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The occurrence of plasminogen activators of the urokinase-type (u-PA) and tissue-type (t-PA) at various stages of the epithelial cycle was studied immunohistochemically in rat seminiferous tubule segments. u-PA immunoreactivity was detected exclusively at stages VII and VIII in Sertoli cells, displaying a distinct granular cytoplasmic staining. t-PA immunoreactivity was found during mid- and late pachytene and diakinesis (stages VII-XIII) in spermatogenic cells, displaying a granular cytoplasmic staining with maximal intensity in stages IX-XIII. The specificity of the stainings was supported by staining controls, including absorption of the antibodies with purified preparations of the activators. It was also supported by zymographic studies of the occurrence of u-PA and t-PA in extracts of tubular segments at different stages of the cycle, isolated by transillumination-assisted microdissection. The possible functions of the two types of plasminogen activators in the seminiferous epithelium are discussed.

Monoclonal and polyclonal antibodies were used in indirect immunofluorescence microscopy to localize vimentin intermediate filaments in the rat seminiferous epithelium. During stages XII-V of the epithelial cycle, the Sertoli cells showed a reaction in the perinuclear area and vimentin-positive extensions, projecting toward the developing spermatid bundles, were also seen. During stages VI-XI these extensions were small and narrow. Monoclonal antibody to vimentin gave a granular reaction in the peripheral region of the flagella of steps 16-19 spermatids. Western blotting indicated a specific reaction with a Mr 58,000 polypeptide in isolated seminiferous tubules and in epididymal spermatozoa. Our results suggest that vimentin filaments in Sertoli cells may be regulated cyclically in a stage-dependent manner. The granular reaction in the spermatid flagellum with the monoclonal antibody suggests that vimentin in germ cells is organized differently from that in somatic Sertoli cells.

The movement and transport of material between intranuclear dense particles, the chromatoid body and the Golgi complex have been studied in early spermatids of the rat. The analyses involved observation of living accurately identified cells, time-lapse cinemicrography and electron microscopy. The chromatoid body establishes transient contacts with intranuclear material during early spermiogenesis. The chromatoid body also makes contacts with the Golgi complex. It is suggested that the chromatoid body receives material from the nucleus during the postmeiotic period and particites in the early formation of the acrosomic system.

Mutagen effect on male germ cells can be analyzed by micronucleus induction during meiotic divisions. These can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the formation of micronuclei in this test system using adriamycin as a model mutagen. Micronuclei were induced in a dose-dependent manner at concentrations of 1-10 ng/ml that were far below the dose that caused morphologically or biochemically detectable cytotoxic effects. The meiotic micronucleus induction in vitro is a potentially sensitive test system of male germ cell mutagenesis.

Chromosome breakage caused by mutagens in male germ cells can be analyzed by micronucleus induction during meiotic division. This can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the mutagenic potential of a male contraceptive, gossypol, in this test system using adriamycin (10 ng/ml) as a reference mutagen. A small but significant increase in the frequency of micronuclei was induced with concentrations of 10 and 20 micrograms/ml of gossypol, while cytotoxic effects appeared at concentration of 20 micrograms/ml and were evident at 50 micrograms/ml. Analysis of meiotic micronucleus induction in vitro seems to be a sensitive test system of male germ-cell mutagenesis, but further studies on the possible mutagenic effects of gossypol are needed.

The variation of the specific Mn2+-dependent adenylyl cyclase (AC activity in spermatids and follicle stimulating hormone (FSH)-responsive AC activities in Sertoli cells in different stages (I-XIV) of the seminiferous epithelial cycle has been investigated. Maximal Mn2+-dependent AC activity was observed in stages II-III while minimal activity was encountered in stages VII-VIII (spermiation). FSH-responsive AC activity exhibited a pattern that coincided with that of the Mn2+-dependent AC. The stage-dependent variation in spermatid AC activity cannot be explained by altered numbers of haploid cells. This raises the question whether the Sertoli cells may regulate the spermatid AC activity. Sertoli cells in various stages are all exposed to the same concentration of circulatory hormones. Hence the stage-dependent difference in FSH-responsiveness indicates that local influences (from germ cells?) may regulate the response of the AC in Sertoli cells to FSH.

