massive degeneration the olfactory bulb, piriform cortex, neocortex, and thalamus between postnatal days 5 and 8 which is due, at least in part, to apoptosis. Some of the neuronal populations that degenerate do not normally express EGF-R, indicating an indirect mechanism of neuronal death. There were also delays in GFAP expression within the glia limitans and within structures outside the germinal zones in early postnatal ages. At or just prior to the onset of the degeneration, however, there was an increase in GFAP expression in these areas. The brains of EGF-R (-/-) animals were smaller but cytoarchitecturally normal at birth and neuronal populations appeared to be intact, including striatal GABAergic and midbrain dopaminergic neurons which have previously been shown to express EGF-R. Multipotent progenitors and astrocytes derived from EGF-R (-/-) mice were capable of proliferating in response to FGF-2. These data demonstrate that EGF-R expression is critical for the maintenance of large portions of the postnatal mouse forebrain as well as the normal development of astrocytes.     

Four frameshift mutations in neurofibromatosis type 1 caused by small insertions

We have been using heteroduplex analysis to assay individual exons within the NF1 gene in an effort to identify disease causing constitutional mutations in neurofibromatosis type 1 patients. Here we report the identification and characterisation of four insertional NF1 frameshift mutations in an analysis of exons 28-39 in a set of 78 patients. These include three 1 base pair insertions and one 2 base pair insertion. Three of these mutations can be attributed to replication slippage errors, while the mechanism behind the fourth may be related to formation of secondary structure during replication. It may be of significance that a majority of the previously reported small insertions in NF1 also lie within exons 28-39.     

Increased apoB100 mRNA in inbred strains of mice by estrogen is caused by decreased RNA editing protein mRNA

Estrogen administration to rats diminishes all apoproteins and lipoproteins from plasma. In contrast, some inbred strains of mice raise their plasma apoB and LDL levels by more than 2-fold (Srivastava et al, 1993, Eur. J. Biochem. 216, 527-538). Further studies with 13 inbred strains of mice given 3 micrograms beta-estradiol/g body weight/day for 5 consecutive days suggest that some mouse strains increased their apoB and LDL levels and some did not. To examine the mechanism of influence of genetic factors on apoB regulation, two strains, C57L and C57BL, that increased their VLDL- and LDL-cholesterol, and 2 strains, BALB and C3H, that did not, were chosen. Estrogen increased plasma apoB levels selectively in the strains C57L and C57BL, termed as 'responders,' but did not change in BALB and C3H, termed as 'non-responders.' One of the mechanisms for increased plasma apoB levels could be through increased production of apoB-containing particles. This possibility was investigated. ApoB and REPR mRNA were quantified by RNase protection assay, and apoB-100 mRNA by apoB mRNA editing assay. Hepatic apoB mRNA increased by 30% in 'non-responders,' but decreased by 20% in the 'responders.' However, apoB-100 mRNA increased relative to apoB-48 mRNA in all the 4 strains by 50%. The mRNA for RNA editing protein (REPR) decreased in all strains, suggesting that apoB-100 mRNA increased as a result of decreased apoB mRNA editing activity. These results suggest that:(a) modulation of apoB mRNA by estrogen was strain-specific;(b) increased apoB100 mRNA in inbred strains of mice were caused by decreased apoB mRNA editing activity; and (c) the differences in the plasma apoB levels among 'responder' and 'nonresponder' strains of mice occur through mechanisms other than the apoB mRNA editing.     

Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1

Mice completely deficient for Dvl1, one of three mouse homologs of the Drosophila segment polarity gene Dishevelled, were created by gene targeting. Dvl1-deficient mice are viable, fertile, and structurally normal. Surprisingly, these mice exhibited reduced social interaction, including differences in whisker trimming, deficits in nest-building, less huddling contact during home cage sleeping, and subordinate responses in a social dominance test. Sensorimotor gating was abnormal, as measured by deficits in prepulse inhibition of acoustic and tactile startle. Thus, Dvl1 mutants may provide a model for aspects of several human psychiatric disorders. These results are consistent with an interpretation that common genetic mechanisms underlie abnormal social behavior and sensorimotor gating deficits and implicate Dvl1 in processes underlying complex behaviors.     

5 alpha-reductase isozymes in the central nervous system

The enzyme 5 alpha-reductase (5 alpha-R) activates several delta 4-3keto steroids to more potent derivatives which may also acquire new biological actions. Testosterone gives rise to the most potent natural androgen dihydrotestosterone (DHT), and progesterone to dihydroprogesterone (DHP), a precursor of the endogenous anxiolytic/anesthetic steroid tetrahydroprogesterone (THP). Two isoforms of 5 alpha-R, with a limited degree of homology, different biochemical properties and distinct tissue distribution have been cloned: 5 alpha-R type 1 and type 2. In androgen-dependent structures DHT is almost exclusively formed by 5 alpha-R type 2; 5 alpha-R type 1 is widely distributed in the body, with the highest levels in the liver, and may be involved in steroid catabolism. In the brain, the roles of the two isozymes are still largely unknown. This brief review will summarize recent experimental data from our laboratory which try to assign possible functional roles to the process of 5 alpha-reduction, and to the two 5 alpha-R isoforms in the CNS.