AZFa protein DDX3Y is differentially expressed in human male germ cells during development and in testicular tumours: new evidence for phenotypic plasticity of germ cells.

BACKGROUND: DDX3Y (DBY), located within AZoospermia Factor a (AZFa) region of the human Y chromosome (Yq11), encodes a conserved DEAD-box RNA helicase expressed only in germ cells and with a putative function at G1-S phase of the cell cycle. Deletion of AZFa results most often in germ cell aplasia, i.e. Sertoli-cell-only syndrome. To investigate the function of DDX3Y during human spermatogenesis, we examined its expression during development and maturation of the testis and in several types of testicular germ cell tumours (TGCTs), including the pre-invasive carcinoma in situ (CIS) precursor cells which are believed to originate from fetal gonocytes. METHODS: DDX3Y protein expression was analysed during development in different tissues by western blotting. The localization of DDX3Y in normal fetal and prepubertal testis tissue of different ages as well as in a series of distinct TGCT tissue samples (CIS, classical seminoma, spermatocytic seminoma, teratoma and embryonal carcinoma) was performed by immunohistochemistry. RESULTS: Germ cell-specific expression of DDX3Y protein was revealed in fetal prospermatogonia but not in gonocytes and not before the 17th gestational week. After birth, DDX3Y was expressed at first only in the nuclei of Ap spermatogonia, then also in the cytoplasm similarly to that seen after puberty. In CIS cells, DDX3Y was highly expressed and located predominantly in the nuclei. In invasive TGCT, significant DDX3Y expression was found in seminomas of the classical and spermatocytic type, but not in somatically differentiated non-seminomas, consistent with its germ-cell specific function. CONCLUSIONS: The fetal germ cell DDX3Y expression suggests a role in early spermatogonial proliferation and implies that, in men with AZFa deletion, germ cell depletion may begin prenatally. The strong expression of DDX3Y in CIS cells, but not in gonocytes, indicates phenotypic plasticity of CIS cells and suggests partial maturation to spermatogonia, likely due to their postpubertal microenvironment.

Enzyme histochemical studies on the pathological changes in human Sertoli cells.

Two forms of human Sertoli cell disorders were characterized enzyme histochemically from the testicular biopsy material of infertile and subfertile patients. Sertoli cell asthenia: a slight injury of the Sertoli cell with exfoliation of individual germ cells; marked by the rarefaction of reaction zones of thiamine pyrophosphatase (TPPase) and a decrease in lactate dehydrogenase (LDH). Sertoli cell insufficiency: severe Sertoli cell damage with the formation of a "puff" and a heavy exfoliation of germ cells (dislocation of Sertoli cell nucleus and cytoplasm along with the related germ cells into the lumen of seminiferous tubule); marked by a heterogeneous activity pattern of TPPase, the disappearance of LDH, maintenance of a slightly weakened activity of alkaline phosphatase, and an increase of acid phosphatase. In the case of Sertoli-cell-only syndrome, the high prismatic Sertoli cells showed strong acid phosphatase activity with scattered weak TPPase reaction, whereas the flat or cube-like Sertoli cells exhibited weak acid phosphatase activity with only one small round reaction zone of TPPase in each cell. In addition, the frequency of the occurrence of Sertoli cell asthenia, Sertoli cell insufficiency, and Sertoli-cell-only syndrome is reported, and its correlation with the andrological diseases discussed.

Pulmonary alveolar proteinosis in rats after administration of quartz: its possible role in morphogenesis of lung cancer.