Cyclic (c) AMP production was measured in vitro in dissected pieces of adult rat seminiferous tubules of defined stages of the seminiferous epithelial cycle (VII-VIII and XIV-IV) in the absence and presence of human follicle-stimulating hormone (FSH) (Metrodin, Serono). The basal rate of cAMP production was stage dependent, being about 2-fold higher in stages XIV-IV. FSH stimulated cAMP output in a time- and dose-dependent (stimulation at doses greater than or equal to 3 mg/l) fashion, and also the stimulability of stages XIV-IV (on average 2-fold) was greater than that of stages VII-VIII. When the tubular pieces were incubated in the presence of pertussis toxin (PT, 100 micrograms/l), the FSH-stimulated cAMP production of stages VII-VIII was enhanced by about 2-fold (P less than 0.01) whereas the basal rate was unaffected. In contrast, neither the basal nor the FSH-stimulated cAMP production of stages XIV-IV were affected by PT. Presence of the Gi-protein in both stages studied was demonstrated with PT-induced ADP ribosylation. However, no release of a putative activator of the Gi-protein could be demonstrated into spent media of the seminiferous tubules when incubated with freshly separated tubules. It is concluded that the poor FSH stimulability in cAMP production of certain spermatogenic stages of adult seminiferous tubules is at least partly due to endogenous inhibitors acting through the inhibitory Gi-protein. This inhibition could be demonstrated in a stage-dependent manner, and was present in stages with the lowest production and least stimulability of cAMP production by FSH.

The stages of the rat seminiferous epithelial cycle have been isolated for flow cytometric analysis of DNA and for culture, using transillumination-assisted microdissection. Precise stages have been identified by phase contrast microscopy of live cell squashes from adjacent segments. Each stage of the cycle showed a characteristic flow cytometric pattern with haploid (1C), diploid (2C) and tetraploid (4C) peaks. Stages I to VIII of the cycle showed an additional hypofluorescent (0.25-0.70C) peak due to a reduced dye-binding capacity of maturation phase-spermatids at steps 15 through 19. The appearance of the hypofluorescent haploid peak coincided with the second nucleoprotein transition at step 15 of spermiogenesis and the homogeneous condensation of the chromatin seen in electron microscopy. As a concomitant of the formation of disulphide bonds during epididymal maturation, the fluorescence intensity decreased further to reach a relative value of 0.07C in the cauda epididymidis. The constant 1C peak was raised by round and elongating spermatids (steps 1-14), 2C by spermatogonia, secondary spermatocytes and Sertoli cells, and the 4C peak by primary spermatocytes and spermatogonia at G2 or M phase of the mitotic cycle. The proportion of each peak accurately reflected the relative proportion of cells in most stages of the cycle when compared with morphometric measurements of histologic preparations. DNA flow cytometry is a suitable method for quantitative evaluation of cultured seminiferous tubule segment DNA. Although the relative yield of the meiotic reductive divisions in vitro is comparable with that observed in vivo, steps 9 and 15 of spermiogenesis involving nucleoprotein transitions and spermiation itself did not occur under the present culture conditions.

The cyclic secretion of plasminogen activator (PA) by Sertoli cells in stages VII and VIII of the rat seminiferous epithelial cycle is influenced by hormones and adjacent spermatogenic cells. To understand this interaction more in detail, we have analyzed the effects of FSH, (Bu)2cAMP, testosterone, insulin, and retinoic acid (RA) on staged seminiferous tubule segments in vitro. FSH stimulated stages VIIcd to XI of the cycle; similar results were obtained with (BU)2cAMP. RA stimulated PA secretion in stages I-VIIab, but testosterone and insulin had no effect in any stage. The secreted PA was mainly of the urokinase type, although small amounts of the tissue-type PA were found after stimulation by FSH and cAMP. These results suggest that spermatogenic cells modify the responsiveness of Sertoli cells to hormonal stimulation. Stages I-VIIab are sensitive to stimulation by RA whereas stages VIIcd-XI are preferentially stimulated by FSH and (Bu)2cAMP.

The distribution and role of the calcium-activated, phospholipid-dependent protein kinase C (PK-C) was studied in rat testis. When testis tissue was homogenized in the presence of 2 mmol/l EDTA and EGTA, the majority (greater than 70%) of the PK-C activity was soluble, the rest was released from the particulate fraction by solubilization with 0.3% Triton X-100. Without chelating agents the soluble PK-C activity was undetectable, and only partially recovered from solubilized membranes. Preincubation of the tissue with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7) mol/l) translocated PK-C to the membranes, and the majority of this activity was recovered by solubilization. Mobility of testicular soluble PK-C activity in HPLC-DEAE cellulose chromatography was similar to that of the brain enzyme. This single step purified testicular PK-C activity 140-fold. The specific activity and subcellular distribution of PK-C was similar in whole testis tissue and separated seminiferous tubules (160-210 pmol 32P X mg protein-1 X min-1 in the soluble and particulate fractions), but 2- to 3-fold higher in purified Leydig cells. However, the majority of total testicular PK-C activity appeared to be of tubular origin. Unilateral cryptorchidism for 1 week reduced PK-C of the abdominal testis by 50%, and the activity of dissected seminiferous tubules varied according to the epithelial wave. Both findings suggest that the bulk of the activity resides in the seminiferous epithelium. Involvement of PK-C in Leydig cell function was demonstrated using the TPA, which at 10(-7) mol/l inhibited basal cAMP production by 50% (P less than 0.01) but increased that of testosterone by 2- to 3-fold (P less than 0.01). On the other hand, when incubated with hCG, TPA inhibited both cAMP and testosterone production; the ED50s of hCG stimulation increased 4- to 10-fold with both parameters. It is concluded that PK-C activity is present in both the seminiferous tubules and Leydig cells, and is involved in the regulation of these testicular compartments. Its total activity and subcellular distribution are at variance according to the functional state and endocrine milieu of the testis.

Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.

Cells at various stages of spermatogenesis show remarkable differences in their sensitivities to toxic and mutagenic agents. For accurate analyses of toxicity and mutagenicity, the most sensitive cell types should be obtained for studies soon after treatment before development of disturbing secondary alterations. The transillumination phase contrast microscopic technique has proved to be useful for this purpose. It allows recognition of rat and mouse seminiferous tubules in living unstained conditions for accurate selection of the desired stages of the cycle of the seminiferous epithelium for morphological and biochemical studies. Specific cell damage can be frequently recognized by transillumination only, whereas in most cases phase contrast microscopy is useful in screening the early stage-specific toxic alterations. A summary is presented of the results obtained by treatment with heat and with anticancer drugs. A meiotic micronucleus method has been developed for direct estimation of the severity of mutagenic insult on rat spermatogenesis. The segment that contains the second meiotic division and the early postmeiotic cells (stages XIV and I) is selected for study. Chromosome damage induced by mutagens may result in the formation of acentric fragments that after meiotic divisions are seen in early spermatids as separate micronuclei. They can be quantitated in squash preparations; the sensitivity of this method is comparable with that of the meiotic metaphase scoring. Recently, an in vitro technique has been developed for stage and cell specific measurements of replicative and repair syntheses of DNA during rat spermatogenesis after mutagen treatments.

Plasminogen activator (PA) is secreted cyclically (in stages VII and VIII) by rat seminiferous tubules. To investigate whether this can be maintained and influenced in vitro, tubule segments from stages VI and VIII of the epithelial cycle were cultured for 3 days in chemically defined medium supplemented with testosterone, FSH, or a combination of testosterone, FSH, insulin and retinoic acid (4F). Morphological and flow cytometric analyses of stage VI tubules suggested a roughly normal differentiation to stage VIII. They developed an increased PA secretion on day 3 of culture. Stage VIII tubules, however, did not develop all the characteristics of stage XII. Step 8 spermatids did not elongate and step 19 spermatids failed to develop into spermatozoa. Secretion of PA on day 3 was not significantly different to that on day 1. The 4F combination very significantly stimulated PA secretion in both stages, but FSH alone was effective only in stage VIII. Most of the secreted PA had a molecular weight of 43,000 in both stages, suggesting that it is of urokinase type. The results suggest that stage VI is more able to differentiate in vitro for 3 days than stage VIII; the cyclic secretion pattern of PA was partially maintained in tubule segments from stage VI. Follicle-stimulating hormone had an effect on PA secretion only in stage VIII, whereas the 4F combination was stimulatory in both stages. The retinoic acid in this combination may be of importance in the regulation of PA secretion by seminiferous tubules.

Spermatogenesis is dependent on stimulation by pituitary gonadotropins, FSH and LH. Targets for these hormones are Sertoli and Leydig cells, respectively. The effect of LH on spermatogenesis is mediated by testosterone. In addition to hormones, interactions between neighbouring cells seem to regulate spermatogenesis. This is reflected by cyclic secretion of several proteins by the seminiferous epithelium, of which plasminogen activator is a good example. While it is controlled by FSH a factor in preleptotene spermatocytes may also influence its cyclic secretion pattern. Both testosterone and FSH have a cyclic action in the seminiferous epithelium. The androgens seem to predominate in stages where spermiation, onset of meiosis and the highest rate of RNA transcription occur (VII-XI). FSH is most active in stages that contain meiotic divisions and early spermiogenesis (XIII-V), greatly stimulating the production of cyclic AMP. To investigate further the "second messengers" of FSH action in the seminiferous epithelium, the cellular distribution of calmodulin was analyzed using an indirect immunocytochemical method. In addition to their clear cyclic distribution in primary spermatocytes and in spermatids, Sertoli cells also showed a bright calmodulin immunofluorescence that was apparently cyclic. These observations suggest a local calmodulin and calcium regulation of spermatogenesis.

