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Testosterone-estradiol-binding globulin, unbound and total estradiol and testosterone, and total progesterone during the menstrual cycle in women with epilepsy taking antiepileptic drugs.

Total plasma estradiol, progesterone, testosterone, and the binding capacity of testosterone-estradiol-binding globulin (TeBG) were measured and plasma unbound estradiol and testosterone calculated, every second day during nine cycles in six patients with epilepsy. They all received phenytoin as one medication. Some had additionally, phenobarbital or ethosuximide. Plasma concentrations were compared with those of a group of healthy women without medication. Total plasma testosterone and TeBG binding capacity were significantly increased in the treated group, as compared to the control group. However, the unbound testosterone concentration was within the range of the controls. The percentage unbound testosterone of total plasma concentration was clearly below that of the control group. This indicates that there was a primarily increased TeBG binding capacity in patients taking antiepileptic drugs.

Adolescent

A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal anti-testosterone immunoglobulins.

An enzyme immunoassay for plasma testosterone was developed based on competition between an immobilised testosterone-casein conjugate and the analyte for polyclonal anti-testosterone immunoglobulins, followed by the use of enzyme-labelled second antibodies to determine the degree of competition. The quantity of immobilised testosterone-casein conjugate was optimised so that the lower affinity anti-testosterone antibody populations present did not affect the assay. The assay standard curve covered a range of 11-300 fmol/well. Testosterone levels in small amounts of male and female plasma could be assayed with good reproducibility and correlated well with results obtained by radioimmunoassay. By comparison with an analogous assay using monoclonal antibodies it appears that, given that the assay sensitivity is the most important criterion for choice, the use a polyclonal antiserum with this type of reactive antibody selection is preferable to the use of monoclonal antibodies.

Antibodies

Comparative studies of the ethynyl estrogens used in oral contraceptives: effects with and without progestational agents on plasma androstenedione, testosterone, and testosterone binding in humans, baboons, and beagles.

The effects of ethynylestradiol or mestranol given in cyclic fashion, with and without a progestational compound (norethindrone acetate, dl-norgestrel, or megestrol acetate), on plasma androgens and their binding were examined in adult women, female baboons, and beagles. The two estrogens are equivalent in their effect, and there were essentially no dose-related differences over the range examined. In human subjects, the estrogens increased total testosterone and testosterone binding, and decreased free testosterone. In baboons, estrogen produced a transient decrease in total testosterone and an increase in binding. The levels of progestational agents used did not affect total testosterone in humans, as is commonly observed with commercial agents, but did decrease it in baboons. Percentage binding was decreased in both species by the 19-nor compounds, but not by megestrol. Androstenedione levels were unaffected in human subjects, but effects of both estrogens and progestins were seen in baboons. Because of the very low levels of androgens in female beagles, this species did not lend itself well to a study of this kind. However, an increase in testosterone binding was induced by estrogen even in the absence of testosterone/estrogen-binding globulin.

Androstenedione

Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard. Comparison with radioimmunoassay.

Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.

Gas Chromatography-Mass Spectrometry

The biological activity of dimeric testosterone, a new long-acting androgen, and of testosterone enanthate in the castrated male rat.

The long-term effect of single intramuscular injections of various doses of dimeric testosterone and of testosterone enanthate into castrated male rats upon serum testosterone, luteinizing hormone (LH), pituitary LH, and on the weight of the seminal vesicles, the ventral prostate and the levator ani muscle, was investigated. The effect of the enanthate was characterized by a rapid onset and a protracted androgenic action and a suppression of serum LH, while the dimeric testosterone brought about only a moderate but very even depot effect. The injection of 5 mg of the dimeric testosterone caused a positive feedback effectu upon LH release for 16 weeks. The results indicate that the dimeric testosterone may exert is hormonal effects as intact ester.

Animals

Endogenous and exogenous testosterone levels after administration of deuterium-labelled testosterone propionate in hypogonadotropic hypogonadism.

A gas chromatography-mass spectrometry-selected ion monitoring technique was employed to determine simultaneously the plasma concentrations of endogenous and exogenous testosterone in three patients with hypogonadotropic hypogonadism after a single i.m. dose of testosterone propionate-19,19,19-d3. The plasma levels of testosterone-19,19,19-d3 derived from testosterone propionate-19,19,19-d3 were maintained above the normal testosterone levels (greater than 4 ng/ml) for 48 h, while the plasma levels of endogenous testosterone changed little.

