Male-Specific Differences in Proliferation,Neurogenesis, and Sensitivity to Oxidative Stress in Neural Progenitor Cells Derived from a Rat Model of ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1^G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1^G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1^G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1^G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1^G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1^G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.     

The effect of sex on the uptake of very low density lipoprotein triglyceride fatty acid from the plasma of the rat in vivo.

Radioactive very low density lipoprotein (VLDL) was prepared by perfusion in vitro of livers isolated from normal fed male and female rats with [1-¹⁴C] oleate or [9, 10-³H] oleate, respectively. These VLDL, whose properties differed due to sex, were mixed. Aliquots of the mixture were injected intravenously into fasted male and female rats and the decay of the radioactivity (¹⁴C and ³H) was measured. Disappearance of radioactivity from plasma triglyceride was more rapid in female animals. Plasma half-life of ¹⁴C and ³H was 42.5±3.7 and 49.7±4.4 minutes, respectively, when the VLDL mixture was injected into male rats. The corresponding values in female rats were 28.3±1.1 and 30.7±1.7, respectively. These data suggest strongly that the rate of utilization of VLDL triglyceride fatty acid is more rapid in the female than in the male, and that the properties of the VLDL particles are of less importance than innate sex differences in the recipient for the rate of clearance of triglyceride fatty acids.     

Sharing of an HLA-B27-restricted H-Y antigen between rat and mouse

The purpose of this work was twofold: 1 to learn whether rats transgenic for HLA-B27 and the human ₂-microglobulin gene HB2M can mount B27-restricted cytolytic T lymphocyte (CTL) responses to the male H-Y antigen, and 2 to learn whether such CTLs would recognize both rat and mouse H-Y in the context of HLA-B27. Female rats of the B27/HB2M transgenic line 21-4L were primed in vivo with cells from males of the same line. CTL effectors were generated from lymph node cells of these females following culture with irradiated antigen-presenting cells from either male 21-4L rats or male mice of the B27/HB2M transgenic 56-3 line. The CTLs showed male-specific, B27-specific lysis of both rat and mouse targets. Lysis of B27 targets was inhibitable by monoclonal antibodies specific for B27 or rat CD8. Specific lysis of male B27 rat and mouse targets was inhibitable equally by either rat or mouse male B27 cold targets, but not significantly by female or nontransgenic cold targets. The B27-restricted CTLs neither recognized nor were inhibited by B27⁺ or B27^- male or female human targets. These results demonstrate that CD8⁺, B27-restricted, anti-H-Y CTLs recognize and evolutionarily conserved H-Y peptide antigen in both rats and mice. In addition, they establish the transgenic rat as a model system for examining the T-cell response to antigen presented by class I HLA molecules. Correspondence to: J. D. Taurog.     

A sex-specific form of cytochrome P-450 catalyzing propoxycoumarin O-depropylation and its identity with testosterone 6 beta-hydroxylase in untreated rat livers: reconstitution of the activity with microsomal lipids

Characteristics of a typical male-dominant reaction, dealkylation of n-propoxycoumarin, in rat livers were studied in relation to microsomal testosterone 6 beta-hydroxylase. The depropylation was more than 10-fold higher in the liver of male than female adult rats, but the sex-related difference was eliminated by neonatal castration. Hypophysectomy of adult male rats, which decreased the rates of male-specific P-450-male-dependent reactions, increased the depropylation of propoxycoumarin, while the rate was decreased by either intermittent injection or continuous infusion of human growth hormone to hypophysectomized rats. With regard to age-related difference, microsomal depropylation was detectable at neonate and reached a maximal level at 14 to 20 d of age, but was abruptly diminished only in female rats at puberty. These changes are in good agreement with those of testosterone 6 beta-hydroxylation and the content of a male-specific P-450(6)beta-1/PB-1. In reconstituted systems using extracted microsomal lipids, P-450(6)beta-1/PB-1 and P-450-male catalyzed the depropylation of propoxycoumarin. However, the microsomal depropylation was inhibited by antibodies which recognize P-450(6)beta-1/PB-1, but not P-450-male. These results indicate that microsomal depropylation of propoxycoumarin is catalyzed mainly by a male-specific P-450(6)beta-1/PB-1 in livers of untreated rats.     

