Intercellular communication in rat seminiferous tubules

Intercellular electrical coupling in seminiferous tubules from prepubescent and adult Wistar rats has been studied by using conventional techniques. It is found that cells in the seminiferous epithelium are electrically coupled. Experiments performed using "Sertoli cell-enriched" seminiferous tubules indicate the existence of intercellular ionic communication between Sertoli cells. Junctional conductance is independent of the direction of electrical field and it is affected by A23187 Ca ionophore (5 microM) but not by exposure to the neurotransmitter norepinephrine (1-5 X 10(-5) M). Intracellular resistivity (including junctional resistance) is higher in mature as compared to immature germinal epithelium. These findings suggest that cell metabolites or second messenger molecules could be transferred via the low-resistance pathways between epithelium cells to coordinate cellular activity.     

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

Comparative ability of rat seminiferous tubules, interstitium, and whole testes to utilize cholesterol-1,5-3H as a substrate for testosterone synthesis and the intratesticular distribution of cholesterol side-chain cleavage enzyme.

Homogenates of rat seminiferous tubules, interstitium and intact testis tissues were assessed for their ability to convert cholesterol -1,2-3H to testosterone in vitro. While 3H-testosterone synthesis was observed in incubates of interstitial and whole testis homogenates, no synthesis was detectable in homogenates of seminiferous tubules. To determine whether cholesterol side-chain cleavage enzyme (CSCCE) was deficient or absent in tubules, mitochondria from tubules, interstitium and whole testes were analyzed for CSCCE activity by measuring conversion of cholesterol -26-14C to 14C-isocaproate (+pregnenolone). Interstitial mitochondrial preparations from each of six testes were found to be approximately 200 times more active in CSCCE than the corresponding tubule mitochondria, and 1600-1800 times more active on a specific activity basis. Although caution is required in extrapolation of in vitro data to the in vivo state, these findings suggest rat seminiferous tubules may be incapable of de novo testosterone biosynthesis and that this lack of synthetic ability may be due to a deficiency of CSCCE.     

The luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, has no direct effect on spermatogenesis in the rat

Treatment of intact rats with luteinizing hormone-releasing hormone (LHRH) agonists has been shown to produce atrophy of a variable number of testicular seminiferous tubules. These findings raised the question of a possible direct versus indirect action of LHRH agonists on spermatogenesis. To answer this question, we treated hypophysectomized rats with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, dihydrotestosterone (DHT), or a combination of these two compounds for a period of 1 mo. Treatment of hypophysectomized animals with the LHRH agonist alone had no significant effect on the atrophy of seminiferous tubules found after hypophysectomy. DHT, however, maintained spermatogenesis at 80% of the level seen in intact animals. When DHT and the LHRH agonist were administered in combination, the stimulatory effects of DHT were observed with no significant interference caused by the LHRH agonist. This study shows that an LHRH agonist has no direct effect on the morphology of the seminiferous tubules in the absence of the pituitary gland and strongly suggests that the atrophy observed in the testis after LHRH agonist treatment in intact animals is mediated by the LHRH agonist-induced changes in luteinizing hormone secretion and/or direct action of the peptide on Leydig cells.     

Testosterone micromilieu in staged rat seminiferous tubules

Endogenous testosterone concentrations in rat seminiferous tubules were measured in relation to different stages of the cycle of the seminiferous epithelium. For this purpose, the seminiferous tubules were mechanically separated from the interstitial tissue on a cooled (1 degree C) petri dish under a stereomicroscope without added medium. After recognition of the stages of the cycle by transillumination, the specimens were rapidly transferred by dry forceps into test tubes for testosterone radioimmunoassay. The results of the dry dissection method were compared with measurements on tubules that were kept after separation in phosphate buffered saline (PBS, pH 7.4), in order to reveal the possible leakage of testosterone from the tubules. The maximal concentration of testosterone per unit length of seminiferous tubule was found in stages VII and VIII of the cycle (288 +/- 60 fmol/cm, mean +/- SEM, n = 12), and the minimal in stages IX-XII (219 +/- 57 fmol/cm, P less than 0.01). If the levels were correlated with unit volumes of the seminiferous tubules, identical concentrations of testosterone (521-542 fmol/mm3, approx. 500 nmol/l) were found in the different stages of the cycle. Despite the similarity of testosterone concentrations in the different parts of the seminiferous tubules the local concentrations of biologically active (i.e. free) testosterone may be modulated by extracellular and intracellular androgen binding components.     

Contractility of rat testicular seminiferous tubules in vitro: prostaglandin f 1 alpha and indomethacin-1,2.

