Quantification of proteins in discrete brain regions of androgen-insensitive testicular feminized Tfm mice

The effect of the testicular feminization mutation (Tfm) on the concentration of specific proteins in the medial preoptic area (MPO), ventromedial hypothalamus (VMH) and parietal cortex (CX) was examined. Adult Tfm and Swiss-Webster male mice were decapitated, the brains were removed and sectioned. Proteins from the three microdissected areas were separated by two-dimensional gel electrophoresis. Gels were stained with silver and then analyzed by quantitative computerized scanning densitometry. Of the 195 proteins quantified, the Tfm mutation significantly influenced the concentration of 16 proteins measured from gels of MPO tissue, 21 from VMH gels and 11 from CX. Of these, three proteins were affected in all brain regions; and three additional proteins were shown to vary in both MPO and VMH. One protein higher in the MPO and VMH of Tfm mice was identified as the glial fibrillary acidic protein. It is suggested that the proteins influenced by the Tfm mutation are regulated by steroids, most likely androgens. Thus, these proteins may prove to be important in hormone-regulated physiological functions.     

Growth hormone-secretory patterns in androgen-resistant (testicular feminized) rats

To investigate the role of androgen receptors in the expression of the male GH-secretory pattern in adult rats, the GH-secretory patterns in androgen-resistant (testicular feminized) rats were compared with their normal male and female littermates. All animals were prepared with intraatrial Silastic catheters and bled every 15 min for 8 h (0800-1600 h). Normal male littermates displayed a characteristic low frequency, high amplitude pattern of GH secretion with bursts of GH occurring every 2.5-3 h and separated by prolonged trough periods where GH values remained low or undetectable (less than 5 ng/ml) for 45-90 min. Normal female littermates showed a characteristic high frequency, low amplitude pattern of GH secretion with pulses of GH occurring every hour. Compared to normal male littermates, females had lower individual GH peak amplitudes and shortened GH-trough periods which contain higher GH levels. GH-secretory profiles displayed by testicular-feminized animals qualitatively and quantitatively resembled those of the normal female littermates. These data suggest that androgen receptors are necessary for the expression of masculine GH-secretory patterns.     

Endocrine status of the testicular feminized male (TFM) rat

The plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), androstenedione (A), dihydrotestosterone (DHT), corticosterone and corticosterone binding globulin (CBG) were determined in 160- and 350-day testicular feminized male (tfm) and normal littermate (NL) rats. In the younger group the concentrations of T, A, DHT and 5α-androstan-3α,17β-diol (Adiol) were also determined in testis tissue.Tfm rats showed a greatly elevated plasma LH indicating lack of androgen feedback. Plasma FSH, however, was normal in both age groups, suggesting that inhibin production from tfm testes was relatively unaffected.Intratesticular concentrations of A were greatly elevated in the tfm animals at both ages whilst the testicular levels of T were highly reduced when compared to NL rats. This indicates a gonadal deficiency of the 17β-hydroxy steroid dehydrogenase (17β-HSD). Furthermore, the relatively low T: DHT ratio and high concentrations of Adiol in the tfm testes confirmed previous reports that the high 5α-reductase typical for the immature rat testis is maintained into adulthood in rats with the tfm condition. A considerable degree of peripheral conversion (A → T) probably helps to maintain normal or supranormal plasma levels of T. Gonadectomy rendered all plasma androgens undetectable, indicating that the adrenal contribution was negligible.Increasing age (350 days) was associated with a marked increase in circulating androgens in tfm rats. This is probably the reason for the observed significant reduction in circulating LH in this age group when compared to the 160-day animals. The aging tfm rat is predisposed to testicular tumors which, on the basis of histology, specific [¹²⁵I]hLH binding and in vitro responsiveness to hCG, appear to be of Leydig cell origin.Microflow fluorometry (MFF) of tfm testis cell suspensions revealed the presence of haploid cells, suggesting that meiosis proceeds to a limited extent.Plasma corticosterone levels in the tfm rat were normal although plasma CBG levels were highly significantly elevated at both ages when compared to the levels in NL rats. We suggest that the adrenal hyperplasia observed in tfm rats is secondary to reduced corticosterone production and diminished free corticosterone in the circulation.     

Decreased Leydig cell responsiveness in the testicular feminized male rat.

