Co-segregation of intermale aggression with the pseudoautosomal region of the Y chromosome in mice.

The sexual dimorphism of aggression has led to a search for its Y chromosomal correlates. We have previously confirmed that initiation of attack behavior against a conspecific male is Y-dependent in two strains of laboratory mice (NZB and CBA/H). We provide evidence that the non-pseudoautosomal region of the Y is not involved and that only the pseudoautosomal region of the Y is correlated with initiation of attack behavior. The autosomal correlates also contribute to this behavior in an additive or interactive manner with the pseudoautosomal correlates.

Genetic architecture of testis and seminal vesicle weights in mice.

Comparisons across 13 inbred strains of laboratory mice for reproductive organ (paired seminal vesicles and paired testes) weights indicated a very marked contrast between the C57BL/6By and NZB/BINJ mice. Subsequently these strains were selected to perform a quantitative genetic analysis and full genome scan for seminal vesicle and testis weights. An F(2) population was generated. The quantitative genetic analyses indicated that each was linked to several genes. Sixty-six short sequences for length polymorphism were used as markers in the wide genome scan strategy. For weight of paired testes, heritability was 82.3% of the total variance and five QTL contributed to 72.8% of the total variance. Three reached a highly significant threshold (>4.5) and were mapped on chromosome X (LOD score 9.11), chromosome 4 (LOD score 5.96), chromosome 10 (LOD score 5.81); two QTL were suggested: chromosome 13 (LOD score 3.10) and chromosome 18 (LOD score 2.80). Heritability for weight of seminal vesicles was 50.7%. One QTL was mapped on chromosome 4 (LOD score 9.21) and contributed to 24.2% of the total variance. The distance of this QTL to the centromere encompassed the distance of the QTL linked with testicular weight on chromosome 4, suggesting common genetic mechanisms as expected from correlations in the F(2). Both testis and seminal vesicle weights were associated with a reduction in the NZB/BINJ when this strain carried the Y(NPAR) from CBA/H whereas the Y(NPAR) from NZB/BINJ in the CBA/H strain did not modify reproductive organ weights, indicating that the Y(NPAR) interacts with the non-Y(NPAR) genes. The effects generated by this chromosomal region were significant but small in size.

Identification of the primary translation product of the sex steroid-binding protein from monkey liver mRNA in a cell-free system.

The synthesis of monkey (Macaca fascicularis) Sex steroid-Binding Protein (mSBP) in a wheat germ cell-free system in response to liver RNA was demonstrated by use of a specific antiserum raised against purified native human SBP. Antibodies precipitate a single translation product behaving as a 42 kDa protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blots of monkey sera subjected to SDS-PAGE and immunorevelation show that the native mSBP migrates as 2 molecular species (50 and 53 kDa) present in the approximate ratio of 1:10, respectively. The difference in apparent molecular weights of the primary translation product and the reduced mature mSBP may represent glycosylation that occurs post translationally. We describe for the first time the biosynthesis of mSBP at the molecular level and suggest that both components of mSBP derive from a common differentially processed precursor. Its mRNA is poorly represented, since the neosynthesized mSBP represents about 0.005% of the total proteins encoded by liver mRNA.

Effect of testosterone on regulation of the level of sex steroid-binding protein mRNA in monkey (Macaca fascicularis) liver.

Two specific oligonucleotide probes complementary to different regions of human sex steroid-binding protein (SBP) cDNA were used to study the levels of hepatic monkey SBP mRNA during different hormonal states. In females the SBP mRNA level was higher than in males and paralleled the serum SBP level. After castration, the SBP concentration increased in the serum but was reduced after testosterone treatment. In contrast, the hepatic SBP mRNA level decreased after castration and was restored by testosterone treatment. These results suggest a high homology of the nucleotide sequence between human and monkey SBP mRNAs. The changes in liver SBP mRNA levels may explain the sex difference in plasma SBP concentrations, but mechanisms other than the regulation of transcription may regulate the plasma concentration in monkeys.

Internalization of human sex steroid-binding protein in the monkey epididymis.