In rats, primary peripheral lung tumors composed predominantly of alveolar type II cells have been induced by inhalation of alpha quartz. In our retrospective study on proliferation markers we evaluated lung specimens of 140 Wistar rats from larger experiments, which had been exposed to Dörentrup quartz (DQ12) by inhalation (10 mg/m3, 56 Weeks, 5 days/week, 7 h/day: n = 27) or intratracheal instillations (5 mg: n = 38; 20 mg: n = 10; 50 mg: n = 28; 15 x 3 mg: n = 12). In the last group 8/12 animals developed lung tumors. Animals were sacrificed 1-32 months after administration. For identification of an increased proliferation of alveolar type II cells the DNA content was monitored by microscopic (static) cytophotometry in histological slides. The argyrophil (AgNOR) method for the demonstration of nucleolar organizer regions (NOR) was used as second marker of type II cell proliferation. Measurements made 24 months after inhalation of DQ12 showed a slight increase of pneumocytic proliferation with 1.64 +/- 0.14 AgNOR/nucleus compared to the controls (1.23 +/- 0.04 mean AgNOR/nucleus). After intratracheal instillation of DQ12 a significant increase of AgNOR was found, e.g. 5 mg: 1.93 +/- 0.23 AgNOR/nucleus (6 months) and 1.96 +/- 0.19 (12 months); 50 mg: 1.77 +/- 15 (6 months) and 2.18 +/- 0.05 (12 months); 15 x 3 mg (+2 ml 2% polyvinylpyridine N-oxide s.c.): 1.81 +/- 0.13 AgNOR/nucleus (27-32 months). With the aid of the 2 c deviation index, i.e. the mean square deviation from the diploid DNA value, it was possible also to identify the pathologically increased proliferation of type II cells after intratracheal instillation of quartz: 0.02 +/- 0.01-0.06 +/- 0.04 c2 (controls); 0.07 +/- 0.04 c2 (5 mg/12 months); 0.12 +/- 0.08 c2 (15 x 3 mg/>27 months) and 0.68 +/- 0.48 c2 (50 mg/12 months). Only in the last group were nearly triploid values detected. Summarizing our results, intratracheal instillation and inhalation of quartz in rats regularly induces alveolar proteinosis and interstitial fibrosis in combination with a dose- and time-dependent increase of the type II cell proliferation rate. As mitogenesis increases carcinogenesis, alveolar proteinosis with increased pneumocytic proliferative activity might be a prerequisite for enhanced tumor development.

Germ cell kinetics during early ovarian differentiation: an analysis of the oogonial cell cycle and the subsequent changes in oocyte development during the onset of meiosis in the rat.

The aim of this study was the comparison between the mitoses of oogonia and the initial stages of oocyte meiosis. The structural alterations that the germ cell chromatin undergoes during the oogonial mitosis have been compared with those occurring during the G1- and S-phase just before meiosis. Using plastic embedded 1-microm sections of fetal rat ovaries (embryonic days = ED 14-20) labeled with 3H-thymidine and re-embedded for electron microscopy, a study of the structural conditions of the nuclear chromatin has been combined with a kinetic analysis of the oogonial cell cycle and the transitional period into the meiotic prophase. After ovarian differentiation (ED 14) the oogonia show a non-clonal, but strong proliferation. On ED 16, proliferation changes to a clonal pattern and decreases during ED 17. A final increase in 3H-thymidine incorporation on ED 18 characterizes the meiotic S-phase. On ED 19 the nuclear labeling drops to zero. The mitotic cycle of the oogonia lasts 16.5 hr and can be divided into 11 stages according to the concept of El-Alfy and Leblond [(1988) Am. J. Anat., 183:45-56] on the basis of the chromatin pattern. The S-phase (10.0 hours) extends from the telophase-interphase transition through the interphase to early prophase. The postmitotic G1- and S-phases show a more extensive duration, respectively 10 and 11.5 hours, and differ from their oogonial counterparts by the spherical shape of the nuclei from the very beginning. The chromatin pattern is similar until the end of the S-phase and lacks any prophase-like, preleptotenal chromatin condensation before the oocytes exhibit (pre-) leptotenal structures. Once the germ cell has completed a sequence of clonal mitotic divisions, it irrevocably progresses into meiosis. During an extended postmitotic period, the structural characteristics of meiosis emerge stepwise.

Engineering enzymes for clinical diagnosis.

The enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3.) is a major limiting factor in the enzymatic creatinine determination because of its comparatively poor catalytic activity and stability in the native form. The gene from Pseudomonas putida coding for creatinase was cloned and used for overexpression of the protein in E coli and Pseudomonas. In addition, it was possible by means of 'random' mutagenesis in vivo and subsequent screening using an activity plate assay to isolate creatinase derivatives that are more stable towards detergents and elevated temperature under test kit conditions. This example shows that enzymes can be optimized for use in given assay conditions by mutagenesis of cloned DNA and suitable screening methods.

Some remarks on the female and male Keimbahn in the light of evolution and history.