A monoclonal antibody C 11 H was produced against human acrosomal antigen. It also cross-reacted with acrosomes of the boar and the mouse. In boar spermatozoa the antibody reacted, in immunoblotting analysis, with polypeptides of Mr 55,000 and 53,000. The proteolytic activity was detected by zymographic casein overlay assay. Partially purified boar sperm acrosin was bound by the C 11 H affinity column, and acrosin activity was detected in the eluate. These experiments indicate that C 11 H antibody recognizes sperm acrosin. The acrosin expression during spermatogenesis was studied with C 11 H antibody, using mouse testis as a model. Immunocytochemical analysis revealed that step 9 spermatids were the first cells to react with C 11 H antibody. During step 14, the perinuclear pattern of C 11 H-binding disintegrated into small particles around the spermatid nuclei for the period of close association between spermatid bundles and Sertoli cells. During late step 15, the antigen became located at the site in the acrosome typical for step 16 spermatids and spermatozoa. These results indicate that monoclonal C 11 H antibody recognizes acrosin that is first expressed in haploid cells coincident with the onset of nuclear elongation and cessation of RNA transcription. The changes in the distribution pattern suggest that acrosin may be modified by Sertoli cells. In addition to studies on acrosin, this antibody may be useful in investigations of transcription and translation and their regulation during spermatogenesis in general.

Ornithine decarboxylase (ODCase) is the first and rate-limiting enzyme in the polyamine biosynthetic pathway and it is androgen regulated in the mouse. The expression of ODCase transcripts during testicular development was examined by Northern blot analysis with a mouse ODCase cDNA probe. Total RNA was isolated from the testes of prepubertal mice at 6, 8, 12, 16, 18, 20, 22, and 30 days of age, from enriched populations of germinal cells obtained from the testis of immature (8 days old) and mature (45 days old) mice and from several mouse somatic tissues. The level of the two ODCase transcripts (2.2 and 2.7 kilobases) was low but detectable in the testes of 6- to 16-day-old mice and increased substantially as the first spermatogenic wave proceeded into spermiogenesis. The low ODCase mRNA levels observed in prepubertal mouse testes were confirmed with RNA samples obtained from enriched germ cell populations of type A and type B spermatogonia and interstitial cells obtained from Day 8 mouse testes. In agreement with the developmental studies, ODCase mRNA levels increased substantially in enriched populations of pachytene spermatocytes, round spermatids, and residual bodies/cytoplasts isolated from mature testes. Similar results were obtained by in situ hybridization of sections of rat testes. Reduced levels of ODCase transcripts were detected in RNA obtained from cultured mouse Sertoli cells obtained from the testes of 21-day-old mice and in RNA from liver, brain, heart, spleen, seminal vesicle, and aorta. In contrast, ODCase transcript levels from kidneys of male mice were as high as those detected in testis RNA. Substantial levels of ODCase mRNAs were also found in the epididymis. Analysis of polysome gradients prepared from total testis extracts revealed a distribution of ODCase mRNA in both nonpolysomal and polysomal fractions of the gradient, suggesting that ODCase is translationally regulated in the mouse testis.

Parts of human seminiferous tubules containing late spermatids with condensed nuclei (Stage II) absorbed more transmitted light than did parts at other stages. Spermatogenic stages I, III, IV and VI were identifiable by phase-contrast microscopy.

Rat seminiferous tubule segments have been cultured in chemically defined medium (F12/DMEM 1:1) without added hormones or growth factors. The segments (1-2 mm) were isolated from defined stages of the cycle of the seminiferous epithelium (VIII and XII) by transillumination-assisted microdissection. The precise stages were examined by phase contrast microscopy of live cells squashed carefully out from the adjacent segments between glass slides. The squash technique was also used for a primary screening of the cultured tubules. Pachytene primary spermatocytes from stages VIII to XII of the cycle were able to complete meiotic divisions in vitro. From stage XII, they differentiated up to step 5 spermatids, expressed their specific antigens, and developed characteristic movement patterns of the flagellum and of the chromatoid body. Preleptotene and zygotene spermatocytes from the same cell association differentiated synchronously, as judged by chromosome morphology, characteristic chromosome rotation in zygotene and early pachytene, and by development of specific antigen expression. The elongation phase of spermiogenesis did not proceed normally in vitro. The rate of differentiation was the same as observed earlier in vivo. Earlier studies with [3H]thymidine labeling and autoradiography only permitted follow-up of the development of preleptotene spermatocytes. With the present method, all stages of spermatogenesis can be traced in culture with great accuracy in experiments relating to local regulation of spermatogenesis.