Human sex steroid-binding protein (hSBP) has been purified from late-pregnancy serum and labelled either by iodination (125I) or by photoaffinity with [3H]delta 6-testosterone. Using a micromanipulator, each labelled protein was separately injected into the lumen of epididymal tubules isolated from the head epididymis of the cynomologus monkey (Macaca fascicularis). Tubules were sampled from 3 to 90 min after the injection and processed for electron microscope autoradiography. Localization of the label occurred over the epididymal epithelium whichever tracer was used. The labelling was not randomly distributed over the different cell types constituting the epithelium, since only the 'principal cells' exhibited a silver grain count significantly greater than the background count. In these cells, labelled protein was found over endocytic organelles (coated structures, endosomes, multivesicular bodies and the trans Golgi network) and nuclei (including the nuclear envelope). Quantitative analysis demonstrated the same pattern of cellular and subcellular distribution for each tracer. Pretreatment with excess unlabelled protein significantly reduced the uptake of radioactivity by the principal cells, demonstrating the specificity of this phenomenon. This is the first study to show direct histological evidence for the internalization of hSBP in the primate epididymis, consistent with earlier immunohistochemical or biochemical localization of this protein. It is concluded that head epididymal cells are able to take up labelled hSBP across their apical membrane. The mechanism of internalization seems to involve endocytosis by the principal cells and leads to labelling of the nuclear compartment. This is strikingly similar to the pattern of uptake of rat androgen-binding protein (rABP) by rat epididymal cells previously demonstrated by our group. To what extent the chemical and structural homology between hSBP and rABP can be held responsible for the common cytophysiological behaviour of these sex steroid-binding proteins remains to be determined.

Effect of the Tfm mutation on handedness in mice.

The hyposthesis has been proposed that testosterone is involved in the determination of handedness in man: a high sensitivity to testosterone being associated with left handedness. Handedness in mice is tested according to Collins' paradigm: most mice present either a right or a left paw preference but others are ambilateral. The hypothesis that there is an association between a low neonatal imprinting by testosterone and a strong handedness (right or left) is tested here using Tfm male mice which are testosterone insensitive. Our results confirmed the hypothesis, since Tfm males were as well lateralized as their female siblings and significantly more strongly lateralized than their male siblings not carrying the mutation.

Hormone-associated variation of the glycan microheterogeneity pattern of human sex steroid-binding protein (hSBP).

hSBP is a steroid-binding protein (human) whose serum concentration is increased by estrogens and decreased by androgens. This regulation is independent of a direct effect on the hSBP gene transcription. The purpose of this work was to study the glycan microheterogeneous composition of the mature protein under physiological estrogen stimulation, by means of crossed affinoimmunoelectrophoresis using concanavalin-A. In men hSBP always divided into 2 fractions, both retarded. In women hSBP showed two other components, still more retarded. An explanation for these differences is given and the role of the glycan moiety of hSBP is discussed.

Murine steroid sulfatase (mSTS): purification, characterization and measurement by ELISA.

The murine steroid sulfatase (mSTS) is a microsomal enzyme, important in steroid metabolism. In the mouse, the gene encoding mSTS is pseudoautosomal and thus escapes X-inactivation. We have purified steroid sulfatase approximately 30-fold from mouse liver microsomes and its properties have been investigated. The major steps in the purification procedure included solubilization with Triton X-100, gel filtration chromatography, DEAE-Sephadex chromatography and HPLC gel filtration chromatography. The purified sulfatase showed a relative molecular weight of 128 kDa on HPLC gel filtration, whereas the enzyme migrated as two bands of 60 and 68 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.2 by column chromatofocusing. Polyclonal antibodies to the purified protein were prepared. An Enzyme Linked Immunosorbent Assay (ELISA) was developed using purified monospecific anti-mSTS antibodies labelled with peroxidase. The standard criteria of precision and reproducibility were satisfied. The assay was applicable to routine determination of mSTS samples in research laboratories. Differences in mSTS liver concentrations were used to identify putative alleles for the mSTS gene (Sts). Results in ELISA confirmed the polymorphism previously demonstrated for an enzymatic mSTS activity assay in two inbred mouse strains.

Endocytosis of human sex steroid-binding protein in monkey germ cells.