The Stanley-Gumbreck pseudohermaphrodite or testicular feminized male (tfm) rat exhibits a decreased Leydig cell sensitivity to human CG (hCG) measured by androgen and cyclic-3',5',-adenosine monophosphate production in vitro. These changes were associated with an 80% reduction in the number of LH receptors in the tfm testis, when compared on the basis of equivalent amounts of testis particle protein or per 10(6) isolated Leydig cells. Androstenedione and not testosterone is the major androgen secreted by the tfm Leydig cell and androstenedione secretion is, therefore, a more appropriate end point than testosterone secretion for Leydig cell function in tfm animals. A dose of hCG (3 ng/2 ml) which elicited a near maximal response in androgen production from the decapsulated testes and Leydig cell suspensions of normals rats, did not significantly stimulate androgen production from Leydig cells of the tfm animals. A much higher dose of hCG (200 ng/2 ml) gave a response from the tfm Leydig cells which was comparable to that obtained with 3 ng from Leydig cells of normal littermates. This indicates that the small number of LH receptors on the tfm Leydig cell membrane are functional and that the reduction in receptor number results in a decrease in the sensitivity of response to LH rather than a reduction in the maximum steroid response.     

The cytochrome P450(17) alpha (17 alpha-hydroxylase/C17,20-lyase) activity of the junctional zone of the rat placenta

The cytochrome P450 responsible for androgen synthesis by the placenta during the second half of pregnancy in the rat was studied in intact and hypophysectomized animals. The two activities of P450(17) alpha, 17 alpha-hydroxylase and C17,20-lyase, were limited to the junctional zone. C17,20-Lyase activity was greater with progesterone than with 17-hydroxyprogesterone as substrate. Although the apparent Michaelis constants were similar, progesterone had a higher maximum velocity than 17-hydroxyprogesterone. Regardless of substrate, C17,20-lyase activity was greater with NADPH than with NADH as an electron donor, and there was no additive effect using both cofactors. Administration of human chorionic gonadotrophin (hCG; 10 IU at 09.00 and 21.00 h on days 13 and 14 and at 09.00 h on day 15) to intact females resulted in more than a 50% reduction of enzyme activity when measured on day 15. The same dose of hCG given to hypophysectomized animals with delayed implantation, i.e. pituitary removal on day 3 and implantation induced by oestrone 5 days later, had no effect on placental enzyme activity, but increased that in the ovary. Administration of ovine LH by osmotic minipump (days 11-15) to intact females resulted in abortion in all animals. The same treatment to animals hypophysectomized on day 11 produced abortion in three of four rats; enzyme activity was greatly reduced in the single animal with placentas. In contrast, infusion of LH into hypophysectomized animals with delayed implantation increased placental enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel compound heterozygous mutation in the CYP17 (P450 17alpha-hydroxylase) gene leading to 17alpha-hydroxylase/17,20-lyase deficiency

Mutations in the CYP17 gene impair steroid biosynthesis in the adrenals and gonads and often cause 17alpha-hydroxylase/17,20-lyase deficiency, leading to amenorrhea, sexual infantilism, and hypokalemic low aldosterone hypertension. Several CYP17 mutations resulting in 17alpha-hydroxylase/17,20-lyase deficiency have been reported previously. In the present study, we found a novel CYP17 mutation from the molecular analysis of a Korean patient with primary amenorrhea with a 46,XX karyotype, and hypokalemic hypertension. We sequenced all 8 exons of the CYP17 gene that were amplified from patient's genomic DNA using polymerase chain reaction (PCR) and found a compound heterozygous mutation in the CYP17 structural gene; a 1-base deletion and a 1-base transversion (TAC-->AA) at codon 329, leading to the production of a truncated protein (1-417 amino acids), and a 3-base deletion (TCC, either 350-351 or 351-352 codon) in the other allele. Restriction enzyme digestion analysis of patient's and parental DNA showed that the 1-base deletion and the 3-base deletion are inherited from mother and father, respectively. Here we conclude that these novel compound heterozygous mutations might account for the patient's clinical manifestations of 17alpha-hydroxylase/17,20-lyase deficiency.     

Testicular trauma resulting in spermatic vessel thrombosis and testicular loss: a case report

Testicular ischemia is typically seen with cases of testicular torsion. Twisting of the spermatic cord and compromise of testicular blood supply can induce testicular loss if not promptly discovered and treated. Non-torsion causes of testicular ischemia are uncommon with rare citations in the literature. Herein, we present a case of testicular ischemia induced by traumatic thrombosis of the spermatic vessels.     