Male sterility belongs to the recently recognized complications of cancer chemotherapy and has an increasing importance. Therefore, more information about the mode of action of anticancer drugs on mammalian spermatogenesis is needed. We have developed a technique based on transillumination of living, freshly isolated unstained rat seminiferous tubules, where the cells specifically killed by the drugs are recognized as dull zones. Early stages of cell degeneration can be rapidly analyzed by phase contrast microscopy of living cells. Because the transillumination technique, in addition, permits an accurate recognition of the segments of the seminiferous epithelial wave, the cells representing various stages of the mitotic and meiotic cell cycles during spermatogenesis can be isolated in living state for morphological analysis. Vinblastine and vincristine cause an arrest of mitotic and meiotic divisions to metaphase followed by cell death, which was more rapid after vincristine administration. Both alkaloids had a slight damaging effect on the pachytene spermatocytes. Large doses of both drugs primarily affected the Sertoli cells by destroying their microtubules and mitochondria. Vincristine specifically damaged the acrosomic system and the cytoplasmic bridges of the young spermatids.

Testicular androgen receptors were measured with a recently developed exchange assay using [3H]methyltrienolone from samples of interstitial tissue, whole seminiferous tubules, and segments of tubules in different stages of the cycle of the seminiferous epithelium. The hexylene glycol method was used to isolate nuclei and pyridoxal 5'-phosphate for extraction of the androgen receptors. This method proved superior to other techniques employed by our own and other laboratories. As a consequence, higher levels of androgen receptors were detected in seminiferous tubules than previously reported. Androgen receptors in seminiferous tubules and interstitial tissue had high affinity for methyltrienolone (Kd = approximately 3 nM) and steroid binding specificity similar to that of these receptors in other tissues. The concentration of nuclear androgen receptors in whole seminiferous tubules was 670 +/- 100 fmol/mg DNA (mean +/- SE), while that of the interstitial tissue was 1070 +/- 295 fmol/mg DNA. Tubules in stages IX-XII and XIII-I of the epithelial cycle contained significantly more nuclear androgen receptors (900 +/- 170 and 805 +/- 125 fmol/mg DNA, respectively) than those in stages II-VI and VII-VIII (485 +/- 95 and 485 +/- 65 fmol/mg DNA, respectively). These results suggest that there are local differences in androgen receptor concentration along the length of the seminiferous tubule. A high concentration of nuclear androgen receptors was also present in interstitial tissue. Androgen receptors were measurable in cytosol prepared from interstitial tissue, but such measurements were obscured in cytosol from tubules, because of a high capacity binding protein for the 3H-labeled ligand. We conclude that nuclear androgen receptors can be measured in various testicular compartments, including different stages of the seminiferous tubules, using an exchange assay that maximizes recovery. The concentration of nuclear androgen receptors in the tubules varies with the cycle of the seminiferous epithelium.

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.

In a survey of all malignant soft tissue tumors in the extremities and limb girdles in Finland between 1960 and 1969, only one alveolar soft part sarcoma was found among 246 tumors (0.4%). Another alveolar soft part sarcoma, diagnosed in 1976, was more thoroughly studied. There was evidence that the characteristic crystals of alveolar soft part sarcoma are formed from the dense granules. Both were PASM-positive at ultrastructural level. No monoamines were detected in the cells by formaldehyde-induced fluorescence. This is a further fact to nullify the theory of the paraganglionic origin of alveolar soft part sarcoma, but the question of the histogenesis of the tumor still remains open.

The interaction of seminiferous tubules and Leydig cells was investigated in the rat in vitro. Crude collagenase-dispersed, or Percoll-purified, Leydig cells were incubated together with seminiferous tubules at different stages of the cycle, isolated by transillumination-assisted microdissection. The testosterone production was measured. Seminiferous tubules inhibited testosterone production of crude Leydig cell preparations, but induced a clear stimulation (30-100%) in Percoll-purified cells. The stimulation was maximal at stages VII and VIII of the cycle, significantly higher than at stages II-VI (P less than 0.05). The stimulation by seminiferous tubules was observed both in basal and hCG-stimulated testosterone production. The effect was independent of FSH or GnRH action. These results demonstrate the presence of paracrine regulatory interaction between seminiferous tubules and Leydig cells, and are in agreement with the concept of a preferential androgen requirement of stages VII and VIII of the cycle.