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All germ cells throughout the animal kingdom contain cytoplasmic cloud-like accumulations of material called nuage. Polar bodies in Drosophila oocytes are probably the best known forms of nuage. In spermatogenic cells, the nuage is called chromatoid body (CB). In early spermatids of the rat, it has a diameter of 1-1.5 microm and a finely filamentous lobular structure. Typically, it is associated with a multitude of vesicles. It is first clearly seen in mid- and late pachytene spermatocytes as an intermitochondrial dense material. During early spermiogenesis it is seen near the Golgi complex and frequently connected by material continuities through nuclear pore complexes with intranuclear particles. In living cells, the CB moves around the Golgi complex and has frequent contacts with it. The CB also moves perpendicularly to the nuclear envelope, and even through cytoplasmic bridges to the neighbour spermatids. One of the major components of the CB is a DEAD-box RNA helicase VASA that belongs to a class of proteins thought to act as RNA chaperones. It is a general marker of all germ cells and best characterized in Drosophila. The mouse VASA homologue was recently used as a marker of sperm formation from embryonic stem cells. It becomes generally accepted that the CB with its associated structures constitute a mechanism of post-transcriptional processing and storage of several mRNA species that are shared between neighbour cells and used for translation when the genome of the spermatids becomes inactive.

A microtubule-associated protein E-MAP-115 has been originally isolated and characterized from HeLa cells. Because of its predominant expression in cultured cells of epithelial origin, it has been suggested to be involved in the regulation of cell polarization. The present immunocytochemical, Northern blot and in situ hybridization analysis of E-MAP-115 in the mouse and rat seminiferous epithelium indicates its distinct association with the spermatid manchette, a unique microtubular structure which appears in the cytoplasm of spermatids at step 8 when nuclear polarization and elongation starts. At steps 15-16 when manchette has been disassembled, immunoreactivity for E-MAP-115 disappeared. At immunoelectron microscopical level, E-MAP-15 was associated with the microtubules of the manchette. In the Western and Northern blot analysis, a distinct stage-dependent expression of a single E-MAP-115 polypeptide and two mRNA species (3.4 and 2.4 kb) could be identified. MTEST 60, a spermatid-specific transcript, showed a 100% homology over region of 68-193 bp of E-MAP-115 sequence. The reported specific localization of E-MAP-115 to the spermatid manchette strongly supports its role as a regulator of cell polarization. This, in turn, supports the hypotheses concerning the dynamic function of the manchette during spermiogenesis.

Interleukin-1alpha (IL-1alpha) is constitutively expressed in an age- and stage-dependent manner by rat Sertoli cells. However, the mechanism of regulation of IL-1alpha is unclear in testis. We studied this regulation at the level of the enzyme calpain, a potential regulator that cleaves 32 kDa proIL-1alpha to produce mature 17 kDa IL-1alpha. Both calpain I and II were found to cleave recombinant rat testis 32proIL-1alpha in vitro. A temporary age-related increase in messenger RNA (mRNA) levels of calpain I was found in testis of 20- and 25-day-old rats, coinciding with important events of spermatogenesis and a gradual increase in IL-1alpha, while calpain II expression was constant. In response to lipopolysaccharide (LPS), calpain I protein levels were down-regulated in the seminiferous tubules, while calpain II was less affected. By contrast, the liver after LPS treatment showed up-regulated calpain I and II immunoreactive protein and reverse transcriptase chain reaction (RT-PCR) signal. Depleting Leydig cells by ethane 1,2-dimethane sulphonate treatment resulted in down-regulated calpain I mRNA and protein expression, whereas calpain II remained unchanged. In summary, there is a differential expression of calpain I and II under pathological conditions induced either by endotoxin stimuli or Leydig cell depletion, which may produce a differential effect on IL-1alpha processing.

