7α-Hydroxypregnenolone regulating locomotor behavior identified in the brain and pineal gland across vertebrates.

The brain synthesizes steroids de novo from cholesterol, which are called neurosteroids. Based on extensive studies on neurosteroids over the past thirty years, it is now accepted that neurosteroidogenesis in the brain is a conserved property across vertebrates. However, the formation of bioactive neurosteroids in the brain is still incompletely elucidated in vertebrates. In fact, we recently identified 7α-hydroxypregnenolone (7α-OH PREG) as a novel bioactive neurosteroid stimulating locomotor behavior in the brain of several vertebrates. The follow-up studies have demonstrated that the stimulatory action of brain 7α-OH PREG on locomotor behavior is mediated by the dopaminergic system across vertebrates. More recently, we have further demonstrated that the pineal gland, an endocrine organ located close to the brain, is a major site of the formation of bioactive neurosteroids. In addition to the brain, the pineal gland actively produces 7α-OH PREG de novo from cholesterol as a major pineal neurosteroid that acts on the brain to control locomotor rhythms. This review summarizes the identification, biosynthesis and mode of action of brain and pineal 7α-OH PREG, a new bioactive neurosteroid regulating locomotor behavior, across vertebrates.

Nongenomic actions of neurosteroid pregnenolone and its metabolites.

Steroids have been widely used in the clinical setting. They bind and activate nuclear receptors to regulate gene expression. In addition to activating genomic transcription, steroids also exert nongenomic actions. The current article focuses on the nongenomic actions of neurosteroids, including pregnenolone (P5), 7α-hydroxypregnenolone, pregnenolone sulfate and allopregnanolone. Pregnenolone and its derivatives promote neuronal activity by enhancing learning and memory, relieving depression, enhancing locomotor activity, and promoting neuronal cell survival. They exert these effects by activating various target proteins located in the cytoplasm or cell membrane. Pregnenolone and its metabolites bind to receptors such as microtubule-associated proteins and neurotransmitter receptors to elicit a series of reactions including stabilization of microtubules, increase of ion flux into cells, and dopamine release. The wide actions of neurosteroids indicate that pregnenolone derivatives have great potential in future treatment of neurological diseases.

7α-Hydroxypregnenolone regulates diurnal changes in sexual behavior of male quail.

In the Japanese quail, 7α-hydroxypregnenolone, a previously undescribed avian neurosteroid, is actively produced in the brain. 7α-Hydroxypregnenolone acts as a novel neuronal activator to stimulate locomotor activity of quail. Therefore, in this study, we determined whether 7α-hydroxypregnenolone changes the expression of sexual behavior in Japanese quail. We first measured diurnal changes in sexual behavior of male quail exposed to a long-day photoperiod. We found that sexual behavior of male quail was high in the morning when endogenous 7α-hydroxypregnenolone level is high. Subsequently, we centrally administered 7α-hydroxypregnenolone in the evening when endogenous 7α-hydroxypregnenolone level is low. In the 30 min after intracerebroventricular (ICV) injection, 7α-hydroxypregnenolone dose dependently increased the frequency of sexual behavior of male quail. However, 7ß-hydroxypregnenolone, a stereoisomer of 7α-hydroxypregnenolone, did not effect on the frequency of sexual behavior of male quail. In addition, to confirm the action of 7α-hydroxypregnenolone on sexual behavior, male birds received an ICV injection of ketoconazole, an inhibitor of cytochrome P450s, and behavioral experiments were performed in the morning. Ketoconazole significantly decreased the frequency of sexual behavior of male quail, whereas administration of 7α-hydroxypregnenolone to ketoconazole-treated males increased the frequency of their sexual behavior. These results indicate that 7α-hydroxypregnenolone regulates diurnal changes in sexual behavior of male quail.

7α-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon.