The frequency of spontaneous in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dose-dependent manner by prostaglandin F1alpha. PGF1alpha treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGF1alpha (10(-7) M). Preinjecting ther rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. in vitro was more effective than pretreatment in vivo in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGF1alpha restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the in vitro contractility of the tubules.     

Isolation and staging of horse seminiferous tubules by transillumination

Stages of the spermatogenic cycle in the horse were determined by trans-illumination of enzymically isolated, seminiferous tubules and were verified by whole-mounted tubules observed by Nomarski optics and by conventional histology. Isolated tubules were obtained from young (less than 2 years) and adult (4-10 years) horses by enzymic digestion. Dispersed tubules were separated into three different groups based on the presence, size, and intensity of a dark region in the centre of the tubules: (1) pale--homogeneously light, (2) spotty--light on the periphery with a wide spotty region in the central two-thirds, or (3) dark--an intensely dark, narrow region through the central one-third. Seminiferous tubules from young stallions separated easily, but were only of the homogeneously light pattern as they lacked mature spermatids. After observation by Nomarski optics and bright-field microscopy, pale tubules under transillumination largely contained Stages I and II, spotty tubules contained Stages V and VI, and dark tubules contained Stages VII and VIII of the spermatogenic cycle. In-vitro incorporation of [3H]thymidine in spermatogonia and preleptotene/leptotene primary spermatocytes of these tubules confirmed the viability of germ cells in isolated tubules, and ultrastructural analysis confirmed excellent preservation of normal structure of seminiferous epithelium in isolated tubules. Hence, segments of seminiferous tubules in specific stages of the spermatogenic cycle can be obtained from enzymically digested horse testes when viewed by transillumination.     

Plasma prolactin levels in maturing intact and cryptorchid male rats: development of stress response.

In order to determine the possible role of the seminiferous tubules in the regulation of prolactin secretion during sexual development, male rats were rendered cryptorchid at 22 days of age, and thereafter different groups of animals were decapitated at 8-10 day intervals between Day 32 and Day 70. Cryptorchid rats showed destruction of the germinal epithelium accompanied by increased plasma FSH and, to a much lesser extent, increased plasma LH titers. Nevertheless, plasma prolactin levels were similar to those of intact controls throughout the entire period studied. Plasma prolactin titers in intact controls remained uniformly low from Day 20 to Day 70, contrasting with previous reports in which increasing prolactin levels have been observed during sexual development. To determine the reason for this apparent discrepancy, a longitudinal experiment was conducted in which intact and cryptorchid male rats were bled every 10 days from Day 30 to Day 70, following a 3-min period of exposure to ether fumes. The prolactin response to this stress increased markedly with age. A similar pattern of prolactin was observed in a cross-sectional study in which different groups of intact animals were bled following a 3-min period of ether exposure, at ages ranging from 20 to 70 daysmthe results indicate that unlike FSH secretion, prolactin secretion is not controlled by the seminiferous tubules. In addition, they suggest that the pattern of increasing plasma prolactin previously described in the developing male rat is at least in part caused by an age-dependent increase in responsiveness of prolactin to stress.     

Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes.

Recently a system was developed in which transplanted donor spermatogonial stem cells establish complete spermatogenesis in the testes of an infertile recipient. To obtain insight into stem cell activity and the behavior of donor germ cells, the pattern and kinetics of mouse spermatogonial colonization in recipient seminiferous tubules were analyzed during the 4 mo following transplantation. The colonization process can be divided into three continuous phases. First, during the initial week, transplanted cells were randomly distributed throughout the tubules, and a small number reached the basement membrane. Second, from 1 wk to 1 mo, donor cells on the basement membrane divided and formed a monolayer network. Third, beginning at about 1 mo and continuing throughout the observation period, cells in the center of the network differentiated extensively and established a colony of spermatogenesis, which expanded laterally by repeating phase two and then three. An average of 19 donor cell-derived colonies developed from 10(6) cells transplanted to the seminiferous tubules of a recipient testis; the number of colonized sites did not change between 1 and 4 mo. However, the length of the colonies increased from 0.73 to 5.78 mm between 1 and 4 mo. These experiments establish the feasibility of studying in a systematic and quantitative manner the pattern and kinetics of the colonization process. Using spermatogonial transplantation as a functional assay, it should be possible to assess the effects of various treatments on stem cells and on recipient seminiferous tubules to provide unique insight into the process of spermatogenesis.     