Renal excretion of perfluorooctanoic acid in male rats: Inhibitory effect of testosterone

There is a marked sex difference in the whole-body elimination of perfluorooctanoic acid (PFOA) in rats, with females excreting the perfluorinated acid much more rapidly (half life [t_1/2] < 1 day) than males (t_1/2=15 days). Our objective was to determine if androgens or estrogens are involved in causing this sex difference in PFOA elimination. Castration of males greatly increased the elimination of [1-¹⁴C]PFOA (9.4 μmiol/kg, i.p.) in urine, demonstrating that a factor produced by the testis was responsible for the slow elimination of PFOA in male rats. Castration plus 17β-estradiol had no further effect on PFOA elimination whereas castration plus testosterone replacement at the physiologic level reduced PFOA elimination to the same level as rats with intact testes. Thus, in male rats, testosterone exerts an inhibitory effect on renal excretion of PFOA. In female rats, neither ovariectomy nor ovariectomy plus testosterone affected the PFOA urinary elimination, demonstrating that the inhibitory effect of testosterone on PFOA renal excretion is a male-specific response. Probenecid decreased the high rate of PFOA renal excretion in castrated males but had no effect on male rats with intact testes. We conclude that testosterone is a key determinant of the sex difference in PFOA elimination in rats.     

Age-dependent exression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2α-, 2β-, 15α-, 16α-, and 16β-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7α-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible form) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6β-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Studies on the Sex-Specific Lethals of DROSOPHILA MELANOGASTER. V. Sex Transformation Caused by Interactions between a Female-Specific Lethal, Sxlf#1, and the Male-Specific Lethals mle(3)132, msl-227, and mle

Interactions between a female-specific lethal mutant, Sxl^f#1, and each of three male-specific lethal mutants, mle(3)132, msl-2²⁷ and mle, of Drosophila melanogaster were observed to produce morphological changes in various sexually dimorphic external characters. Chromosomal females heterozygous for Sxl^f#1 and homozygous for any one of the male-specific lethals (and to a lesser degree heterozygous for male-specific lethals) sometimes had sex combs, male-type tergites, male-type sternites, male-type anal plates or male-type external genitalia. Penetrance was not high and expression was often incomplete; single individuals never had all the sexually dimorphic structures transformed. When mothers were homozygous for male-specific lethals, higher proportions of female progeny were affected than when mothers were heterozygous, suggesting a maternal effect.     

One amino acid change in rat SMR1 polypeptide induces a 1 kDa difference in its apparent molecular mass determined by electrophoretic analysis

SMR1 is a male-specific, 19 kDa, in vitro translation product of Wistar rat submaxillary glands, which may be the precursor of a small hormone resembling the TRH. In Sprague-Dawley and Fischer rats, instead of SMR1, a male-specific 18 kDa polypeptide may be found. We have cloned the cDNA encoding the 18 kDa polypeptide. We show that the 19 and the 18 kDa polypeptides have the same sequence except for one amino and change.     

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bg ^j/bg ^j) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10^–5 M testosterone or 5-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.     

Regulation of leptin by steroid hormones in rat adipose tissue.

We investigated if steroid hormones regulate the secretion and the expression of leptin in female and male rat adipose tissue fragments in vitro. Dexamethasone time and dose-dependently increased the secretion and mRNA expression of leptin with a half-maximal stimulation of approximately 1 nM. A time-course revealed a maximal stimulatory effect of 17 beta-estradiol after 24 hours. In male adipose tissue 17 beta-estradiol increased leptin secretion (32% by 50 nM 17 beta-estradiol, P = 0.07 and 34% by 500 nM 17 beta-estradiol, P < 1780.05) after 24 hours. An additional effect of estrogen was seen in the dexamethasone (50 nM) stimulated cells (38% with 50 nM 17 beta-estradiol, P < 0.05 and 48% by 500 nM 17 beta-estradiol, P < 0.05). Basal secretion of leptin was equal in female and male adipose tissue, whereas the effects of 17 beta-estradiol (50 nM) and dexamethasone were significantly increased in female as compared with male adipose tissue. Progesterone, testosterone, dihydrotestosterone and dehydroepiandrostendione-sulfate neither affected leptin secretion in male nor female adipose tissue in vitro. Furthermore, to investigate the effect of estrogen female rats were ovariectomized (OVX) and the adipose tissue was incubated in vitro and compared with adipose tissue leptin secretion from sham operated rats (SHAM), and with ovariectomized rats treated with 17 beta-estradiol (EST). A decreased basal and dexamethasone-stimulated leptin secretion from OVX rats compared with SHAM rats was found (P < 0.005) whereas 17 beta-estradiol treatment of ovariectomized rats maintained a normal leptin secretion. However, the dexamethasone stimulation was equally increased above basal levels in SHAM, OVX and EST rats (3.7 +/- 1.2, 2.9 +/- 0.8, 4.2 +/- 1.4, NS, ANOVA) respectively.     