Adult

Serum LH and FSH following passive immunization against circulating testosterone in the intact male rat and in orchidectomized rats bearing subcutaneous silastic implants of testosterone.

Subcutaneous Silastic implants were designed to release quantities of testosterone approximating that produced by the rat testis and appropriate testosterone treatment was found to produce a physiologic inhibition of both LH and FSH secretion in orchidectomized rats. In rats bearing such testosterone implants, intravenous injection of an ovine anti-testosterone serum (0.3 cc) was sufficient to completely abolish the inhibitory effects of the testosterone implant for a period of 3 days as judged by the development of typical post-castration increases in serum LH and FSH. When this dose of antiserum was administered to intact adult male rats, the increases in serum LH on days 1, 2, and 3 post-injection were approximately 19%, 15%, and 11% of those observed in untreated castrates. Increases in serum FSH in antiserum injected rats followed a pattern similar to that for serum LH but the response was slightly greater (19%, 23%, and 22% of the castrate response) and there was no indication of a decreasing effect during the three days following injection. In these experiments, passive immunization against testosterone in intact male rats produced a clearcut stimulation of both LH and FSH release, but produced no data to support the suggestion that the intact testis can secrete nonsteroidal compounds capable of producing a differential inhibition of FSH secretion.

Animals

[Effect of neonatal injections of estradiol, testosterone and cryproterone acetate on plasma and testicular testosterone and on the genital system in adult male mice].

On day old male mice received a single injection of oestradiol benzoate, testosterone propionate or cyproterone acetate in order to study their action on testicular development, particularly testosterone secretion. Oestrogenization of newborn males leads, when the animals mature, to a high proportion or cryptorchidism, to atrophy of testes and seminal vesicles, and inhibition of spermatogenesis. Testosterone levels were reduced in the plasma. Testosterone propionate produced moderate reduction of testicular weight but spermatogenesis was not impaired. Plasma testosterone level was reduced. Cyproterone acetate increased significantly testicular testosterone level.

Animals

[Testosterone and dihydrotestosterone radioimmunoassays in Müllerian ducts of control and testosterone propionate injected quail embryos (author's transl)].

Testosterone (T) and dihydrotestosterone (DHT) have been quantitated by means of radioimmunoassay in Müllerian ducts (CM) from control quail embryos (6 to 8-day male and 6 to 15-day female) and from female embryos injected with 50 nanograms of testosterone propionate (PT) on the 8th day. These hormones are demonstrable in undifferentiated CM from 6-day control embryos. In control males although a highly significant decrease of the CM weight occurs during the CM involution, the detected amounts of androgens remain at a constant level. In control females, the right CM shows a slight increase of the androgen content during the rudimentation, i.e. from day 8 on; in the left CM: the highest steroid levels are found on the 6th day; while the CM differentiate, concentrations decrease and become similar to those found in neutral tissues. Given testosterone propionate on day 8 female embryos: right CM: both testosterone and DHT levels highly increase until day 14; the CM of treated embryos contain 8 times as much steroid as control; left CM: testosterone and DHT increase after injection until day 9,5; they slightly decrease between days 9,5 and 12, and then remain constant on day 14. Differences in concentrations are highly significant between CM of control and of PT - injected embryos. It seems that the binding sites of these androgens are more numerous during the involution and that bound testosterone or DHT could take a part in CM regression in male embryos and right CM rudimentation in female embryos.

Animals

Measurement of the testosterone binding parameters for both testosterone-estradiol binding globulin and albumin in individual serum samples.

