5 alpha-reductase activity in rat adipose tissue

We measured the 5 alpha-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [3H]dihydrotestosterone from [3H]testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5 alpha-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10(-8) M), when added to the medium, caused a 90% decrease in 5 alpha-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5 alpha-reductase activity in each tissue studied.     

Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis

The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.     

Partial characterization of testosterone 5 alpha-reductase solubilized from rat testicular microsomes

Testosterone 5 alpha-reductase was successfully solubilized by the use of digitonin from rat testicular microsomes and then partially purified by polyethylene glycol fractionation and DEAE-Sephacel column chromatography. The 5 alpha-reductase activity of the partially purified preparation was significantly stimulated by addition of phosphatidylserine (bovine brain). Synthetic dilauroylphosphatidylcholine also increased the reductase activity to a somewhat lesser extent than did phosphatidylserine, whereas natural phosphatidylcholine from bovine liver did not exhibit any stimulation. When synthetic phosphatidylcholines with varying acyl chain lengths were tested for their stimulatory effects on the reductase activity, dilauroylphosphatidylcholine was most active; dimyristoylphosphatidylcholine was less active; dioleoylphosphatidylcholine was almost inactive.     

Hypothalamic 5 alpha-reductase and 3 alpha-oxidoreductase activity in the male rat

The aim of the present study was to assess the progesterone (Pr) transforming 5 alpha-reductase (5 alpha-R) and 3 alpha-oxidoreductase (3 alpha-OR) activities in the hypothalamus of the male rat as a function of age and following castration and/or adrenalectomy performed at the sixth day of life. The hypothalamic activity of these enzymes was estimated from the sum of the 5 alpha- or 3 alpha-reduced metabolites produced from 14C-labeled Pr incubated "in vitro" with hypothalamic tissue. Plasma levels of testosterone (T), progesterone (Pr), estrone (E1), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured simultaneously. Special attention was paid to the GC/MS analysis of the endogenous content of the hypothalamic Pr-metabolites 3 alpha-hydroxy-pregn-4-en-20-one (3 alpha-Pr), 5 alpha-pregnane-3,20-dione (5 alpha-Pr) and 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha,3 alpha-Pr).The high 5 alpha-R and 3 alpha-OR activities estimated in the hypothalamus of prepubertal rats are not related to the action of gonadal or adrenal steroids. Substantial and comparable endogenous 3 alpha- and/or 5 alpha-Pr-metabolites were found in hypothalami from both prepubertal and mature rats. The results of the present study do not provide evidence for a contributory role of the 3 alpha-hydroxylated Pr derivative to the regulation of gonadotropin secretion in the male rat.     

Prolactin and its involvement in fluid regulation in the bovine

Plasma prolactin (PRL) concentrations, osmolality, water consumption, feed intake, urine excretion, and fecal water output were determined in twelve steers of 3 breeds exposed to 5 feed and water regimes. Breed differences were found in water intake and plasma PRL concentrations when feed and water were ad lib., however, during any of the other 4 treatments, responses were similar between breeds. During dehydration and feed restriction, water intake, urine, fecal water, and plasma prolactin decreased; however, during hydration and refeeding such changes were not as clearly related. No consistent relationships between plasma prolactin and osmolality were found. Data suggests that PRL's role in fluid regulation in the bovine is most likely associated with alterations in renal hemodynamics rather than by changes in plasma osmolality.     

Testosterone 5 alpha-reductase in rat brain

We describe a simple procedure for the microassay of testosterone 5 alpha-reductase in homogenates of rat brain. This enzyme converts testosterone to dihydrotestosterone. We have used this assay to characterize the enzymatic activity and to map its distribution. The apparent Km is 4.1 x 10(-6) M and the Vmax is 85.6 pmol/mg protein/h. The pH optimum is broad and extends from pH 6.0 to 8.0. For the brain regions surveyed, testosterone 5 alpha-reductase activity varied over a 10-fold range. The highest activities were observed in homogenates of the midbrain and pons (37-39 pmol/mg protein/h). The lowest were seen in homogenates of the thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, olfactory tubercle, and preoptic area (3-7 pmol/mg protein/h).     

Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat

The accumulation of mast cells in the rat testicular interstitium was studied under different experimental conditions in order to correlate this accumulation with the alterations of specific testicular tissue compartments or cell types. Estrogen treatment was effective in inducing mast cell proliferation when administered on Day 1 or at higher doses at 10 days of age. Estrogens were ineffective beyond 20 days of age. Postnatal treatment of neonatal-estrogen-treated rats with FSH and LH prevented the appearance of mast cells. In contrast, treatment with the Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) was effective in inducing mast cell accumulation only when administered to adult rats, inducing small numbers of mast cells at 45 days of age; it was ineffective on 30-day-old rats. Hypophysectomy alone did not determine the appearance of mast cells. However, when atrophic Leydig cells were destroyed with EDS, high numbers of mast cells accumulated in the testis. These results support the existence of Leydig cell-related inhibitory factors for mast cells in the rat testicular interstitium.     

Hyperprolactinemia enhances LH-stimulated 4-ene-5 alpha-reductase activity but inhibits LH-induced 17-hydroxylase activity in testes of hypophysectomized immature rats

Two pituitaries from 7-week old female rats (Sprague-Dawley strain) were grafted under the capsule of the left kidney of 21-day old male rat. The pituitary grafted and sham-operated rats were hypophysectomized at 27 days of age. The hypophysectomized rats, in groups of 4, were given daily injections of 9 micrograms NIAMDD-oLH-23 (minimum effective dose) or saline for 3 days starting from day 29. Testicular homogenates were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione, and enzyme activities per testes were estimated. Testicular HCG-binding sites were also measured. Hypophysectomy caused significant decreases in activities of testicular 5 alpha-reductase, 17-hydroxylase, and 17 beta-hydroxysteroid oxidoreductase. These decreased enzyme activities were significantly stimulated by LH treatment. Although pituitary grafts alone showed no effects on these enzyme activities in the testes of the hypophysectomized rats, the grafts significantly enhanced LH-stimulated 5 alpha-reductase activities but inhibited LH-stimulated 17-hydroxylase activity. Testicular LH/HCG receptors were significantly increased by the grafts, especially in the presence of LH, without affecting affinity for HCG. The present results demonstrate for the first time that hyperprolactinemia directly stimulates LH-stimulated 5 alpha-reductase activity in rat testes. The results also show that the same grafts directly inhibit LH-stimulated 17-hydroxylase activity, probably via postreceptor mechanisms.     

Assessment of freezing procedures for rat immature testicular tissue

Fertility preservation has been included in the management of childhood cancer treatment. Cryopreservation of immature testicular tissue is the only available solution for pre-pubertal boys. Different freezing protocols have been developed in several species but without a clearly identified procedure. We tried to evaluate several protocols for cryopreservation of rat immature testicular tissue. Twelve different freezing protocols using different (i) cryoprotectant (dimethylsulphoxide [DMSO] or 1,2-propanediol [PROH]), (ii) cryoprotectant concentration (1.5M or 3M), (iii) equilibration time (30 or 60 min), (iv) equilibration temperature (4 °C or room temperature), (v) size of testicular fragment (7.5mg or 15mg), (vi) package (straws or cryovials), were compared using cord morphological damage evaluation. A testicular tissue piece of 7.5mg cryopreserved in cryovial using 1.5M DMSO, an equilibration time of 30 min at 4 °C showed fewer morphological alterations than the other protocols tested. The selected freezing protocol was able to maintain rat immature testicular tissue architecture, functionality after testicular pieces organotypic culture, and could be proposed in a human application.     