From the existence of two types of cells for reproduction-the female and male germ cells (GCs)-and by recombination of the genome, evolution proceeded dramatically. Unicellular and multicellular plants frequently are characterized by a sequence of haploid and diploid phases, or generations, with gametes and spores as reproductive cells. Isogamy, anisogamy, and oogamy can be distinguished depending on the GCs that correspond, differ in size, or impose as egg cell and sperm cell. In protozoans, too, species are found in which GCs differ clearly from each other. In the female lineage of angiosperms, a "Keimbahn chain" consisting of five successive germ line cells can be observed. Oogenesis and spermatogenesis are complete in coelenterates and similar in mammals. However, the controlling mechanisms are by far more complex in the latter. This means that the balance of hormonal and vegetative nervous influences (stimulation, inhibition) on gametogenesis is not primarily orientated on the germ line cells themselves, but mostly on the structural and functional situation of the gonads and the individual carriers. This becomes particularly evident in insects, where gametogenesis, on the one side, depends on the development of the rest of the organism but on the other side represents an independent developmental process. The point at which germ line cells and somatic cells separate correlates more or less with the degree of phylogenetic development. In worms, insects, and up to the anurans, a part of the cytoplasm, the so-called germ plasma, is separated for the development of GCs during oogenesis (preformistic development). However, in urodeles, reptiles, birds, and mammals, GCs and somatic cells cannot be distinguished before gastrulation (epigenetic development). In various species (e.g., in some oligochaetes and snails), there exist "double spermatogenic lines." In mammals (probably in other vertebrates and perhaps in various phyla of animals, too), the female Keimbahn is provided with only one proliferation system. The male gametogenesis is equipped with two systems: the first corresponds to the female germ line, the second is responsible for the immense number of gametes produced in the mature testes. In mammals the message to become male lies on the Y-chromosome (on its short arm in man and mouse) and was identified as the gene SRY in human and Sry in mouse. The fertility genes that are responsible for an uninterrupted spermatogenesis, up to fertilizing spermatozoa, are sitting on the long arm of the human Y-chromosome. J. Exp. Zool. (Mol. Dev. Evol.) 285:197-214, 1999.

["Environment and fertility". A contribution to the sensitivity of male germ cells to environmental pollutants]. / "Umwelt und Fertilität". Ein Beitrag zur Sensibilität der männlichen Keimbahn gegenüber Umweltnoxen.

If "early cells of the germ line" or stem cells of a man or other male mammals are irreversibly damaged by noxious agents, such as ionizing rays, cytostatic drugs or others, you will find Sertoli-cells-only-syndrome. In contrast, the loss of differentiating spermatogonia, spermatocytes, or spermatids is a temporal phenomenon, as long as the systems of the somatic cells in the testis are intact. Irreversible damage of these cell systems, however - e.g. Leydig cell system and vascular system which are both situated in the extratubular compartment, the boundary tissue, or the Sertoli cells - results in permanent infertility just as in case of irreversible damage of cells of the "early germ line" or stem cells. On the other hand, a temporary damage of the somatic cell systems in the testis brought about by irritations of the extra- or intratesticular control systems may be neutralized by cure of the impairment. Mutations of the genetic matter caused by exogenous agents in the cells of the germ line are of limited importance to the fertility of the individual but have considerable significance for the genetic state of the population as a whole. Genetic damage of "early cells of the germ line" or stem cells is of higher consequence than negative alteration of the genome in cells belonging to the differentiating part of the germ line. On the other hand, the sensitivity of the genome is especially pronounced in spermatocytes and spermatids, the most advanced cells of the male germ line. The sensitivity of the cell (CS) and the sensitivity of the genome (GMS) frequently do not coincide.

The genetic control and germ cell kinetics of the female and male germ line in mammals including man.