This report describes a solid phase method for the characterization of testosterone binding to both albumin and testosterone-estradiol binding globulin (TeBG). TeBG is adsorbed from serum samples onto a solid phase matrix of concanavalin A covalently linked to 4B Sepharose. The binding of testosterone is then examined both in the presence and absence of the endogenous serum albumin. Analysis of the resulting Scatchard plots permits determination of the TeBG binding capacity, TeBG association constant and a parameter of albumin binding equivalent to the product of its affinity and capacity for binding testosterone. Results showed that the TeBG capacity was lower in men than in women (18.4 +/- 5.8 vs. 33.1 +/- 19.2 nM, p less than 0.01). The association constant was greater in men (1.59 +/- 0.35 vs. 1.19 +/- 0.32 x 10(9)M-1, 10(9)M-1, p less than 0.01). There was no difference in the albumin binding parameter (43.8 +/- 18.3 vs. 46.6 +/- 15.5, NS). These parameters can then be used to calculate the distribution of the circulating testosterone into albumin bound, TeBG bound and unbound fractions.

Adult

A comparison of the effects of neonatally administered testosterone, testosterone propionate and dihydrotestosterone on aggressive and sexual behaviour in the female golden hamster.

Female golden hamsters received one of the following treatments on the day following birth: (i) 300 mug testosterone propionate in arachis oil, (ii) 300 mug testosterone in oil, (iii) 300 mug dihydrotestosterone in oil, or (iv) oil alone. As adults all animals, underwent three tests for behaviour. First, while intact, females were observed in aggressive interactions with males. Secondly, after ovariectomy, females were primed with oestrogen + progesterone and tested for receptivity with a stud male. Thirdly, all ovariectomized females were primed with testosterone propionate and tested for male patterns of behaviour with a receptive female. Compared with the effects of oil administration (control), testosterone propionate administration resulted in increased aggressiveness and the capacity to show male patterns of sexual behaviour, together with a decreased capacity to show female patterns of sexual behaviour. Testosterone increased aggression and male sexual behaviour, but had no effects on receptivity, while dihydrotestosterone decreased some components of receptivity but had no effects on aggressiveness or the capacity to show male mating behaviour.

Aggression

Effects of testosterone, testosterone metabolites and anti-androgens on the function of the male accessory glands in the rabbit and rat.

The androgenic potencies of testosterone, 5alpha-dihydrotestosterone, 5alpha-androstane-3alpha,17beta-diol and 5alpha-androstane-3beta,17beta-diol towards the prostate, glandula seminalis+glandula vesicularis, ampullae and epididymis were evaluated after administration to castrated rabbits. The influence of cyproterone acetate, stilboestrol and medrogestone on accessory gland function was also investigated in rabbits and rats. In the rabbit it was found that the minimum dose of testosterone propionate that would maintain the function of all accessroy glands at normal levels was approximately 200 microgram/animal/day. Higher levels of testosterone propionate overstimulated the function of the prostate, glandula seminalis+glandula vesicularis and ampullae, but did not affect the epidiymis. Whereas testosterone propionate and 5alpha-dihydrotestosterone propionate were essentially equipotent in their capcity to support growth and secretory activity and stimulated all the accessory glands, 5alpha-androstane-3alpha,17beta-diol dipropionate had a pronounced differential effect; it was considerably more potent than testosterone propionate in promoting secretion in the prostate, but was ineffective in maintaining the function of the epididymis. 5alpha-Androstane-3beta,17beta-diol dipropionate was the weakest androgen tested. Evidence also indicated that the potency of a steroid can depend on whether it is administered as its free or esterified form. Cyproterone acetate suppressed fructose secretion in the prostate of the rabbit but had no adverse effects on the function of the epididymis in either the rabbit or rat. Stilboestrol was the most potent anti-androgen tested and medrogestone the least effective.

Androgen Antagonists

Effects of FSH and testosterone on highly purified rat Sertoli cells: inhibin alpha-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone.

The effects of FSH and testosterone on inhibin mRNA expression and inhibin production by highly purified Sertoli cell preparations were examined. Sertoli cells were isolated from testes of 22-day-old rats by sequential trypsin, collagenase and hyaluronidase treatments, with subsequent osmotic shock treatment on day 3 of culture. Contamination by peritubular and germ cells was less than 0.5 and 1-3% respectively. Intracellular and secreted inhibin levels were measured by radioimmunoassay, using Sertoli cells which were incubated for 24 h in the absence or presence of FSH and testosterone from days 4 to 5 of culture. FSH stimulated the cellular inhibin content and the secreted inhibin level by four- and sevenfold respectively, with a half-maximal effective dose of 5-50 ng/ml. Under the present incubation conditions, testosterone (1 mumol/l) had no effect on immunoreactive inhibin levels in either the presence or absence of FSH. Similarly, the expression of inhibin alpha-subunit mRNA was increased following FSH stimulation, whereas testosterone had no effect. The expression of inhibin beta B-subunit mRNAs was not influenced by FSH or testosterone. It is concluded that highly purified Sertoli cell preparations, with a very low number of peritubular or germ cells, are fully responsive to FSH with respect to inhibin mRNA expression and inhibin production.