We investigate in this study the hypothesis of human sex steroid-binding protein hSBP internalization into germ cells in a primate model. Human SBP was purified from late-pregnancy serum and labeled either with colloidal gold particles (18 nm) or with [3H]delta 6-testosterone by photoaffinity treatment. The germ cells were isolated from sexually mature monkey testis or caput epididymis (Macaca fascicularis) by mechanical means and cell suspensions (4 x 10(6) per 100 microliters culture medium) were incubated in presence of hSBP-gold complex (60 ng/100 microliters) or hSBP-[3H]delta 6-testosterone complex (66 ng/100 microliters, 20,000 cpm) for 2, 5, 15, 45, and 60 min. The samples were processed for electron microscopy followed by autoradiographic treatment for the radiolabeled samples. Localization of the label occurred over the whole germ cell lineage whichever tracer was used. Spermatogonia, spermatocytes, spermatids, testicular and epididymal spermatozoa exhibited specific binding sites over the plasma membrane associated with clathrin-like coated pits and vesicles. At 34 degrees C, intracellular localization of the labeled ligand was found within coated vesicles, in early and late endosomes. In addition, in early spermatogenic cells, labeled ligand was detected in the nuclei and/or associated with the nuclear envelope whereas in late spermatids and residual bodies, the labeling was accumulated in multivesicular, prelysosomal structures. Quantitative analysis of the "labeled cells/total cells" ratio exhibited a negative correlation to the maturation steps, epididymal spermatozoa being the least labeled. The cellular distribution is similar with one or the other protein in the same spermatogenic cells. Unlabeled hSBP treatment prior to labeled hSBP reduced significantly the internalization. Lowering the temperature to 4 degrees C prevented endocytosis and enhanced membrane binding. EDTA pretreatment strongly decreased hSBP internalization and modified the early endocytic steps, namely, the pinching off of the coated vesicles. It is concluded that monkey germ cells are able to internalize the human sex steroid-binding protein through specific endocytic organelles. This endocytosis leads to the labeling of the nuclei in the early spermatogenic cells and of the multivesicular bodies in the late germ cells. This strongly suggests that steroid-binding proteins may be required for spermatogenesis in acting at the germ cell lineage level either by themselves or by serving as steroid transmembrane carriers.

Cardiovascular risk factors and combined estrogen-progestin replacement therapy: a placebo-controlled study with nomegestrol acetate and estradiol.

OBJECTIVE: To assess the effects of oral E2 replacement therapy combined with nomegestrol acetate, a 19-norprogesterone derivative, on cardiovascular risk factors. DESIGN: A double-blind randomized prospective study comparing the effect of a placebo and two oral E2-nomegestrol acetate combinations (1 mg-2.5 mg and 1.5 mg-3.75 mg) over a three-cycle trial. SETTING: Department of Internal Medicine and Nutrition, Hotel-Dieu, Paris, France. PATIENTS: Fifty-seven nonhysterectomized women with natural menopause. MAIN OUTCOME MEASURES: Blood pressure, renin substrate, glucose, total cholesterol, high-density and low-density lipoprotein cholesterol, triglycerides, apoproteins A1 and B, lipoprotein(a), antithrombin III, fibrinogen, plasminogen, prothrombin fragment 1 + 2, protein C, and total and free protein S. RESULTS: Both treatments significantly reduced menopausal complaints, total cholesterol, low-density lipoprotein cholesterol and lipoprotein(a). Treatment with the 1.5 mg-3.75 mg combination resulted in a significant increase in apolipoprotein A1. No significant change were observed in other parameters. CONCLUSIONS: Sequentially combined with oral E2 in hormone replacement therapy, nomegestrol acetate had favorable effects on plasma lipids and lipoproteins. This nonandrogenic progestin decreased lipoprotein(a) levels as observed previously with medroxyprogesterone acetate combined with conjugated equine estrogens.

Murine steroid sulfatase gene expression in the brain during postnatal development and adulthood.

The microsomal enzyme steroid sulfatase (STS, E.C.3.1.6.2) plays a central function in the neurosteroid mode of action, since it is responsible for the switch between the sulfated and the free forms of steroids which have opposite effects. In this study, using an enzyme linked immunosorbent assay (ELISA) for the STS, we have investigated the brain expression of STS in mice during development. We confirm that STS is present in the brain as previously shown by the measurement of the enzymatic activity. At birth, the STS level is clearly higher than in adults. We observed differences between physiological stages in females brain. The STS level is the same in pregnant and non-pregnant females, whereas STS concentration dramatically increased after delivery and during lactation.

Linkage between brain serotonin concentration and the sex-specific part of the Y-chromosome in mice.