Sex differences in the uptake and retention of imipramine and desmethylimipramine in the rat lung

¹⁴C-Imipramine was administered to male and female Sprague-Dawley rats and animals were sacrificed at 4, 8, 12, and 20 hours later. At all time points, total radioactivity in female lung was several-fold higher than in males. In addition, female lungs had a higher concentration of desmethylimipramine (DMI) as compared to imipramine than did male rat lungs. This was reflected by a higher conversion of imipramine to DMI by hepatic and pulmonary microsomes from female rats. Finally male rats cleared both imipramine and DMI from their lungs at a slower rate than did female rats.     

Does neonatal androgen modulate uptake and retention of 3H-estradiol in gonadotrophs and lactotrophs?

Summary Castrated adult male and female and androgenized female rats (AS rats) were injected i.v. with ³H-estradiol (E₂). Nuclear uptake and retention of the ³H-steroid was examined in pituitary luteinizing hormone (LH) and prolactin (PRL) cells by the combined techniques of autoradiography and immunocytochemistry. About 80% of PRL cells were found to concentrate the radioactive steroid compound in all experimental groups, while 89%, 82% and 68% of LH cells were found to be labeled in AS rats, normal female and male rats, respectively. This suggests that there are subpopulations of LH or PRL cells that contain no or, if any, small numbers of E₂ receptor. Statistical analysis revealed that PRL cells take up more radioactivity than LH cells in male rats, while there is no significant difference between female and AS rats. Variations in E₂ uptake (coefficient of variation) was higher in LH cells than in PRL cells in male rats and in AS rats. In females, on the contrary, coefficient of variation was larger in PRL cells. Thus the characteristics of nuclear uptake and retention of estradiol in LH and PRL cells appear to be modulated in part by neonatal androgen since the pattern found in AS rats is different than that found in normal male and female rats.     

Sexual differentiation in Asparagus officinalis L.

Summary As a first approach in investigating the genetical bases of sexual dimorphism in the dioecious plant Asparagus offcinalis L. at the molecular level, we have determined DNA content per cell, DNA sequence complexity and mRNA activities in both developing and mature male and female flowers of Asparagus. 2C DNA content (around 3.9 pg) was independent of sex and rather low when compared to other Liliiflorae; sequence complexity, however, showed a high proportion of repeated sequences. Polyadenylated mRNA from male and female flowers at young and mature stages of development were assayed by in vitro translation in the presence of [³⁵S]methionine, and the synthesized proteins were analysed by two-dimensional gel electrophoresis. Results have shown that there are no appreciable differences in polypeptide patterns from male and female flowers at a young stage of development, while specific sequences of mRNA are produced only very late during the development, most likely linked to the appearance of mature pollen grains and mature megagametophy tes.     

Effects of Female Hormones (17β-Estradiol and Progesterone) on Nitric Oxide Production by Alveolar Macrophages in Rats

The effect of female sex hormones on nitric oxide (NO) production was studied in alveolar macrophages (AMs). Male rats were treated with endotoxin (LPS) intratracheally or saline as control. AMs were obtained by bronchoalveolar lavage 90 min later and were cultured in the presence or in the absence of LPS and 17β-estradiol or progesterone (10⁻⁹to 10⁻⁴M). NO production was assessed by measurement of nitrites in the medium. In some experiments, NO production by AMs was measured in intratracheally LPS-treated orchidectomized rats or in female control and ovariectomized rats. Both spontaneous and stimulated NO production were higher in AMs from female than from male rats, but without statistical significance. However, ovariectomy induced significant inhibition in spontaneous production of NO by AMs. In orchidectomized rats, the NO response by AMs to LPS stimulation relative to spontaneous NO production was significantly downregulated. Female sex hormones in physiological concentrations seem to be necessary for spontaneous NO production in female rats. Pharmacological doses of estradiol inhibitedin vitroLPS-stimulated NO production in AMs of both saline- and LPS-treated rats, and basal NO production only in LPS-treated male rats. Progesterone at 10⁻⁴M inhibited basal andin vitroLPS-stimulated NO generation by AMs of both saline- and LPS-treated male rats. In LPS-treated female ratsin vitroLPS-stimulated NO production was not affected by estradiol treatment. In ovariectomized LPS-treated female rats progesterone at 10⁻⁵M significantly inhibited NO production byin vitro-stimulated AMs. Thus female sex hormones may contribute to the gender-related differences in the immune response.     