Animals

Effect of actinomycin D or puromycin on microsomal testosterone hydroxylase activity enhanced by testosterone in female rat liver.

The injection of testosterone propionate for 4 successive days into female rats resulted in an increase of the in vitro conversion of the hydroxylated testosterones from testosterone by the hepatic microsomal fraction, but no change in the content of microsomal cytochrome P-450 occurred. Actinomycin D or puromycin, which was administered for 4 days with injections of testosterone propionate, prevented the enzyme induction.

Animals

Plasma testosterone levels and ovarian testosterone content in adult mice treated with diethylstilbestrol neonatally.

Neonatal female NMRI mice (n = 16) were treated with 5 micrograms diethylstilbestrol (DES) per day, for the first 5 days after birth and killed postpubertally. Control females (n = 52) were injected with vehicle only and killed in different stages of the estrous cycle. The plasma testosterone level was significantly lower in DES females than in control females in any of the estrous phases. Ovariectomy (n = 5), adrenalectomy (n = 5) or a combination of both ablations (n = 3) did not affect the plasma testosterone in DES treated females while it was significantly reduced in control females (ovariectomy n = 5; adrenalectomy n = 9); most effective was the combination ovariectomy-adrenalectomy (n = 7). Ovarian homogenates from DES treated females (n = 10) had a significantly lower testosterone content than homogenates from control females in any phase of the estrous cycle (6-10 females per phase), which held true on both a per ovary basis and when related to ovarian weight. After a 2 h incubation in vitro, the testosterone levels had increased significantly in DES homogenates (n = 6) and to a lower extent in homogenates from control females in estrus (n = 9). No similar effect was found in homogenates from diestrus (n = 10) or proestrus (n = 9) females. The results are discussed in relation to the special ovarian morphology of adult but neonatally DES treated females and also with respect to endocrine control mechanisms.

Adrenalectomy

Testicular testosterone concentration and in vitro response to HCG in normal and in testosterone immunized rabbits.

The testosterone concentration, the in vitro response to HCG and the percentage Leydig cells in testes of normal and of testosterone-3-BSA immunized rabbits were determined. Following immunization all three parameters increased in the same order of magnitude (1.8-2.6fold). The results indicate that active immunization with testosterone has no deleterious effects on the endocrine capacity of the Leydig cells. The observed functional and morpholigical alterations of the testes are due solely to increased trophic hormone secretion from the pituitary caused by antibody binding of circulating androgens. The basic testosterone concentration in the testes of the control rabbits were in the range of values reported for other species.

Animals

The use of 6alpha-and 6beta-carboxymethyl testosterone-bovine serum albumin conjugates in radioimmunoassay for testosterone.

The synthesis of 6alpha- and 6beta-testosterone-bovine serum albumin (BSA) conjugates is described. 6beta-Carboxymethyl-4-androstene-3,17-dione was prepared by a route analogous to that described earlier for 6beta-carboxymethyl progesterone. Sodium borohydride reduction of the 3 and 17 keto groups and subsequent selective oxidation of the resulting 3beta, 17beta-diol using MnO2 provided 6 -carboxymethyl testosterone. Further acid catalyzed epimerization of the C-6 center gave the isomeric 6alpha-carboxymethyl testosterone. The 6alpha- and 6beta-testosterone derivatives were attached to BSA via a mixed anhydride coupling employing tributylamine and i minus-butylchlorocarbonate. For each molecule of BSA, the 6alpha- and 6beta-conjugates contained an average of 23 and 20 steroid residues, respectively. Antisera to the conjugates exhibited similar high specificities toward various steroids, the only incidence of serious cross-reaction being the expected case of dihydrotestosterone.

Androstenedione