R. Vihko

Endocrine characteristics of adolescent menstrual cycles: impact of early menarche.

An initial group of 200 girls, 7-17 years old, was investigated longitudinally 4 times at 1.5-, 1.5- and 5-year intervals. The present study gives information of the impact of early menarche, a risk factor for breast cancer, on some physical and endocrine characteristics in these subjects. The frequency of ovulation depended significantly on both the time since menarche and the age at menarche. Early menarche was associated with early onset of ovulatory cycles. Even in early puberty, before menarche, the subjects who displayed early menarche during follow-up had higher serum FSH and estradiol concentrations than the girls whose menarche took place after the age of 13.0 years. Adrenal androgen secretion (dehydroepiandrosterone) was not influenced by age at menarche but it increased, as expected, on the basis of chronological age. The group with early menarche was characterized by high circulating estradiol concentrations also after menarche, even in the oldest subjects so far studied, 17-25 years of chronological age. At these ages, the differences in the frequencies of ovulatory cycles were disappearing between the groups formed on the basis of age at menarche. The present findings in pre- and postmenarcheal subjects suggest that the increased risk of breast cancer associated with early menarche is created over several years of exposure to high-level estrogen stimulus.

Oestrogen-induced progesterone receptor in human uterus.

Abstract High affinity progesterone binding proteins having the characteristics of steroid receptors were found in the endometrial and myometrial cytosols of human uteri obtained after hysterectomy from fertile females and estrogen-treated postmenopausal women. Only traces, if any, of this type of binding protein were present in the uterine cytosols of untreated postmenopausal women. The steroid-binding properties of the progesterone receptors in both uterine compartments were very similar. Both proteins were capable of binding a great variety of potent progestins, derivatives of 19-norprogesterone and norethisterone, whereas cortisol was not bound by either receptor. The following physicochemical characteristics were determined at +4°C for the myometrial progesterone receptor(s): Sedimentation rate 3.8S and 7.5S (hypoionic medium), 3.8S (hyperionic medium, 0.3 M KCl); equilibrium association constant 1–4 × 109 l/mol; rate of association 6.7 × 104 l/mol × s−1; rate of dissociation 4.4 × 10−5 s−1; isoelectric points at pH 4.8 and 5.2. The binding process was heat and acid labile had a pH optimum of 8.3. An involvement of SH-groups in the steroid binding process was indicated by a loss of binding ability in the presence of p-hydroxymercuribenzoate. Furthermore, some divalent cations interfered with hormone binding, possibly by interacting with SH-groups on the protein. In the myometria of estrogen-treated postmenopausal women, the binding site concentration varied from 0.35 to 3.6 × 10−12 mol/mg of cytosolic protein. In fertile females, the concentration was 0.06-0.55 × 10−12 mol/mg protein. In normally menstruating women, the myometrial binding site concentration seemed to correlate directly with plasma estradiol-17β and indirectly with plasma progesteone concentrations, as in some animal species.

Assay of estosterone progesterone and 17α-hydroxyprogesterone in human plasma by radioimmunoassay after separation on hydroxyalkoxypropyl sephade

Abstract A method for the determination of testosterone, progesterone and 17α-hydroxyprogesterone in 1–2 ml samples of male and female blood plasma is described. After extraction of the unconjugated steroids, they are chromatographed on a column of a highly lipophilic Sephadex derivative, hydroxyalkoxypropyi Sephadex, in light petroleum-chloroform, 95:5. The final measurement of the individual steroids is made by radioimmunoassay using dextran-coated charcoal in the separation of bound and unbound radioactivity. Using 1–2 ml plasma samples the limit of sensitivity for progesterone and testosterone determination was about 0.1 ng/ml and for 17α-hydroxyprogesterone about 0.15 ng/ml. The coefficients of variation in the determination of these steroids ranged from 7.9–15.4%. The blank values, obtained by analysis of quartz-distilled water by the method described, were negligible in the tesosterone and 17α-hydroxyprogesterone determinations. Values below 10 pg were sometimes observed in the progesterone analysis. Plasma concentrations of these three steroids in a group of females both in the follicular and luteal phase of the menstrual cycle and in connexion with the intake of oral contraceptive of the combination type are presented. In addition, values obtained in a group of normal males are also given. The concentration values can be obtained within two days of drawing the blood samples and one technician can analyze 20 plasma samples in a 5-day period.