Salmon migrate upstream against an opposing current in their natal river. However, the molecular mechanisms that stimulate upstream migratory behavior are poorly understood. Here, we show that 7α-hydroxypregnenolone (7α-OH PREG), a newly identified neuronal modulator of locomotion, acts as a key factor for upstream migration in salmon. We first identified 7α-OH PREG and cytochrome P450 7α-hydroxylase (P4507α), a steroidogenic enzyme producing 7α-OH PREG, in the salmon brain and then found that 7α-OH PREG synthesis in the brain increases during upstream migration. Subsequently, we demonstrated that 7α-OH PREG increases upstream migratory behavior of salmon. We further found that 7α-OH PREG acts on dopamine neurons in the magnocellular preoptic nucleus during upstream migration. Thus, 7α-OH PREG stimulates upstream migratory behavior through the dopaminergic system in salmon. These findings provide new insights into the molecular mechanisms of fish upstream migration.

Possible hormonal interaction for eliciting courtship behavior in the male newt, Cynops pyrrhogaster.

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7α-hydroxypregnenolone (7α-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7α-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7α-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results.

Effect of 17α-hydroxyprogesterone and 17α-hydroxypregnenolone on sperm quality and sperm quantity in male mud spiny lobster (Panulirus polyphagus).

The present study aimed to determine the effect of 17α-hydroxyprogesterone (17α-OHP) and 17α-hydroxypregnenolone (17α-OHPL) on sperm quality and sperm quantity in male mud spiny lobster (Panulirus polyphagus). The mean of sperm quality was increased in 17α-OHP and 17α-OHPL treated hormones. In 17α-OHP injected animals, the mean of sperm quantity of dose 0.01 µg g(-1) b.wt. was increased than 17α-OHPL. Meanwhile, 17α-OHP and 17α-OHPL concentrations were lower when injected with the hormones but 17α-OHP was higher at only day 15 (dose 0.01 and 0.1 µg g(-1) b.wt.). For 17α-OHPL, the hormone was a prohormone in the body of P. polyphagus and only required smaller to increase the sperm quantity. Besides, when the higher dose of 17α-OHPL (0.1 µg g(-1) b.wt.) was used in P. polyphagus, the development of P. polyphagus was inhibited and decreased the sperm quantity and 17α-OHPL concentration in hemolymph was lower. Injection of 17α-OHP in P. polyphagus has increased the sperm quality and quantity for both 17α-OHP dosage of 0.01 and 0.1 µg g(-1) b.wt. However, injection of 17α-OHPL in P. polyphagus has decreased the sperm quantity only, also for both dosage of 0.01 and 0.1 µg g(-1) b.wt. and lower on hormone concentration.

Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster.

Reproductive behavior in amphibians, as in other vertebrate animals, is controlled by multiple hormones. A neurosteroid, 7α-hydroxypregnenolone, has recently been found to enhance locomotor activity in the male newt, Cynops pyrrhogaster. Here, we show that this neurosteroid is also involved in enhancing the expression of courtship behavior. Intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone enhanced courtship behavior dose-dependently in the sexually undeveloped males that had been pretreated with prolactin and gonadotropin, which is known to bring the males to a sexually developed state. But, unlike the case in the locomotion activity, 7α-hydroxypregnenolone did not elicit the behavior in males receiving no prior injections of these hormones. ICV administration of ketoconazole, an inhibitor of the steroidogenic enzyme cytochrome P450s, suppressed the spontaneously occurring courtship behavior in sexually active males. Supplementation with 7α-hydroxypregnenolone reversed the effect of ketoconazole in these animals. It was also demonstrated that the effect of the neurosteroid on the courtship behavior was blocked by a dopamine D2-like, but not by a D1-like, receptor antagonist. These results indicate that endogenous 7α-hydroxypregnenolone enhances the expression of the male courtship behavior through a dopaminergic system mediated by a D2-like receptor in the brain.

Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways.

The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

Role of progestogens for the prevention of premature birth.

Premature birth represents a major cause of perinatal morbidity and mortality. The short- and long-term sequelae of prematurity have serious consequences for newborn survival and health in later life. In addition, prematurity is a major problem with regard to health expenditure. Despite major progress in obstetrics, perinatology and neonatology, the percentage of premature birth persists and there is even a tendency towards a slight increase. Therefore, besides screening programmes for the detection of vaginal infections, additional therapeutic opportunities must be sought. According to previously published data, vaginal progesterone and intramuscular 17alpha-hydroxyprogesterone caproate should be considered possible treatment options for the prevention of preterm delivery.

7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system.