The implication of the sex-specific part of the Y-chromosome (YS-SP) on brain serotonin (5-HT) level was investigated using congenic strains for this chromosomal region. The 5-HT level, which was higher in the NZB than in the CBA/H strain of mice, was depleted by the transfer of the YS-SP from NZB on CBA/H whereas the transfer of the YS-SP from CBA/H on NZB had no effect. The variations of 5-HT levels were not correlated with plasma testosterone concentration which is also dependent of the YS-SP.

[Semi-automated determination of urinary 17-oxosteroids in a continuous flow system (author's transl)]. / Dosage semi-automatique en flux continu des 17-cetosteroides urinaires

A semi-automated determination of 17-oxosteroids in urinary extracts is described using a modified Zimmerman reaction in aqueous phase according to Epstein. In order to eliminate the interfering chromogens a double manifold and a double-beam colorimeter are used in continuous flow. The spectra and the chromogenecity of seven different 17-oxosteroids are presented. The specificity and the reproducibility of this technique are good and the comparison with gas-liquid chromatography and manual Zimmerman reaction shows a good correlation.

Immunochemical characterization of corticosteroid-binding globulin in human bronchoalveolar fluid.

The corticosteroid-binding globulin (CBG) is a plasma protein which is present both in liver, where it is mainly synthesized, and in cells of different target tissues for glucocorticoids. Using monospecific antibodies raised against human CGB, we could demonstrate the antigenic identity of the protein in human bronchoalveolar fluid. We found that the bronchoalveolar fluid/serum concentration ratio of CBG was similar to that of albumin. Since albumin is not synthesized in pulmonary cells, it was concluded that, in healthy human, CBG enters bronchoalveolar fluid by diffusion through alveolar cells. It is suggested that the expression of the CBG gene in pulmonary cells could occur during the pathological state.

Immunochemical characterization and quantification of the sex steroid-binding protein (SBP) in human amniotic fluid.

The sex steroid-binding protein (SBP) is a plasma protein whose concentration in the maternal circulation increases during pregnancy. Using monospecific antibodies raised against human SBP, we could demonstrate the antigenic identity of the protein in human amniotic fluid. In this fluid, we found that the SBP concentration was correlated with the total protein concentration throughout gestation. The concentration gradient of SBP between maternal serum and amniotic fluid was compared to that of other serum proteins, in relation to their relative molecular mass, and it was concluded that SBP enters amniotic fluid in a non-specific manner similar to that of other serum proteins. It is suggested that SBP could act to sequester the sex steroid hormones in amniotic fluid.

Immunochemical characterization and quantitation of human sex steroid binding plasma protein.

The interest in the measurement of human sex steroid binding plasma protein (h-SBP) is now increasing since it allows the estimation of the free fraction of circulating hormones in plasma. Up to this date, this protein could only be determined by measuring the total binding capacity of serum for dihydrotestosterone (DHT). The purpose of the present work was to purify the protein, to prepare a rabbit monospecific antiserum and to develop an immunoelectrophoretic assay of h-SBP. The immunological assay is specific, accurate and sensitive. A good correlation with the radioligand assay was found. The h-SBP levels obtained by immunoelectrophoretic assay of different serum samples were 5.3 +/- 1.4 (SEM) mg/L in normal men and 13.4 +/- 2.6 (SEM) mg/L in normal women.

Opponent strain effect on eliciting attacks in NZB mice: physiological correlates.

In agonistic encounters between male mice, the characteristics of the opponent may influence the attacking behavior of its partner. The present study shows that the opponent's ability to elicit attacking behavior in NZB males is strain dependent. BALB/c opponents elicit attacks more frequently, earlier and more intensively than C57BL/6 males. Plasma testosterone concentration was found to be higher in BALB/c than in C57BL/6 intact males. The weight of seminal vesicles in castrated males of both strains increased with injections of either 10- or 250-micrograms testosterone propionate (TP). This response was greater in BALB/c with the higher TP dose. The submandibular glands reacted to TP only in castrated BALB/c males with the higher dose. Furthermore, BALB/c males produced more marking secretions than C57BL/6 males. These results suggest that for these two strains, a higher testosterone sensitivity and a greater production of secretions are associated with a higher probability of opponents to elicit attacks. Genetic hypotheses on the underlying mechanisms are discussed.