Metaphase-specific cell death in meiotic spermatocytes in mice

Apoptosis of male germ cells is a widespread but little-understood phenomenon in many animal species. The elucidation of its mechanisms could be useful in the understanding of male infertility. We have examined the distribution of dying cells with the terminal transferase-mediated nick-end labeling (TUNEL) method and by an electron-microscopic procedure in the testes of 10 mouse strains, viz., C57BL/10 (B10), SL/NiA (SL), C57BL/6 (B6), C3H/He (C3H), BALB/c (BALB), DBA2 (DBA), CBA/J (CBA), MRL/MpJ(-)+/+ (M+), MRL/MpJ-lpr/lpr (lpr), and wild-type NJL mice (Mus musculus musculus). In the testes of the B10, NJL, SL, B6, C3H, BALB, DBA, and CBA mice, very few TUNEL-positive cells are distributed in the seminiferous tubules, whereas in the testes of the M+ and lpr mice, many TUNEL-positive cells, which are restricted to stage XII seminiferous tubules, have been identified. The most important finding is that many metaphases of meiotic spermatocytes show a marked TUNEL-positive reaction. Some metaphases show apoptotic morphology electron-microscopically. These results suggest that the testes of MRL strains will provide a useful model for the study of the mechanism of metaphase-specific apoptosis in meiotic spermatocytes.     

Effects of cholinergic and adrenergic drugs on intraluminal pressures and contractility of the rat testis and epididymis in vivo

Intravenous administration of methacholine (200 micrograms/kg) caused no changes in the seminiferous tubules of rats, but significantly increased intraluminal pressures and contractility of the caput, the corpus and the cauda epididymidis. The effect of methacholine was abolished by pretreatment with atropine (500 micrograms/kg), but not by phentolamine (400 micrograms/kg) or propranolol (400 micrograms/kg). Adrenaline (5-40 micrograms/kg), noradrenaline (5-40 micrograms/kg) and phenylephrine (100-400 micrograms/kg) had no effect on the seminiferous tubules, but dose-dependently elevated intraluminal pressures and enhanced the contractility of all regions of the epididymis. Isoproterenol (100-800 micrograms/kg) did not affect intraluminal pressures of the seminiferous tubules and the epididymal duct. The stimulatory effect of adrenergic agonists was specifically blocked by phentolamine, but not by propranolol or atropine. Cholinergic and adrenergic antagonists did not alter spontaneous contraction of the epididymis. The results suggest that the contractility of all segments of the rat epididymis, but not the seminiferous tubules, can be increased by autonomic drugs. The enhancing effect of adrenergic drugs is probably the result of activation through alpha-adrenergic receptors.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105, 000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1, 2-³h]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M ? 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1, 2-α-methylene-6-chloro-pregn-4, 6-diene-17α-o1–3, 20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Development and application of a system for seminolipid metabolism using mouse seminiferous tubules

A convenient tool for studying metabolism of seminolipid in testis was developed by using mouse isolated seminiferous tubules prepared by collagenase treatment. Because more than 99% of [³⁵S]sulfate-incorporation was distributed in seminolipid, its metabolism in seminiferous tubules can be analyzed without disturbance of the other sulfolipids in this assay system. Furthermore, the contents of seminolipid and its precursor, galactosylalkylacylglycerol, which were determined by liquid chromatography-electrospray ionization mass spectrometry, did not change within a few hours, indicating that the incorporations of [³⁵S]sulfate into seminolipid solely reflects the turnover rate of this sulfolipid. As an initial application of this system, we characterized heat-susceptibility of the seminolipid turnover rate in mouse seminiferous tubules. Severe heating (44°C for 10 min) of the isolated seminiferous tubules suppressed the ³⁵S-incorporation into seminolipid to 47% of heating at scrotal temperature (32°C for 70 min). In contrast, pretreatment of the testis in vivo under the same condition (44°C for 10 min) did not decrease the seminolipid turnover rate in the isolated seminiferous tubules. In addition, the activity of galactocerebroside sulfotransferase decreased in the temperature-dependent manner in seminiferous tubules as well as crude tubular homogenates, where the activity is significantly more stable in the former than the latter. The newly developed system could provide useful basic data for further analyses of seminolipid metabolism in the testis.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105,000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1,2-³H]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M — 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1,2-α-methylene-6-chloro-pregn-4, 6-diene-17α-ol-3,20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Transferrin receptors of isolated rat seminiferous tubules bind both rat and human transferrin

The binding and uptake of rat and human transferrin by isolated rat seminiferous tubules was studied. During the isolation and incubation of the tubules, the blood-testis barrier remained intact. Iron-saturated and iron-free (apo-) transferrin use the same binding sites on the surface of the tubules, but the dissociation constant is about two times higher for apotransferrin than for iron-saturated transferrin. The affinity of the receptors is equal for rat and human transferrin, but human transferrin binds to more surface binding sites (2.6 X 10(10) per 10 cm tubule length) than rat transferrin (1.1 X 10(10) per 10 cm tubule length) at 0 degrees C. At 33 degrees C equal numbers of human and rat transferrin molecules are taken up (about 8 X 10(10)) per 10 cm tubule length. The quantitative difference between 0 degrees C and 33 degrees C is caused by the fact that at 33 degrees C receptor-mediated endocytosis and recycling occur. As a consequence, both surface and intracellular transferrin receptors are detected at 33 degrees C. The dissociation constants are not temperature-dependent.     