The female germ line (germ cell lineage, Keimbahn) is provided with only one proliferation wave, the oogenic, whereas male gametogenesis involves two successive waves: prespermatogenic, which corresponds to the female proliferation wave, and spermatogenesis, which is responsible for the immense number of male gametes produced in mature testes. Both male proliferation systems are linked by the transitional or T prospermatogonia. Using the reverse percentage of labelled metaphases method, it has been shown that the first differences between female and male germ cells can be identified by the end of the first wave, when oogonia and multiplying or M prospermatogonia are proliferating. This prenatal first wave of proliferation of male germ cells was also demonstrated in man and ceases around the 22nd week of pregnancy. Spermatogenesis involves a stock of stem cells (stem spermatogonia), a flexibly reacting pool of undifferentiated spermatogonia and several generations of differentiating spermatogonia, which proliferate almost exponentially. Furthermore, it consists of spermatocytes and haploid spermatids transforming into spermatozoa. The oocytes pass through the preleptotene stage, synthesizing DNA, and thereafter traverse the meiotic prophase up to the diplotene stage. In mammals they act as 'pre-embryos' in a similar but to a lesser degree than oocytes of amphibia and insects. The maternal chromosomes are largely responsible for the development of the embryo, the paternal genome for the development of the extra-embryonic tissue. The synthesis of transgenic animals is a powerful weapon in the armoury of geneticists, as has recently been demonstrated: a 14 kb genomic DNA fragment (Sry) is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.

Cell progression and radiosensitivity of T1-prospermatogonia in Wistar rats.

T1-prospermatogonia pass through a quiescent stage which lasts from before birth until day 4 after birth (p.n.). They progress into DNA synthesis and mitosis in two synchronous waves which are separated by 24 hours in the evenings of day 4 and 5. The first wave contains about 25 per cent of the total population, the second 75 per cent. The mean duration of S-phase is 10 hours, the mean duration of G2-phase is 4 hours. After irradiation, the capacity of T1-prospermatogonia to produce the normal number of proliferating and differentiating cells in the testes is reduced. During maturation, between day 21 post-conception (p.c.) and day 5 p.n. the radiosensitivity of T1-prospermatogonia decreases by a factor of over 5.

Kinetics of the male gametogenesis.

The kinetics of the male gametogenesis during the pregonadal period, prespermatogenesis, and "early" spermatogenesis has been described in detail. Concerning spermatogenesis in the adult individual reference is made to the articles of Courot, Hochereau-de Reviers and Ortavant (1970) and of Clermont (1972). The comparison of female and male gametogenesis (Fig. 1) shows that the "gonia stage" (asterisks) of the female germ cells is limited to one proliferation wave only, whereas the "gonia stage" of the male germ cells consists of a first proliferation wave, comparable to that of oogonia, a preparative phase to initiate spermatogenesis, and a second proliferation wave with renewal and differentiation of the spermatogonia. Germ cells in the "gonia stage" are highly sensitive towards ionising radiation and cytostatic drugs.

Details of the female and male pathway of the Keimbahn determined by enzyme histochemical and autoradiographic studies.

Autoradiographic studies and the use of enzyme histochemistry have revealed that early germ line cells (female and male PGC, oogonia, prediplotene oocytes and prospermatogonia) as well as the more advanced germ cells (diplotene oocytes, spermatogonia, spermatocytes and spermatids) show specific patterns of their DNA and RNA synthesis and their enzymatic equipment. The female and male germ lines show similar kinetics up to the arise of oocytes and T prospermatogonia (T for transitional), the final products of a first limited multiplication process of primitive gonia. In former studies we supposed that oocytes and T prospermatogonia are the first exponents of the female and male pathway of the germ line (Hilscher and Hilscher, 1989a). Recently, it could be shown--using the reverse PLM method in slides of plastic embedded material--that the first differences between female and male GC can already be stated at the end of the first proliferation wave of oogonia and multiplying prospermatogonia; that means even before the existence of oocytes and T prospermatogonia (Hilscher and Hilscher, 1989b). Oogonia and M prospermatogonia (M for multiplying) are equipped both with only one active X chromosome. While oocytes traverse the prediplotene stages of meiotic prophase T prospermatogonia prepare for a second extensive proliferation process: spermatogenesis. Oocytes in meiosis are provided with two active X chromosomes, T prospermatogonia possess only one, and the presence of the Y chromosome is not vital for them. However, the Y chromosome is required for the normal course of spermatogenesis characterized by a stock of stem cells, that are responsible for the continuous production of male gamets. The mammalian oocyte--similar as that of insects and amphibia but to a lower degree--acts as pre-embryo.

Kinetics of gametogenesis. II. Comparative autoradiographic studies of oogonia and multiplying prospermatogonia of the Wistar rat.