Gender difference in the Oatp1-mediated tubular reabsorption of estradiol 17beta-D-glucuronide in rats

The gender difference in the urinary excretion of estradiol-17beta-glucuronide (E(2)-17betaG) was examined in rats. The urinary clearance of E(2)-17betaG was >250 times lower in male than in female rats. No such major gender difference was observed in its biliary excretion or metabolism in kidney homogenate. Both plasma protein binding and inulin clearance were comparable in male and female rats, suggesting that this gender difference cannot be explained by glomerular filtration. The urinary clearance with respect to the plasma unbound E(2)-17betaG in male rats was <1% of the glomerular filtration rate, indicating its potential reabsorption by the kidney, and this increased to a level comparable with that found in female rats when dibromosulfophthalein was coinfused. A marked increase in E(2)-17betaG urinary excretion was also observed in male rats that had undergone orchidectomy. Testosterone injections given to female rats reduced the urinary excretion to a level comparable with that of control male rats. The concomitant change in the expression of the gene product for organic anion-transporting polypeptide Oatp1, of which E(2)-17betaG is a typical substrate, was found in the kidney membrane fractions after these treatments. These results suggest that urinary E(2)-17betaG excretion is subject to hormonal regulation and that the large gender difference can be explained by regulation in Oatp1-mediated reabsorption.     

Analysis of transposon insertion mutants highlights the diversity of mechanisms underlying male progamic development in Arabidopsis

To identify genes with essential roles in male gametophytic development, including postpollination (progamic) events, we have undertaken a genetic screen based on segregation ratio distortion of a transposon-borne kanamycin-resistance marker. In a population of 3359 Arabidopsis Ds transposon insertion lines, we identified 20 mutants with stably reduced segregation ratios arising from reduced gametophytic transmission. All 20 mutants showed strict cosegregation of Ds and the reduced gametophytic transmission phenotype. Among these, 10 mutants affected both male and female transmission and 10 mutants showed male-specific transmission defects. Four male and female (ungud) mutants and 1 male-specific mutant showed cellular defects in microspores and/or in developing pollen. The 6 remaining ungud mutants and 9 male-specific (seth) mutants affected pollen functions during progamic development. In vitro and in vivo analyses are reported for 5 seth mutants. seth6 completely blocked pollen germination, while seth7 strongly reduced pollen germination efficiency and tube growth. In contrast, seth8, seth9, or seth10 pollen showed reduced competitive ability that was linked to slower rates of pollen tube growth. Gene sequences disrupted in seth insertions suggest essential functions for putative SETH proteins in diverse processes including protein anchoring, cell wall biosynthesis, signaling, and metabolism.     

Sexual dimorphism in obesity-related genes in the epicardial fat during aging

Aging increases the risk of cardiovascular disease and metabolic syndrome. Alterations in epicardial fat play an important pathophysiological role in coronary artery disease and hypertension. We investigated the impact of normal aging on obesity-related genes in epicardial fat. Sex-specific changes in obesity-related genes with aging in epicardial fat (EF) were determined in young (6 months) and old (30/36 months) female and male, Fischer 344 × Brown Norway hybrid (FBN) rats, using a rat obesity RT² PCR Array. Circulating sex hormone levels, body and heart weights were determined. Statistical significance was determined using two-tailed Student’s t test and Pearson’s correlation. Our results revealed sex-specific differences in obesity-related genes with aging. Dramatic changes in the expression profile of obesity-related genes in EF with aging in female, but not in male, FBN rats were observed. The older (30 months) female rats had more significant variations in the abundance of obesity-related genes in the EF compared to that seen in younger female rats or both age groups in male rats. A correlation of changes in obesity-related genes in EF to heart weights was observed in female rats, but not in male rats with aging. No correlation was observed to circulating sex hormone levels. Our findings indicate a dysfunctional EF in female rats with aging compared to male rats. These findings, with further functional validation, might help explain the sex differences in cardiovascular risk and mortality associated with aging observed in humans.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.