Testicular steroidogenesis in the camel (Camelus dromedarius) during the mating and the nonmating seasons

Enzyme reactions in the camel testis involved in androgen synthesis were studied to determine the factors which account for the low testosterone production during the non-mating season (NMS). Testes excised during the NMS were found to have a relatively high activity of the 3 beta-hydroxysteroid dehydrogenase systems of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone, but the 4-ene-17 alpha-hydroxylase and 4-ene-17,20-lyase systems were apparently less active than the 5-ene-17 alpha-hydroxylase and 5-ene-17,20-lyase. On the other hand, testes excised during the mating season (MS) were found to have a relatively high activity of 4-ene-17 alpha-hydroxylase, 4-ene-lyase, and 17 beta-hydroxysteroid oxidoreductase. The 19-hydroxylation and aromatizing activities for testosterone and androstenedione were not detected in testes excised in either season. It is proposed that during the NMS the predominant route of testosterone biosynthesis is pregnenolone leads to 17 alpha-hydroxypregnenolone leads to dehydroepiandrosterone leads to androst-5-en-3 beta, 17 beta-diol leads to testosterone.     

Testicular steroidogenesis in the mature and immature baboon Papio anubis.

The following studies were undertaken to compare testicular steroidogenesis in the mature and immature baboon. Testicular fragments (50 mg) were incubated for 3 hr with [7-³H]pregnenolone, or with [7-³H]progesterone. The mature testis formed more testosterone (4.6%), androstenedione (1.6%), and progesterone (28.5%) from pregnenolone than did the immature testis (0.6, 0.5, and 26.1%). The immature testis formed more 17α-hydroxyprogesterone (34.7%) and 20α-dihydroprogesterone (23.2%) from pregnenolone than did the mature testis. Similar conversions were obtained in progesterone incubates. 5α-Androstanediol was identified only in mature incubates. These results suggest that the mature baboon testis has greater C₁₇-C₂₀ lyase, 17β-hydroxysteroid dehydrogenase, and 5α-reductase activities than the immature testis, while the immature testis has greater 20α-reductase activity.     

The androgen receptor of the testicular-feminized (Tfm) mutant mouse is smaller than the wild-type receptor

The physicochemical and immunological properties of androgen receptors from kidney and brain of testicular-feminized (Tfm) mutant mice and wild-type mice were compared. Analysis by gel filtration and sucrose density gradients revealed that the mol wt of the mutant receptor was 66K (38A; 3.8S) which was significantly smaller than the 110K (53A; 4.6S) size of the wild-type androgen receptor (P less than 0.05). Mixing experiments failed to demonstrate any role for differential proteolysis in the size differences between these receptors. Interaction of the mutant androgen receptor with specific polyclonal antiandrogen receptor antibodies produced significantly smaller immune complexes than that formed with wild-type receptor (12S vs. 17S; P less than 0.01). This confirmed the smaller size of the Tfm mutant androgen receptor and suggested that it contained fewer epitopes. The Tfm kidney cytosols also demonstrated a decreased concentration of androgen receptor-binding activity relative to that of the wild type. Together, these results suggest that the androgen insensitivity associated with the Tfm phenotype is due to a deficiency of androgen receptor in target tissues and a qualitative defect in the androgen receptor protein itself.     

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.     

Inability of Tfm (testicular feminization) epithelial cells to express androgen-dependent seminal vesicle secretory proteins in chimeric tissue recombinants

To assess the role of androgen receptors (ARs) in the expression of androgen-dependent seminal vesicle (SV) secretory proteins, tissue recombinants were prepared with rat seminal vesicle mesenchyme plus ureter epithelium of wild-type or Tfm mice (rat SVM plus wild-type mouse URE and rat SVM plus Tfm mouse URE, respectively). After growth in male hosts, both the wild-type and Tfm ureter epithelia were induced by SVM to differentiate into a simple columnar epithelium exhibiting the complex folded morphology characteristic of the SV. In SVM plus wild-type mouse URE recombinants, epithelial ARs were induced, and the epithelium expressed the full spectrum of SV secretory proteins. By contrast, in SVM plus Tfm mouse URE recombinants, the Tfm epithelium was genetically incapable of producing functional ARs and failed to produce SV secretory proteins. These data demonstrate in vivo that the induction of SV secretory proteins by androgens is an event requiring intraepithelial ARs. In contrast, androgen-dependent epithelial morphogenesis, columnar cytodifferentiation, and probably also proliferation can be expressed in Tfm epithelium grown in association with wild-type mesenchyme, strongly suggesting that these events are indirect effects on the epithelium mediated by mesenchymal ARs.     