It is becoming clear that steroids can be synthesized de novo by the brain and other nervous systems. Such steroids are called neurosteroids, and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. In this study, we show that the newt brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid that stimulates locomotor activity. 7alpha-hydroxypregnenolone was identified as a most abundant amphibian neurosteroid in the newt brain by using biochemical techniques combined with HPLC, TLC, and GC-MS analyses. The production of 7alpha-hydroxypregnenolone in the diencephalon and rhombencephalon was higher than that in the telencephalon and peripheral steroidogenic glands. In addition, 7alpha-hydroxypregnenolone synthesis in the brain showed marked changes during the annual breeding cycle, with a maximal level in the spring breeding period when locomotor activity of the newt increases. Behavioral analysis of newts in the nonbreeding period demonstrated that administration of this previously undescribed amphibian neurosteroid acutely increased locomotor activity. In vitro analysis further revealed that 7alpha-hydroxypregnenolone treatment resulted in a dose-dependent increase in the release of dopamine from cultured brain tissue of nonbreeding newts. The effect of this neurosteroid on locomotion also was abolished by dopamine D(2)-like receptor antagonists. These results indicate that 7alpha-hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts through the dopaminergic system. This study demonstrates a physiological function of 7alpha-hydroxypregnenolone that has not been described previously in any vertebrate class. This study also provides findings on the regulatory mechanism of locomotor activity from a unique standpoint.

Development of sensitivity to maturation-inducing steroids and gonadotropins in the oocytes of the tobinumeri-dragonet, Repomucenus beniteguri, Callionymidae (Teleostei).

The effectiveness of steroids and gonadotropins (GtHs) in inducing final oocyte maturation was examined at different times of the day (0100, 0500, 0700, 0900, 1100, 1300, 1700, 2100 hr) in a daily spawning marine teleost, the tobinumeri-dragonet, Repomucenus beniteguri. The responsiveness of oocytes to GtHs and steroids was different at various times of day. The sensitivity of oocytes to hormones was apparent only during a certain period of the day (0100-0900 hr). Germinal vesicle breakdown (GVBD) could be induced by GtHs but not by steroids at 0100 hr. At 0500 hr, not only GtHs but also steroids at higher doses induced GVBD and ovulation. Oocytes underwent GVBD and ovulation at low doses of steroids at 0900 hr. GVBD and ovulation spontaneously occurred at 1100 and 1300 hr without any hormonal treatment. In addition, diameter and histological changes of ovarian oocytes were investigated. Ovarian oocytes were smaller than 420 microns, and only one oocyte group existed in distribution of oocyte frequency at 2100 and 0100 hr. With the increase in size of oocytes, two distinct groups appeared at 0500 hr. Larger oocytes completed GVBD at 1100 hr. Ovulation occurred between 1300 and 1700 hr and oviposition was completed between 1700 and 2100 hr daily. These results clearly show that the oocyte of the dragonet possesses a daily maturation rhythm. Responsiveness of oocytes to GtHs appeared earlier than responsiveness to steroids. This suggests that sensitivity to steroids is induced by GtH.

Co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in primary cultures of bovine adrenocortical cells in response to ACTH treatment.

Bovine adrenocortical cells in primary culture were used to examine the trophic effect of ACTH on the induction of the 17 alpha-hydroxylase and C-17,20-lyase activities. The addition of exogenous pregnenolone to bovine adrenal microsomes showed the appearance of 17 alpha-hydroxy-pregnenolone before the formation of dehydroepiandrosterone. The same sequence of activities was evident in postmitochondrial supernate from bovine adrenocortical cells cultured 36 h in the presence of 1 microM ACTH but not in postmitochondrial supernate from control cells. In another study, bovine adrenocortical cells were cultured for 36 h after which 30 microM 17 alpha-hydroxypregnenolone was added to the medium and the incubation continued 1 h; there was a 4-fold increase in androgen content in the media from ACTH-treated cells over controls. Measurement of the 17 alpha-hydroxylase and C-17,20-lyase reactions in postmitochondrial supernate from cells cultured 0-72 h in the presence of ACTH or 1 mM dibutyryl cAMP showed concomitant increases in the two activities and both activities were inhibited by the same compounds known to inhibit 17 alpha-hydroxylase activity. These observations support the concept of the co-induction of 17 alpha-hydroxylase and C-17,20-lyase activities in response to ACTH; results in keeping with previous studies indicating that the two activities are catalyzed by a single gene product, the polypeptide chain P-45017a.

