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.

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.

[The effects of flunitrazepam (rohypnol) on respiration (author's transl)]. / Der Einfluss von Flunitrazepam (Rohypnol) auf die Atmung

The effects of flunitrazepam (0.03 mg/kg administered intravenously over a two-minute period) was investigated in 11 healthy volunteers with normal pulmonary function. Spirometer tracings were recorded continuously by the Siregnost FD 40 and blood gas measurements were performed by the Harnoncourt AVL gas analyzer. Flunitrazepam produced a characteristic cyclical hypoventilation/hyperventilation pattern lasting 15 min., followed by quiet sleeping rhythms. The duration of action was 20 min. There was a significant fall in PCO2, whilst the CO2 tension showed a significant rise. Changes in pH were in accordance with respiratory acidosis. Apnoea did not occur after the administration of flunitrazepam.

[Experimental torsion of the spermatic cord. Histological and histochemical studies (author's transl)]. / Experimentelle Hodentorsion. Histologische und enzymhistochemische Untersuchungen.

In 40 male adult rats on unilateral torsion of the spermatic cord was initiated by operation. In the first group (4 x 5 animals) the testes were removed after 3, 6, 12, 24 hours. The treatment of the animals in the second group (4 x 5 animals) was untwisting of the torsion after 3, 6 12, 24 hours. Three months later both testes were removed. Histological and enzymehistochemical results are as follows: 1. The extend of acute damage to the affected testis depends on the length the torsion lasted. 2. The delay of time of untwisting the testis probably does not influence the degree of later regeneration. 3. Even short duration of torsion of the affected testis results in damage to the contralateral testies.

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).

Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men.

Chromatin condensation is vital for the function of the spermatozoon as the motile carrier of the paternal genome. The degree of condensation can be shown with the aid of acidic aniline blue staining, which is able to discriminate between lysine-rich histones and arginine- and cysteine-rich protamines. Using this technique and employing the Düsseldorf classification of sperm morphology in cases of disturbance of spermatogenesis, it was demonstrated that chromatin condensation is impaired not only in malformed but also in morphologically normal spermatozoa. Among morphologically normal spermatozoa, the percentages of spermatozoa with chromatin condensation disturbances increase in patients with different patterns of sperm malformation, if compared with patients with normozoospermia.

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.

Histological and morphometric studies on the kinetics of germ cells and immature Sertoli cells during human prespermatogenesis.

Human prespermatogenesis between the 8th week of pregnancy and six months after birth was studied in testis material of 28 male foetuses from spontaneous abortions and 81 infants who died from sudden infant death. The foetuses and infants were grouped in 10 age groups. A first steep raise in the numbers of germ cells per 20 tubular cross sections from 22.3 in the first group up to 69.5 in group 3 was observed, i.e. up to the end of the 22nd week of pregnancy. Thereafter, a continuous decrease could be observed modulated by a second slighter increase during the first 4 months after birth. The ratio of germ cells and immature Sertoli cells improves from about 1:20 at the beginning to 1:8 in group 3; afterwards it changes in favour of the immature Sertoli cells down to 1:140 at the end of the study. The initial augmentation of germ cells is interpreted as the effect of a first proliferation wave comparable to that of M-prospermatogonia in other species. The decrease of germ cells is due to the stop of germ cell proliferation and simultaneous high proliferative activity of the immature Sertoli cells.

Correlation between multi-headed spermatozoa in the ejaculate and histological findings in the testis.

Semen smears of 163 patients as well as bioptic material of 163 patients (93 cases of paraffin histology and 70 cases of cryostat sections) were grouped by their content of multi-headed spermatozoa or multi-nucleated spermatids, respectively. It could be demonstrated that findings in the ejaculate concerning multi-headed spermatozoa are in good agreement with those in the testis in respect of the presence of double- and multi-nucleated spermatids. Furthermore, it could be shown that the frequency of multi-headed spermatozoa is positively correlated to inflammatory processes either of immunologic origin or caused by micro-organisms.

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...

Stages of the cycle of the seminiferous epithelium in the dog.

In principle, the spermatogenesis of the dog resembles that of other animals. It consists of 6 consecutive, differentiating spermatogonia generations (A1-A4, Im, B). The number of ensuing spermatogenetic cell generations corresponds to that of other mammals. We divided the spermatogenesis of the dog into 10 stages in order to attain a precise systematization. The cell-divisions of the successive spermatogonia occur in stages II, III, V, VI, VII, and VIII. Stages V a and V c are characterized by the meiotic divisions.