Regulation of the pituitary 5 alpha-reductase activity by gonadotropin releasing hormone and testosterone in the adult male rat

Intact or castrated adult male rats were treated for nine days with GnRH (10 micrograms/day), the synthetic GnRH goserelin (100 micrograms/day) or the GnRH-antagonist Org 30276 (250 or 500 micrograms/day). In some series, 1 mg testosterone propionate was administered alone, or in combination with goserelin or Org 30276. The in vitro metabolism of [1 alpha,2 alpha-3H]testosterone by pituitary and hypothalamic homogenates was investigated in combination with the estimation of plasma concentrations of testosterone and gonadotropins. No qualitative or quantitative differences were observed in hypothalamic testosterone metabolism or in the pituitary 17 beta-hydroxysteroid dehydrogenase activity. Testosterone administration to intact male rats decreased the pituitary 5 alpha-reductase activity and LH, while administered to castrated rats, it was able to suppress totally the castration-induced increase of the 5 alpha-reductase activity and of the gonadotropin secretion. The drastic decrease of the plasma levels of testosterone, observed after a prolonged treatment with GnRH, goserelin or Org 30276 was not accompanied by an increased pituitary 5 alpha-reductase activity. Injected to castrated rats, it was observed that the castration-induced increase of the pituitary 5 alpha-reductase was further stimulated by GnRH, totally suppressed by goserelin and partially suppressed by Org 30276. Concomitant administration of goserelin or Org 30276 and testosterone propionate to castrated rats resulted in a further decrease of the pituitary 5 alpha-reductase activity, compared to the castrated, GnRH-analogue treated rats. These data indicate that the pituitary 5 alpha-reductase enzyme system is controlled by both direct steroidal and indirect GnRH-mediated mechanisms.     

Selective inhibition of the 5 alpha-reductase of the rat epididymis.

The effect of several synthetic steroids belonging either to the 4-aza-3-oxo-steroid family or to androstene and androstane derivatives was investigated "in vitro" on the epididymal as well as prostatic 5 alpha-reductase activity. For this purpose rat caput epididymis and prostate were incubated with the different steroidal compounds at molar concentrations of 10(-7), 10(-6), and 10(-5) in the presence of labelled testosterone as substrate. The steroids 4-MA (17 beta, N,N-diethyl-carbamoyl-4-aza-5 alpha-androstan-3-one) and 4-OH-A (4-hydroxy-androstenedione), already known to be effective 5 alpha-reductase inhibitors at the level of the prostate, have been used as reference molecules. The 5 alpha-reductase activity was evaluated by measuring pg of dihydrotestosterone (DHT) formed in 2 h of incubation by mg of tissue. The steroids A, B, C, F, G and I inhibit the formation of DHT in the rat epididymis although to different extents; they are also equally effective on the formation of DHT in the rat prostate. The steroids D, E, H and L are devoid of any inhibitory property on the formation of DHT in both the rat epididymis and prostate. The most interesting results were obtained with compound M which exhibits a dose-dependent and significant inhibitory effect on the formation of DHT in the epididymis, but it is inactive at the level of the prostate. These findings suggest that it is possible (a) to selectively interfere with the 5 alpha-reductase of the epididymis without affecting that present in the prostate, and (b) consequently to envisage new ways to regulate male fertility.     

Circadian rhythm of acid phosphatase activity in rat liver microsomes and dependence of NADH 5 alpha-reductase on phosphatase activity

The specific activity of acid phosphatase in male and female rats follows a circadian rhythm. Preincubation of liver microsomes with testosterone led to an increase of phosphatase activity and a loss of circadian rhythm. NADH 5 alpha-reductase was inactivated by several animal and bacterial acid and alkaline phosphatases while the acid phosphatase from potatoes was ineffective. The extent of inhibition depends on the course of circadian rhythm of NADH 5 alpha-reductase activity. Preincubation of microsomes in the presence of testosterone inhibited the NADH 5 alpha-reduction of testosterone. No such inhibition was observed after preincubation of microsomes with progesterone.     

Prolactin secretion in testicular cancer patients

The role of prolactin (PRL) in testicular function and in its disorders is still obscure. To draw some preliminary conclusions on the relation between the PRL and testis cancer, we assessed the PRL response to thyrotropin-releasing hormone (THR) in 15 patients with testicular cancer (8 seminoma; 6 nonseminoma; 1 leydigioma), and in 11 healthy male subjects as controls. The results showed that 5/15 cancer patients gave no PRL response to TRH; 4 of them had a nonseminoma and the fifth a seminomatous testis carcinoma. Patients with nonseminoma had significantly lower mean peak values of PRL after TRH than controls or patients with seminoma. The biological significance of the altered PRL response to TRH in testicular carcinoma has still to be established.