Role of testosterone in the early stage of spermatocytogenesis in rats.

The role of testosterone in the early stage of spermatocytogenesis was investigated in newborn rats. The testes of rats, either 0 or 6 days of age, were implanted into those of hypophysectomized adult rats that had or had not been injected with testosterone propionate (TP) after hypophysectomy and also into those in intact adult rats. All the animals were autopsied 17 or 11 days later when the implanted testes reached 17 days of age. The implanted testes were examined for cellular components in the seminiferous tubules. In an additional experiment, newborn rats were injected with TP or cyproterone acetate, an antagonistic substance against androgen, daily for the first 17 days of life and examined for testes. Proliferation of supporting cells and development of seminiferous tubules were less remarkable in the testes of newborn rats which had been implanted into the testes of hypophysectomized rats than in those which had been implanted into the testes of intact adult rats. Proliferation of supporting cells was not stimulated by TP, but development of seminiferous tubules was slightly promoted. Progress in spermatocytogenesis from gonocytes to pachytene primary spermatocytes was observed in the testes of newborn rats which had been implanted into the testes of hypophysectiomized rats. It was not so marked after injection with TP. These results suggested that testosterone might have stimulated development of seminiferous tubules and maturation of spermatocytes in the early stage of spermatocytogenesis by its synergistic action with a gonadotropin, possible follicle-stimulating hormone.     

The influence of unilateral castration on testicular morphology and function in adult rams

An experiment was designed to investigate the mechanisms controlling testicular compensatory hypertrophy in rams. Endocrine and histological events were examined, with special attention to Sertoli cell hyperplasia and hypertrophy as contributing factors to the compensatory process. Fifteen sexually mature yearling Targhee rams were allotted to intact control (C, n = 5) and unilateral castrate (UC, n = 10) treatment groups in June. Approximately 150 days after UC, testicular tissue was collected in November after efferent duct cannulation and rete testis fluid (RTF) collection or perfusion fixation. Unilateral castration increased mean testis weight by 56% (p = 0.01) and mean epididymal weight by 15% (p = 0.05). Although the mean volume of RTF collected more than doubled after UC (1.55 +/- 0.86 vs. 0.63 +/- 0.10 ml for UC and C rams, respectively), the difference was not statistically significant. By 150 days after UC, the concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) in jugular venous blood did not differ between the two treatment groups. The concentrations of T. dihydrotestosterone (DHT), and androgen-binding protein (ABP) in RTF were also similar for UC and C rams. However, since the observed mean RTF volume was increased, the amounts of T, DHT, and ABP exiting the testes of these UC rams via the RTF were approximately doubled, although this difference was not statistically significant. UC increased the mean diameter of seminiferous tubules by 21% (p less than 0.01) and of their lumina by 51% (p less than 0.01), but did not significantly increase mean height of seminiferous epithelium or estimated length of seminiferous tubules per testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of transferrin endocytosis and iron uptake by intact isolated rat seminiferous tubules and Sertoli cells in culture

The receptor-mediated endocytotic cycle of rat and human transferrin has been studied in intact, isolated rat seminiferous tubules and Sertoli cells in culture. Double-labeled [( 59Fe125I]) transferrin has been used to study the fate of transferrin and iron. Diferric transferrin binds to the tubules and the cultured Sertoli cells and is internalized. The iron remains inside, while the transferrin recycles and is released into the medium. Although, as reported before (Wauben-Penris et al., 1986), "extra" binding sites for human transferrin exist as compared to rat transferrin, this does not result in extra uptake of transferrin or iron. Both rat and human transferrin transport iron into the cells and recycle back to the surface, and do so with identical kinetics. A striking difference has been found between the mean efficient recycling times of the transferrin receptors in intact tubules (90 min) and in Sertoli cells in culture (21 min). Possible explanations of this difference are discussed. Light-microscopic autoradiography of [125 I]-labeled transferrin has revealed that the transferrin protein is excluded from the adluminal compartment, even after 21 h of incubation. This indicates that externally added transferrin itself does not deliver iron to the postmeiotic germ cells in intact, isolated rat seminiferous tubules.     

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.