Steroid metabolism in testis tissue: concentrations of unconjugated and sulfated neutral steroids in boar testis

Abstract In boar testis tissue, several unconjugated and monosulfated neutral steroids have been identified and most of them quantified. The main steroids present were two 16-androstenes, 5α-androst-16-en-3β-ol and 5α-androst-16-en-3α-ol. A third compound with a similar structure, 5,16-androstadien-3β-ol, was also identified but only in the monosulfate fraction. Testosterone or its sulfate were not found, the level of detection being about 0.5 μg/100 g tissue wet wt. This probably indicates the rapid secretion and/or metabolism of testosterone, once formed, in boar testis. Of significance, was the detection in boar testis tissue of two compounds not previously found in biological material, 19-nortestosterone (17β-hydroxy-4-estren-3-one) and 3β,17α-dihydroxy-5β-pregnan-20-one. The former steroid is probably an intermediate in the testicular biosynthesis of estradiol from testosterone. The presence of several steroids with a 3β-hydroxy-5-ene structure suggests that they might be intermediates in the biosynthesis of testosterone and the 16-androstenes. Their presence in high concentrations as sulfate conjugates points to the possible physiological importance of this type of steroid conjugate in the biosynthesis of steroids.

Progesterone-binding protein in human myometrium. Ligand specificity and some physicochemical characteristics

Abstract After in vitro labelling with radioactive progesterone a steroid-binding macromolecule was isolated from the cytosol fraction of the myometra of two women treated with oestrogen prior to hysterectomy. On density gradient centrifugation in a hypoionic medium, the steroid-protein complex formed two separate peaks with sedimentation coefficients of 3.8 S and 7.5 S, respectively, the former being clearly the major component. Only one peak of bound radioactivity (3.8 S) was seen on gradient centrifugation in 0.3 M KCl. The intrinsic association constant for progesterone was 3 · 10 9 l/mole and the binding site concentration 1 · 10 −12 moles/mg of cytosol protein. The binding process was heat and acid labile and had a pH optimum of 7.5–9. p- Hydroxymercuribenzoate (1 mM) completely abolished binding ability. The lability encountered on prolonged storage could be counteracted by the addition of glycerol (25%) to the buffer. With regard to ligand specificity, 19-nor-4-pregnene-3,20-dione, 16α-ethyl-4-pregnene-3,20-dione, 16α-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione, 16α-ethyl-21-fluoro-19-nor-4-pregnene-3,20-dione, 16α-ethyl-21-fluoro-19-nor-4,6-pregnadiene-3,20-dione, 17α-ethinyl-17β-hydroxy-4-oestren-3-one and 11β-chloro-17α-ethinyl-17β-hydroxy-4-oestren-3-one competed effectively with progesterone for the binding sites. The ligand specificity was close to that found with the progesterone-binding protein from oestrogen-treated, castrated sheep myometrium.

Presence of testosterone and other neutral steroids in human fetal testes.

Abstract Free and sulfate-conjugated neutral steroids were isolated from the pooled testes of 33 human fetuses of 8.5 – 20.0 cm crown-rump length and the compounds were identified by gas-liquid chromatography and gas chromatography — mass spectrometry. Unconjugated pregnenolone and testosterone were the main compounds present. Thus it has been established that under in , vivo conditions the fetal testis synthesizes androgens and that apparently the pathway pregnenolone → 17a-hydroxypregnenolone → dehydroepi-androsterone → androstenedione/5-androstene-3β, 17β-diol → testosterone is preferred.