Interleukin-1 (IL-1), a multifunctional cytokine produced mainly by activated macrophages, is also produced in the intact testis. Rat testicular IL-1 was found to be identical to IL-1alpha, judged by immunoneutralization of the bioactive protein and sequence comparison of cloned rat testicular and macrophage pro-IL-1alpha cDNA. Testicular IL-1alpha mRNA was first demonstrated on postnatal day 15, and the corresponding bioactive protein from day 20. IL-1alpha mRNA was still low on day 20, but then increased rapidly in parallel with the bioactive protein to establish a plateau level from day 25. In adult testes, IL-1alpha mRNA and immunoreactive protein were low in stage VII of the seminiferous epithelial cycle, whereas other stages showed a clearly detectable expression. In the adult testis, the concentration of IL-1alpha was 75 pg/mg testicular protein (approximately 200 pM). In conclusion, production of testicular IL-1alpha is developmentally and stage-dependently regulated, probably at the transcriptional level, emphasizing an important paracrine role in testicular function.

Spermatogenic cells from a mouse strain expressing enhanced green fluorescent protein (EGFP) under chicken beta-actin promoter were studied under living conditions to analyse stage- and cell-specific expression and hormonal regulation of the transgene. The isolated seminiferous tubules were examined by transillumination and the live cell squashes by phase contrast and fluorescence microscopy. FSH effects were measured in whole seminiferous tubules comparing stages I-VI, VII-VIII and IX-XII of the cycle. Beta-actin was highly expressed in spermatogonia, but almost no expression was found at early meiosis (leptotene spermatocytes). A gradual increase in translation of beta-actin was found during later stages of meiosis and early spermiogenesis, with a maximum in elongating spermatids. FSH increased the translation of beta-actin after 4 h and 24 h of incubation at stages I-VI, after 24 h at stages VII-VIII but not at stages IX-XII of the cycle. The results support the view that beta-actin plays a role in the nuclear elongation of spermatids and that its expression is regulated by FSH in a stage-specific fashion. Techniques used in this study give us new insight to study temporal and hormonal regulation of gene products in living spermatogenic cells.

The cause of fertility problems in insulin-dependent diabetes is largely unknown. To evaluate the role of autoimmunity-associated phenomena in the testis as a possible cause of the derangement in spermatogenesis, the stage-specific apoptosis of germ cells in the insulitis phase of pre-diabetes was quantified in the testes of non-obese diabetic (NOD) mice. The seminiferous epithelium of normal BALB/c and NOD mice contained cells positive for in-situ end-labelling (ISEL) of DNA. ISEL-positive germ cells formed clusters in the seminiferous epithelium of the NOD mice in marked contrast to the seminiferous epithelium of the BALB/c mice, which contained only individual cells positive for ISEL. ISEL-positive cells were present in the basal and luminal compartments of the epithelium. Ultrastructural analysis and demonstration of externalized phosphatidyl serine confirmed that the cells were undergoing apoptosis. The ultrastructurally apoptotic cells included spermatogonia, spermatocytes and spermatids. In cytological squash preparations of segments of seminiferous tubules from NOD mice aged 17-20 weeks, the number of ISEL-positive cells/mm tubule was significantly lower in segments at stages I-II of the seminiferous epithelial wave but higher at stages III-IV in comparison to BALB/c mice. The numbers of ISEL-positive cells/mm tubule in the other stages were similar in the two strains of mice. Analysis of 32P-3'-end labelled DNA from the testes showed that the BALB/c mice had relatively more DNA fragmentation than did the NOD mice. These data suggest that autoimmune insulitis in the NOD mice is associated with increased amounts and abnormal stage distribution of apoptosis in the seminiferous epithelium, resulting in derangement of spermatogenesis.

Two techniques have been combined for quantification of apoptotic germ cells in defined stages of the cycle of the seminiferous epithelium: the improved transillumination method and nonradioactive in-situ end-labelling of DNA (ISEL). Segments of rat seminiferous tubules were squashed between a microscope slide and coverslip, and the stage identified under a phase-contrast microscope. After fixation, apoptotic cells were detected by ISEL and scored per 1 mm tubule. In the normal testis apoptotic cells were found in all stages, the highest frequency occurring in stages XII-XIV (19 cells/mm). In short-term (24 and 48 h) experimentally cryptorchid testes, a significant increase in number of apoptotic germ cells was evident in all stages, except for VI and VIII. Apoptosis of germ cells was confirmed by electrophoresis of radioactively labelled DNA from stages VII-VIII and XIII-I. It is proposed that apoptosis is a means of eliminating the most sensitive germ cells after short-term experimental cryptorchidism.