Cytochrome b(5) modulation of 17{alpha} hydroxylase and 17-20 lyase (CYP17) activities in steroidogenesis

CYP17 is a steroidogenic enzyme located in the zona fasciculata and zona reticularis of the adrenal cortex and gonad tissues and which has dual functions - hydroxylation and as a lyase. The first activity gives hydroxylation of pregnenolone and progesterone at the C(17) position to generate 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone, while the second enzymic activity cleaves the C(17)-C(20) bond of 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone to form dehydroepiandro-sterone and androstenedione respectively. The modulation of these two activities occurs through cytochrome b(5). Association of cytochrome b(5) and CYP17 is thought to be based primarily on electrostatic interactions in which the negatively charged residues pair up with positively charged residues on the proximal surface of the CYP17 molecule. Non-specific interactions of the hydrophobic membrane regions of cytochrome b(5) and CYP17 are also thought to play a crucial role in the association of these two haemoproteins. Although cytochrome b(5) is known to stimulate CYP activity by contributing the second electron in the catalytic cycle, in the case of CYP17, the mechanism of cleavage stimulation proceeds via an allosteric mode. It is hypothesised that cytochrome b(5) promotes the cleavage by aligning the iron-oxygen complex attack onto the C(20) rather than the C(17) atom of the steroid substrate molecule. Thus, further understanding of the mechanism of modulation by cytochrome b(5) of the hydroxylase and lyase activities should shed new insights on developing therapeutic targets in CYP17-linked biochemical processes such as adrenarche, polycystic ovary syndrome and prostate cancer.     

Acute effects of aminoglutethimide on testicular steroidogenesis in normal men.

Aminoglutethimide (AG), a known adrenal inhibitor, was administered acutely to normal men in order to study its effects on testicular steroidogenesis. Sixteen subjects between the ages of 21--30 yr received either placebo or 1250 mg AG in divided doses during a 24-h period. To reduce the contribution of adrenal steroids, adrenal function was inhibited in both groups of men by the administration of dexamethasone (2 mg) on the night of the experiment. As a result, mean morning plasma cortisol levels fell to less than 2 micrograms/100 ml. AG blunted the normal diurnal rise in testosterone, which was observed in men receiving placebo, and significantly suppressed the levels of this androgen at 0700 and 0900 h. Estradiol concentrations fell to a greater extent than those of testosterone. The differences between the placebo and drug treatment groups were significant at all time points except 1900 h. A pattern similar to that of estradiol was observed for plasma estrone. When the overall effect of AG administration was examined by analysis of variance, the differences in the levels of all three steroids produced by treatment were highly significant (P less than 0.01 to less than 0.001). After the inhibition of testosterone and estradiol levels, LH and FSH concentrations were significantly (P less than 0.01 and P less than 0.001, respectively) higher in men receiving AG than in those given placebo. However, the data exhibited a large variance due to pulsatile gonadotropin secretion. These observations suggested that AG induces an acute inhibition of testicular steroidogenesis and probably also of the aromatization of testosterone to estradiol.     

Androgen and estrogen receptors in brain cytosol from male, female, and testicular feminized (tfm/y hermaphrodite) mice.

Specific binding of [3H] 5alpha-dihydrotestosterone (DHT) and [3H] estradiol by cytoplasmic extracts from whole brain of castrated male, female, and androgen-insensitive, testicular feminized (tfm/y male-female), mice has been investigated using glycerol gradient centrifugation and charcoal assay. Mouse brain cytosol contains macromolecules with the characteristics of steroid hormone receptors, binding preferentially with high-affinity androgens or estrogens. Both DHT- and estradiol-receptor complexes migrate at 8-9 S in gradients at low ionic strength and at 4-5 S in gradients containing 0.5M KCl. KD's (mean +/- SE) for DHT binding by brain cytosol from castrated males, females, and tfm/y male-female are 1.1 +/- 0.4, 0.9 +/- 0.4, and 0.8 +/- 0.1 X 10(-9)M, respectively. DHT binding activity in brain cytosol from tfm/y male-female mice is reduced to about 20-30% of that from their normal littermates, as is the case for tfm/y male-female kidney cytosol. The residual androgen receptor in tfm/y male-female brain cytosol has normal sedimentation properties. Unlike the situation for androgen binding, the number of estradiol binding sites is comparable in brain cytosol from male, female, and tfm/y male-female mice. KD's (mean +/- SE) for estradiol binding are 1.6 +/- 0.5 X 10(-10)M for castrated males, 2.4 +/- 0.4 X 10(-10)M for females, and 1.8 +/- 0.4 X 10(-10)M for tfm/y male-female. Cross-competition experiments with unlabeled estradiol, DHT, or testosterone, have shown a difference in the degree of specificity of the androgen and estrogen receptors, the estrogen receptor having considerably more specificity. For the interaction of estradiol with the androgen receptor, the Ki is 8-9 X 10(-9)M. The decrease in the number of DHT binding sites in the brain of tfm/y male-female mice without a concomitant decrease in estradiol binding sites, and the different specificities of the two sites, point to the existence of distinct androgen and estrogen receptor molecules in mouse brain cytosol.