Follicular growth in relation to serum hormonal patterns in adolescent compared with adult menstrual cycles

The purpose of this study was to clarify the endocrine regulation of the adolescent menstrual cycle, especially the relationships between ovarian follicular development, luteal phase progesterone secretion, and function of the hypothalamic-pituitary unit. One menstrual cycle of each of 17 women who were 15 and 16 years of age and 12 women who were 25 to 35 years of age was characterized by ultrasonography and hormone measurements. In both groups there was a close correlation between follicle size and serum estradiol concentrations. In the adolescents, follicle development was slower, and an eventual ovulation took place from a smaller follicle than in the older group. The immediate preovulatory follicle size correlated with the maximal serum progesterone concentration during the luteal phase. Late follicular development in adolescents may be related to the slow increase of serum follicle-stimulating hormone concentrations early in the cycle.

Characterisation of 2-amino-2-deoxy-d-glucose, 2-amino-2-deoxy-d-galactose, and related compounds, as their trimethylsilyl derivatives by gas-liquid chromatography-mass spectrometry

Abstract Combined gas-liquid chromatography-mass spectrometry (GC-MS) has been used for the analysis of 2-amino-2-deoxy- d -glucose, 2-amino-2-deoxy- d -galactose, their methyl glycosides, and the corresponding alditols, as well as of the N -acetylated forms. The compounds were analyzed as trimethylsilyl (TMS) derivatives synthesized by using chlorotrimethylsilane and hexamethyldisilazane in pyridine, or using these reagents together with bis(trimethylsilyl)acetamide. In the latter procedure, the free amino groups, in addition to the hydroxyl groups, are also trimethylsilylated. Identification of the derivatives was based on their retention times in gas-liquid chromatography and their mass-spectral characteristics. The value of GC-MS of TMS derivatives in the analysis of polysaccharides is discussed.

Lipidex chromatography in the radioimmunoassay of serum and urinary cortisol.

A highly specific method for the determination of cortisol in human serum and urine is described. The sample is first extracted with diethyl ether/ethyl acetate (1 : 1, by vol.), then chromatographed on a highly lipophilic derivative of Sephadex (hydroxyalkoxypropyl Sephadex, Lipidex) in light petroleum/chloroform (1 : 1, by vol.), and finally cortisol is measured by radioimmunoassay using a cortisol-21-BSA antiserum. Bound and unbound radioactivities are separated using dextran-coated charcoal technique. The 8 a.m. values (mean +/- S.D.) of cortisol among 11 young females and 16 young males were 152 +/- 32 ng/ml (range 111-235) and 185 +/- 21 ng/ml (103-232), respectively. The respective values at 4 p.m. were 84 +/- 29 ng/ml (26-117) and 84 +/- 36 ng/ml (32-172). The importance of Lipidex chromatography was demonstrated with assays of serum samples from children with congenital adrenal hyperplasia; without chromatography cortisol values were 6 times those with chromatography. Specific cortisol assays from pregnancy serum also necessitated Lipidex chromatography. Among 11 young men and 9 young women the mean daily urinary cortisol excretion was 56 +/- 26 mug (32-109) and 60 +/- 24 mug (39-109), respectively. Specific urinary cortisol determination could not be achieved without Lipidex chromatography.

Comparison of serum steroid responses to a single injection of hCG in man and rat

The responses of peripheral serum steroids to a single injection of hCG (80 IU/kg b wt) were compared in adult male rats and humans. Before hCG, the quantitatively dominating steroids were dehydroepiandrosterone, testosterone and 17-hydroxypregnenolone in the men, and testosterone and progesterone in the rats. One hour after hCG the concentrations of testosterone and all its precursors measured except for pregnenolone were significantly elevated in the rat serum, whereas a clear rapid response was not observed in the men. Transient blockade of C21 steroid side-chain cleavage was seen in both species at about 24-36 h after hCG, which occurred at the same time as the maximum concentration of estradiol in the men. No changes in rat serum estradiol concentrations were observed. Both species showed a secondary stimulation of testosterone and androstenedione formation at around 3 days. Our findings are compatible with the concept that the main difference in the gonadotropin-stimulated steroidogenesis in man and rat is the magnitude of the rapid steroidogenic response to hCG, which is very small in man and indicates smaller supply or lesser metabolism of mitochondrial cholesterol in human testis.