Type beta transforming growth factor is a potent modulator of differentiated adrenocortical cell functions.

Whereas TGF-beta exhibited no detectable effect on DNA synthesis, it was found to exert a striking inhibitory effect on the steroidogenic activities of bovine adrenocortical cells in culture. Basal, as well as ACTH- and angiotensin II- activated adrenocortical cortisol productions were inhibited in a time and dose-dependent manner following TGF-beta treatment. Half-maximum inhibition of ACTH- and AII-activated steroidogenesis was observed with TGF-beta concentrations of 0.40 and 0.12 ng/ml, respectively. This effect was half maximal after 6 hours of cell exposure to optimally effective TGF-beta concentrations (1 ng/ml) and reached a plateau after 12-15 hours, resulting in an average 60% inhibition in the steroidogenic response to ACTH and 90% in the case of AII. Supply of different exogenous steroid substrates to support steroidogenesis in adrenocortical cells pointed to a marked loss in steroid-17 alpha hydroxylase activity as a major alteration following TGF-beta treatment. TGF-beta thus appears as a potent modulator of differentiated adrenocortical cell functions in vitro; in this regard it may play a significant role in the development and the regulation of adrenal cortex in vivo.

Effect of adrenocorticotrophin on cortisol and androstenedione secretion from dispersed cells of guinea-pig adrenal zonae fasciculata and reticularis.

We have studied cortisol and androstenedione secretion by dispersed cells of the outer zona fasciculata (ZF) plus zona glomerulosa, and the inner zona reticularis (ZR) plus medulla of the guinea-pig adrenal. The ZF and ZR were microdissected apart, the cells dispersed and incubated (200 000 cells/ml) for 90 min in the presence of adrenocorticotrophin (ACTH; 500 ng/l), dibutyryl cyclic AMP (dbcAMP; 1 mmol/l), pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol. The steroid concentrations were 5-25 mumol/l. Cortisol secretion was assayed by radioimmunoassay. There was no detectable cortisol secretion (less than 50 nmol/l) from the ZR in the controls (no additive) or after dbcAMP stimulation. Adrenocorticotrophin-stimulated cortisol secretion was also low (range less than 50-340 nmol/l). In contrast the ZF secreted 177-379 (control), 828-2052 (dbcAMP) and 2863-9735 (ACTH) nmol cortisol/l. There was no detectable (i.e. less than 2 nmol/l) cAMP production by ZR or ZF either basally (no ACTH) or after ACTH stimulation (500 ng/l). Challenge of the ZR cells with each cortisol precursor steroid (5 mumol/l) increased (P less than 0.05) cortisol secretion over that seen with the corresponding basal and ACTH-stimulated controls. Thus pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol (converted directly to cortisol by 21-hydroxylase) gave rise to (mean +/- S.D., n = 4) 406 +/- 86, 680 +/- 180, 1307 +/- 111, 1141 +/- 234 and 3160 +/- 419 nmol cortisol/l respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Demonstration of testosterone secretion by testicular tissue of hypophysectomized boar as affected by HCG in organ culture]. / Mise en évidence d'une sécrétion de testostérone par le tissu testiculaire de Verrat hypophysectionisé, sous l'action de HCG en culture organotypique.

Boar Leydig cells undergo a strong atrophy from 1 to 3 months after hypophysectomy but can be reactivated by the gonadotropin HCG in organ culture conditions. This reactivation which appeared at histological and ultrastructural level was evidenced by the capacity of testicular tissue to synthesize testosterone as judged by radioimmunoassay. Both synthesis in the tissue and release into the medium increased according the incubation time with HCG; the adjonction of 17 alpha-OH-pregneolone to culture medium led to increase the intra and extra-tissular concentration of testosterone.

Origin of urinary 16 beta-hydroxydehydroepiandrosterone in essential hypertension.