In the rat the last generation of oogonia and multiplying prospermatogonia (M-prospermatogonia), frequently arranged in synchronized clusters, enters mitosis on about day 17 post conception (p.c.). The duration of the S-phase D-S-Duration and the minimal generation time Tmin of both kinds of "gonia" were determined by the method of labeled mitoses (22 female and 22 male fetuses derived from 11 pregnant rats were sacrificed from 2 to 22 h after a single i.p. injection of 3H-thymidine on day 17 p.c.). Three curves, derived from the labeled prophases, metaphases and the postmitotic descendents of oogonia and M-prospermatogonia--oocytes and primary transitional prospermatogonia (T1-prospermatogonia)--were evaluated. It was demonstrated that the curves as well as the calculated values of D-S-Duration and Tmin are very similar for oogonia and M-prospermatogonia. D-S-Duration ranged from about 10 to 12.5h (10 h read off from the curves of labeled metaphases), Tmin from 16.5 to 18 h (16.5 h read off from the curves of labeled metaphases).

Pathogenicity to animals of fine dusts from Ruhr mines.

Twelve different groups of fine mine dusts were examined for their in vivo aggressivity by intraperitoneal injection. Twelve months after injection of the dust, quantitative morphometric analysis of the lymph nodes was carried out to determine (a) the extent of the typical quartz reaction, (b) the in vivo cytotoxicity, (c) the size of the lymph nodes. Different fine dusts showed significant variations in parameter (a) and (c). In the second part of the investigation, the results of the lymph node tests and of in vitro cytotoxicity tests, and also the quartz contents of the dusts were fed into a data matrix and subjected to correlation analysis and principal component analysis. The following hypotheses were arrived at to explain the effect of the fine dust in vivo: (1) The amount of fine dust which penetrates into the lymph nodes is dependent on its in vitro cytotoxicity (Factor A). This is not related to the quartz content of the dust and is not inhibited by PVNO. (2) The specific fibrogenic activity of the fine dust within the lymph node is primarily dependent on its quartz content (Factor B). It appears that this effect is inhibited by PVNO. (3) The total fibrogenic activity depends on a combination of Factor A and B.

Kinetics of the enzymatic pattern in the testis. I. Stage dependence of enzymatic activity and its relation to cellular interactions in the testis of the Wistar rat.

The "morphology" of the enzymatic activities of thiamine pyrophosphatase (TPPase), acid phosphatases (ACPases), adenosine triphosphatase (ATPase) and steroid-3 beta-ol dehydrogenase (St-3 beta-ol DH) has been described using as a basis the classification of the seminiferous epithelium of the rat into 14 stages as proposed by Leblond and Clermont (1952a, b). It was demonstrated (Figs. 1, 2) that 1. the kinetics of the enzymatic pattern is correlated with the developmental stages during spermatocyto- and spermiogenesis, and that therefore the chemocytostructure, especially of the germ cells, shows characteristic changes. 2. the enzymatic pattern yields information on the chemohistostructure of the testis, and thus indicates interactions between the germ cells and the coordinated somatic cells. This is valid especially for the behaviour of the "marker enzymes" TPPase and ACPases. Initially the activity of both enzymes is distributed in the cytoplasm: TPPase appears in stage VII in the preleptotene spermatocytes, and ACPases appear in stage VII in the pachytene spermatocytes. In the following stages the activity of TPPase and ACPases increases and becomes more and more concentrated, i.e. from stage IX to XIV and thereafter from stage I to XIII in the case of TPPase, and from stage I to XIII in the case of ACPases. Finally the enzymatic activity of both TPPase and ACPases is arranged in spherical bodies near the nucleus of the spermatocytes. Thus the late pachytene and diplotene spermatocytes, as well as the spermatocytes in diakinesis, are characterized by deeply stained spherical dots covering the region of the Golgi apparatus. Both enzymes disappear during the maturation divisions--parts of the cytoplasm of the II-spermatocytes during interphase react weakly positive--, reappear in the Golgi region of the newly formed spermatids in stage I, remain there up to stage V in the case of ACPases, and up to stage VII in the case of TPPase. From stages VIII to XIV TPPase is weakly positive in the Golgi apparatus of the elongating spermatids, moving within the cytoplasm from the head region towards the tail. Finally they appear in the cytoplasm of the Sertoli cells: (1) ACPases appear in the borderline region between the Sertoli cells and the elongated spermatids in stages XII to XIV (2) TPPase first appears in the basal region of the Sertoli cells in stages XI to XIV, and becomes positive in the subsequent stages I to IV as "streamer like" bands from the basement membrane up to the heads of the elongated spermatids. Both enzymes disappear gradually during stages I to III and IV to V respectively. Stage dependence of ATPase can be observed in the apical region of the Sertoli cells around the heads and the middle pieces of the elongated spermatids. ATPase appears for the first time in stages IX to X, and becomes increasingly more and more concentrated and condensed up to the point when the newly formed spermatozoa are released in stage VIII...