Morphological studies on the origin of adult-type Leydig cells in rat testis.

The differentiation of adult-type Leydig cells (ATLC) in rat testis was studied with the help of electron microscopy and a combined technique of autoradiography and enzymehistochemistry. Male Wistar rats on each alternative day from postnatal day (pnd) 9 to 25 were perfused in situ, and the testicular sections were observed by electron microscopy. The cytoplasm of the ATLC contained smooth endoplasmic reticulum, mitochondria, a few small lipid droplets and a large well developed Golgi apparatus. In addition to typical fibroblasts/fibrocytes, a group of fibroblast-like cells was observed in the peritubular and perivascular spaces of the interstitial tissue. These cells, containing smooth endoplasmic reticulum and a large number of mitochondria with tubular-type cristae, were named as intermediate-type cells. In the autoradiographic experiments the 3-H-thymidine and 14-C-thymidine uptake behaviour of fetal-type Leydig cells, ATLC, intermediate-type cells and myoid cells was studied from 1 till 25 pnd. The intermediate-type cell, observed predominantly between pnd 9 to 21, showed the highest labelling index. The absolute number of fibroblasts/fibrocytes showed a peak on pnd 13 and decreased gradually with age. The duration of DNA synthesis phase ranged for interstitial fibroblasts: 7.9 h, for intermediate-type cell 10.4 h, for ATLC 17.4 h. The results lead to the conclusion that ATLC most probably originate to a large extent from the peritubular and to a lesser extent from perivascular fibroblast-like cells of the interstitial tissue.

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 effects of some Robertsonian chromosome combinations on the seminiferous epithelium of the mouse.

In Mammals, structural rearrangements of the karyotype cause considerable trouble to the spermatogenic process. Making use of an experimental animal model of Robertsonian chromosomal variation in the house mouse (Gropp, Winking & Redi, 1982a) the effects of these chromosome structural rearrangements on the spermatogenic process were studied in fertile and chromosomally derived subfertile and sterile mice. Each karyotype condition was related to the cytological composition of the twelve stages of the seminiferous epithelium, studied in PAS-haematoxylin-stained testicular sections, with the following results: in subfertile males there is a depletion of spermatogonia in the regenerating compartment but their differentiation is not affected. In the sterile males there is degeneration of primary and secondary spermatocytes and massive spermatid degeneration. Spermatocyte development is retarded in nearly 50% of the spermatocyte population in subfertile males. Moreover the ratio between primary spermatocytes and spermatids is reduced to about 1:2 in subfertile males, while the few spermatids produced in sterile males had degenerated during stages I to VIII. The number of Sertoli cells/100 micron throughout the cycle of spermatogenesis is the same in the three conditions studied. These data indicate that the spermatogenic process is affected by structural changes not only at the meiotic level (primary spermatocyte failure to follow the normal pattern of differentiation and occurrence of defective spermatids) but also at the premeiotic stage, when undifferentiated spermatogonia are regenerating.

Stage-dependent enzymatic activities in spermatogenesis of mice with the standard karyotype and of chromosomal variants with impaired fertility.

The enzymatic activities of thiamine pyrophosphatase (TPPase), acid phosphatases (ACPases), alkaline phosphatases (APases) and steroid-3 beta-ol dehydrogenase (beta-HSDH) in different germ cells and somatic cells in the testis of three cytogenetically determined states of fertility (i.e. normal, impaired fertility and sterility) were studied histochemically in the mouse. The TPPase, ACPases and APases activities showed a characteristic stage dependent pattern when the activities were related to the typical twelve stages of the seminiferous epithelium in the mouse, according to Oakberg (1956). Comparing the enzymatic patterns of the activities in the normal spermatogenic process versus the impaired and sterile conditions, the following conclusions can be drawn: even in impaired and sterile conditions the enzymatic activity patterns retain their characteristic stage dependence; the pattern of beta-HSDH and ACPases is not altered in the impaired and sterile conditions; TPPase and APases patterns are modified in impaired and sterile mice. It is concluded that the kinetics of the enzyme activities can serve as a useful marker for characterizing pathological spermatogenic processes.