The excretion rates and precursors of the 3-sulfate and glucuronide conjugates of 16 beta-hydroxydehydroepiandrosterone (16 beta-OH DHEA) were measured in normotensive controls and in patients with normal and low renin essential hypertension. The hypertensive subjects, and to the greatest degree those of the low renin subgroup, excreted increased amounts of 16 beta-OH DHEA sulfate and glucuronide and lesser amounts of DHEA sulfate and glucuronide than the controls. The major precursor of the urinary 16 beta-OH DHEA sulfate in the hypertensives was circulating DHEA sulfate, whereas the major precursors of 16 beta-OH DHEA glucuronide were DHEA, DHEA sulfate and 17-OH pregnenolone, as determined from their specific activities. Furthermore, both subgroups of hypertensives had similarly elevated DHEA and DHEA sulfate secretory rates compared to the controls. The stimulus to this increased peripheral conversion of circulating 17-OH pregnenolone, DHEA and DHEA sulfate into 16 beta-OH DHEA conjugates in essential hypertension, especially of the low renin type, is unknown.

5-ene-3beta-hydroxysteroid dehydrogenase of human and bovine adrenocortical endoplasmic reticulum: solubilization and fractionation.

Protein moieties of various molecular sizes and possessing 5-ene-3beta-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118 000, with an isoelectric point between 7-2 and 7-4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17alpha-hydroxy-pregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrated specificity.

Pathways of testosterone synthesis in decapsulated testes of mice.

In order to study the temporal relations in the biogenesis of testosterone, decapsulated testes of adult mice were incubated with carbon-14-labelled sodium acetate and attempts were made to isolate the most likely intermediates. Considerable quantities of radiochemically homogeneous squalene, lanosterol, cholesterol, testosterone and androstenedione, but no pregnenolone, progesterone, 17-hydroxypregnenoline, 17-hydroxyprogesterone, dehydroepiandrosterone, pregnenolone sulphate or dehydroepiandrosterone sulphate were isolated. The same pattern of incorporation was found when gradually increasing amounts of non-labelled pregnenolone, progesterone, 17-hydroxypregenenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate or testosterone were added to the system as "trapping agents" or when Leydig cell preparations rather than decapsulated testes were used. The presence of 10 mIU of HCG greatly enhanced the de novo formation of testosterone, androstenedione and 5alpha-dihydrotestosterone but did not change the pattern of acetate incorporation. Radioimmunoassays of the incubation medium with or without added HCG, and carried out at different periods of time indicated the presence of gradually increasing amounts of testosterone and androstenedione together with some 5alpha-dihydrotestosterone, whereas only trace amounts of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and dehydroepiandrosterone were present. An analysis of the incubated testes revealed that the addition of HCG significantly enhanced the content of testosterone, androstenedione and 5alpha-dihydrotestosterone. Little or no increase was observed as far as pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone or dehydroepiandrosterone were concerned. It is concluded that decapsulated testes of mice synthesize de novo testosterone from sodium acetate under conditions in which the formation of pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnenolone sulphate and 17-hydroxypregnenolone sulphate cannot be demonstrated.

Maintenance of rate testis fluid testosterone and dihydrotestosterone levels by pregnenolone and other C21 steroids in hypophysectomized rats.

This study was undertaken to determine whether maintenance of spermatogenesis in hypophysectomized rats by pregnenolone and other C21 steroids may be due to in vivo conversion of these compounds to androgens. Hypophysectomized rats were treated sc with 2 mg of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone or testosterone propionate in 0.2 ml sesame oil daily for 14 days beginning 2 days after hypophysectomy. Rete testis fluid (RTF), peripheral blood, and testicular venous blood were collected on the day of the last injection. Testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay after chromatographic separation. Results demonstrate that T and DHT could be found in the RTF of C21 steroid-treated hypophysectomized rats at levels similar to those seen in the intact rat. Results imply that the maintenance of spermatogenesis by C21 steroids is probably due to the conversion of these compounds to T in the testis. Relatively little T was released from the testis into the peripheral circulation of these rats since T levels in testicular venous plasma were low and peripheral plasma T levels were not distinguishable from those seen in untreated hypophysectomized rats. Histological examination of the testes of C21 steroid-treated hypophysectomized rats showed nearly quantitative maintence of spermatogenesis and atrophy of Leydig cells. These findings suggest that most of the conversion of C21 steroids to androgens occurred in the seminiferous tubules.