Combined use of autoradiography and enzyme histochemistry in studies on the mammalian testis.

Combined application of the methods of enzyme histochemistry and of autoradiography in one and the same section of tissue provides a method to achieve further informations on the individual cells. Prespermatogenesis and spermatogenesis in rodents with their well defined steps of development and very specific enzymatic patterns of each of the stages of the cycle of the seminiferous epithelium were used as a model to study the double labelling with the aid of the enzyme reaction and labelling with 3-H-thymidine. The techniques applied in this study are described and discussed in detail. Possibilities to elucidate the regeneration mode of the seminiferous epithelium by means of this method are discussed.

The effect of local testicular irradiation on testicular histology and plasma hormone levels in the male rat.

Adult male rats were subjected to local testicular irradiation, plasma hormone levels and testicular histology being quantified at intervals up to 52 days thereafter. LH and FSH increased coincidently with spermatid but not with spermatocyte or spermatogonia depletion. Testosterone levels seemed to decrease but this effect was not significant. Oestradiol levels showed no significant changes. From the correlations between the various parameters it was concluded that the lack of inhibin was the main cause of the increase in both LH and FSH and that spermatids provide the signal for production of this non-steroidal inhibitor. The site of inhibin production was not definitively established but the results would be consistent with production of inhibin by the Sertoli cells in association with spermatids.

Enzymhistochemical and morphometrical studies on delta 5-3 beta-hydroxysteroid dehydrogenase during the fetal and neonatal development of rat Leydig cells.

The aim of the present report was to study the course of development of rat Leydig cells from 17 fetal day (f.d.) up to 5 postnatal day (p.n.d.), with the help of enzymhistochemical reaction of delta 5-3 beta Hydroxysteroid-dehydrogenase (3 beta-HSDH). Testes were obtained from 17 to 21 days old embryos and from 1 to 5 days old offsprings. Cryostat sections were cut and processed for enzymhistochemical reaction of 3 beta-HSDH, employing dehydroepiandrosterone as substrate. The areas of the Leydig cells, showing the enzymatic activity, were measured morphometrically. The 3 beta-HSDH positive intertubular Leydig cells appear on 17 f.d., and grow further showing a maximum peak, relative to the size of the testis, on 19 f.d. Thereafter, the percentage of the Leydig cells relative to the size of the testis decreases continuously up to 4 p.n.d.. The Leydig cells did not regress during the period of observation. Postnatally, the large complexes disperse into many small complexes.

Cyproterone acetate induced changes in the behaviour of hydroxysteroid dehydrogenases in rat Leydig cells during perinatal development.

The influence of cyproterone acetate (CA) upon the behaviour of hydroxysteroid dehydrogenases (HSDH) in the Wistar rat Leydig cells was investigated during the perinatal phase with the help of enzymhistochemical cum morphometrical techniques. The pregnant rats as well as their offsprings were injected with CA (dosage: 35 mg/kg body wt) sc, daily from 14 fetal day upto 31 postnatal day (p.n.d.). The animals were killed on 5, 20, and 32 p.n.d.; the enzymhistochemical reactions for 3-beta-HSDH, 11-beta-HSDH, 17-HSDH and 3-alpha-HSDH were performed in the cryostat sections of the testis, and the morphometric evaluation of HSDH positive Leydig cells was carried out. On 5 p.n.d. the activity of 17-beta-HSDH was slightly impaired in the intertubular Leydig cells of the CA treated animals. On 20 p.n.d., CA prevented nearly completely the HSDH activity in the newsly built peritubular Leydig cells; the activities of 3-beta-HSDH, 17-beta-HSDH, and 3-alpha-HSDH resided mainly in the intertubular Leydig cells. On 32 p.n.d. the HSDH activities in the Leydig cells were observed in the control as well as in treated animals. It seems that the differentiation of peritubular Leydig cells, and thereby the steroid production, is delayed by CA, but not entirely blocked.