Stage-specific DNA synthesis and interleukin-1 (IL-1) bioactivity were measured in the rat testis at 2, 6, 12 and 25 days after local irradiation with 3 Gy to investigate whether there was any correlation between germ cell DNA synthesis during repopulation and IL-1-like bioactivity in the seminiferous epithelium. DNA synthesis by intermediate and type-B spermatogonia was reduced significantly at 2, 6 and 12 days after irradiation in seminiferous tubules at stages II-III and IV-V. IL-1 bioactivity was increased significantly at 6 and 25 days after irradiation at stages II-VI during repopulation of spermatogonia. At 2, 6 and 25 days after irradiation at stages VIIa-c a significant increase in IL-1 bioactivity was observed that correlated with repair synthesis of DNA. Increased IL-1 bioactivity was also observed at stages IX-XII at 6 days post-irradiation. These observations support the concept that IL-1 is a spermatogonial growth factor which might also stimulate repair synthesis of DNA.

The effect of combined treatment with a GnRH agonist (buserelin depot, BUS, 6.6 mg every 2 months) with an antiandrogen (cyproterone acetate, CPA, 300 mg day-1) or a prolactin-suppressing agent (bromocriptine, BR, 20 mg day-1) on pituitary-testicular function were studied in patients with advanced prostatic carcinoma. The patients (n = 5-6 per group) were treated in this fashion for 6 months and thereafter orchidectomized. Serum testosterone and gonadotrophin responses were followed during treatment, and histology and certain endocrine parameters were studied using testicular tissue obtained at orchidectomy. Serum LH was suppressed in all treatment groups from mean levels of 4-6 IU l-1 to less than 0.1 IU l-1, whilst serum FSH levels decreased in all groups during the first month of therapy from 4.5-7 to 1-2 IU l-1, but recovered thereafter. Only minor increases in serum gonadotrophin levels were evident 3 months after castration. No differences in gonadotrophin responses were seen between the different treatment groups. Serum levels of testosterone were suppressed from 15-20 nmol l-1 to the castrate range (approximately 1 nmol l-1), in each of the treatment groups. Testicular weight decreased significantly more (P less than 0.05) in the BUS + CPA group, compared to the other treatments. No differences were found in the testicular concentration of testosterone, or LH and FSH receptors between the three treatment groups. On histological examination, spermatogenesis was found to be impaired severely in all groups, with the lowest Johnsen score in the BUS + BR group (2.16 +/- 0.13, vs. 2.73 +/- 0.25 with BUS alone, P less than 0.05). Seminiferous tubular diameters were reduced similarly in all treatment groups. In conclusion, the combination of CPA or BR with BUS in the treatment of prostatic carcinoma does not potentiate the suppression of gonadotrophin or testosterone secretion, evidently because the GnRH agonist exerts a maximal suppressing effect. However, other antigonadal effects were enhanced slightly, including suppressed testicular weights by CPA and further suppression of spermatogenesis by BR.

It has been shown previously that the intact adult rat testis produces large amounts of an interleukin-1 (IL-1)-like growth factor. The present study has investigated whether this testicular IL-1-like factor (tIL-1) is produced and secreted differentially by the fourteen stages of the seminiferous epithelial cycle in the rat testis. Seminiferous tubule segments representing defined stages were identified by transillumination-assisted microscopy and isolated by microdissection. Pooled segments were either homogenized and extracted with aqueous buffer or incubated for 24 h to produce conditioned media (CM). The recovered material was then analysed for IL-1 bioactivity in a sensitive murine thymocyte proliferation assay. When divided into four stage groups, extracts of stages II-VI, IX-XII and XIII-I showed equally high IL-1 activity whereas stage group VII-VIII showed much lower activity. More detailed analysis with 10 different stage groups showed that tIL-1 activity was undetectable in extracts of substages VIIab and VIIcd. The same pattern was seen when CM from cultured tubular segments were analysed. Labelling of seminiferous tubules with tritiated thymidine in vitro and analysis by autoradiography revealed that DNA-synthesizing spermatogonia were absent in substages VIIb and VIIc and sparse in substages VIIa and VIId. The results show that tIL-1 activity is produced in a stage-dependent manner and suggest that tIL-1 might be involved in the regulation of spermatogonial proliferation in vivo.