Effects of nomegestrol acetate (5 mg/d) on hormonal, metabolic and hemostatic parameters in premenopausal women.

The effects of nomegestrol acetate on circulating hormone levels, metabolic and hemostatic parameters and blood pressure were studied in 36 premenopausal women. The progestogen was administered from day 7 to 25 of the cycle during six cycles at a dosage (5 mg/d) known to inhibit ovulation. Analysis were performed before and in the third and sixth cycles. Estradiol and progesterone levels decreased significantly (p less than 0.001) during treatment. Body weight, fasting blood glucose and insulin, total HDL and LDL cholesterol, apolipoprotein B, fibrinogen and plasminogen did not change significantly. Triglycerides in the third cycle (p less than 0.05) and apolipoprotein A1 levels (p less than 0.01) in both periods of sampling decreased significantly. There was a significant increase in antithrombin III (p less than 0.01). These results indicate that nomegestrol acetate has no deleterious effect on blood glucose and lipids. The decrease in apolipoprotein A1 and increase in antithrombin III may be related either to the decrease in estradiol levels induced by the treatment or to the effect of the progestogen itself.

Hemostatic and metabolic effects of lowering the ethinyl-estradiol dose from 30 mcg to 20 mcg in oral contraceptives containing desogestrel.

The metabolic and hemostatic effects of two oral contraceptives containing 150 mcg desogestrel and 20 mcg ethinyl-estradiol (EE) (MERCILON) or 30 mcg EE (MARVELON) were compared in order to examine the effect of reducing the EE dose in contraceptive pills. Forty-nine women participated in this randomized study during 6 cycles. In both groups, there was a significant increase in triglycerides, HDL-cholesterol and apoprotein A1; the same increase was observed for SBP and CBG. Slight and transient variations of fasting blood glucose levels were seen in the 30 mcg EE group and in the two groups for fasting insulin levels. The increase in renin substrate was significantly higher with the 30 mcg EE than with the 20 mcg EE pill. In both groups, plasminogen increased significantly, but antithrombin III, total and free protein S and fibrinogen decreased significantly only in women taking the 30 mcg EE pill, whereas there was no significant change in the 20 mcg EE group. Reducing the dose of EE in oral contraceptives from 30 mcg to 20 mcg minimizes their impact on renin substrate and hemostatic parameters.

Ultradian, circadian and seasonal variations of plasma progesterone and LH concentrations during the luteal phase.

The circadian variations in plasma progesterone (P) and LH concentrations were investigated in six women, aged 23-40 years. All were studied in the mid-luteal phase (7 +/- 2 days after LH mid-cycle surge). Experiments were conducted in autumn and in spring. Blood samples were obtained every 15 min for 24 hr. Plasma P and LH concentrations were measured by RIA. Each subject's time-series was analysed using three methods; visual inspection (chronogram), spectral analysis to estimate component periods of rhythms (tau) and cosinor analysis to quantify the rhythms parameters. Marked temporal variations in plasma P concentration were observed in each subject. The maximal variations over a 24-hr period, ranged between 13-58.5 mmol/l. Differences related to sampling time were statistically validated by ANOVA (p less than 0.00001). Significant harmonic periods were detected by spectral analysis but differed among subjects. In all subjects but one, a circadian rhythm was detected. The acrophase location was similar (about 0700 hr) in the four subjects studied in autumn, but ranged from 1940 to 0320 hr in those studied in spring. An ultradian rhythm with tau = 8 hr was also validated in six time-series with similar acrophases (about 0200, 1000, and 1800 hr). Cosinor analysis of pooled data revealed that the 24-hr, 12-hr, and 8-hr rhythms were statistically significant (p = 0.001) in autumn. algebraic sum of these three cosine functions yielded a circadian waveform with peak-times occurring near 0300 and 1130 hr and a trough-time about 2200 hr. In spring, the circadian pattern appeared quite different, and peak-times were found near 0700 and 2000 hr, and trough-times near 0300 and 1500 hr. Furthermore, the 24-hr mean of P was higher in autumn (28.9 +/- 0.4 nmol/l) than in spring (17.2 +/- 0.4 nmol/l), p from ANOVA less than 0.00001. The evidence for a similar circadian LH pattern is not as strong. Seasonal, circadian and ultradian rhythms characterize the physiologic time structure of plasma P concentration in mid-luteal phase.