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The effects of 3 Gy local X-irradiation on the adult rat testis were studied together with exact determination of the radiation dose distribution in the testis. Seminiferous tubule segments were isolated 8-66 hours postirradiation (p.i.), squashed between a microscope slide and a coverslip, and the exact stage of the seminiferous epithelial cycle was identified under a phase-contrast microscope. The squash preparations were subjected to in situ end labeling (ISEL) for visualization and quantification of apoptotic cells. In controls, the highest numbers of apoptotic cells were scored in stages XII-XIV and I. In situ end-label staining of cells was observed in A3-A4 spermatogonia, spermatocytes at zygotene, pachytene, and meiotic division phases, as well as in early spermatids. In irradiated testes, from 8 hours p.i. and onward, intermediate- and B-type spermatogonia were sensitive at stages II-VI. At 42 hours, in stage I, elevated numbers of degenerating spermatocytes were seen. Most of them had not undergone meiotic divisions at stage XIV and showed an apoptotic type of degeneration at stage I. At the time of irradiation, the cells were in stage XIII, suggesting that diakinetic spermatocytes are particularly sensitive to irradiation. Also, preleptotene-zygotene spermatocytes in stages VII-XII were sensitive to irradiation. Apoptotic-type of cell degeneration was confirmed by living cell squash preparations, electron microscopy, and DNA electrophoresis. In conclusion, irradiation may provide a useful model system for studying apoptosis, and its control in spermatogonia and meiotically dividing cells.

The prooxidant effect of X-irradiation on rat testis and liver tissue was studied with doses of 0.5 and 3.0 Gy; the latter dose kills the proliferating spermatogonia and causes a maturation-depletion process in the germ cells. The level of lipid peroxidation, measured by the formation of diene conjugates and thiobarbituric acid-reactive substances (TBARS) and the activities of the antioxidant enzymes were determined 0.5 hours, 1 day, 7 days, and 31 days after the exposure. In the liver, increased levels of diene conjugation (+36%, P < 0.05) in the group of 3.0 Gy at 0.5 hours indicated increased lipid peroxidation. At the same time, TBARS were increased (+25%, P < 0.05) in the group of 0.5 Gy, but not in the 3.0-Gy group. In the testis, diene conjugation was not determined at 0.5 hours postirradiation, and at day 1 it was at the control level. The level of TBARS in the testis was below control (-11%, P < 0.01) in the 3.0-Gy group at day 1. At day 31 after 3.0 Gy in the testis, an increase in the amount of conjugated dienes (+24%, P < 0.01) was observed in parallel with a decreased level of TBARS (-15%, P < 0.01). The activity of superoxide dismutase (SOD) was decreased in the testis at 0.5 hours postirradiation (-28%, P < 0.05, and -29%, P < 0.05, in the groups of 0.5 and 3.0 Gy), whereafter it returned to normal by day 7. In the liver, such inactivation of SOD was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium. The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin. Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05). Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05). Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle. Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I. Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05). The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.

Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Testes of adult Sprague-Dawley rats were irradiated locally by 3 Gy of 4 MeV X-rays produced by a linear accelerator. This type and dose of radiation gives an even distribution through the testis and selectively kills the proliferating spermatogonia. The seminiferous tubular cells were quantified by DNA flow cytometry at defined stages of the epithelial cycle at 7, 17, 22, 38, 52, and 80 days after irradiation. The flow cytometric technique was modified by using frozen instead of fresh samples. Freezing did not alter cell numbers when compared with fresh samples. At 7 days post-irradiation no significant changes were observed in any cell population by DNA flow cytometry, whereas histological analysis revealed a reduction in intermediate and type B spermatogonia. At 17 and 22 days post-irradiation, the number of cells at meiotic prophase (4C) was decreased, particularly in stages II-V of the cycle. In stages VII-VIII, cell numbers were 40 and 31%, and in stages IX-XIII, 24 and 43% of that in non-irradiated controls at 17 and 22 days, respectively. At 38 days after irradiation, both 4C and 1C (haploid) cells were decreased in number. The 4C cells were reduced to 24, 17, and 13% of that in non-irradiated controls in stages II-V, VII-VIII, and IX-XIII of the cycle, respectively. The corresponding numbers of 1C cells were 5, 17, and 4%. At 52 days after irradiation, 1C cells had declined to 38 and 19% of control values in stages II-V and IX-XIII, respectively. In stages II-V, 1C' cells (haploid cells with condensed nuclei) declined to 28% of controls at 52 days. The present data provide a quantitative basis for the use of X-ray-irradiated rat testes as a model system in experiments pursuing interactions between Sertoli cells and spermatogenic cells.

Endogenous testosterone (T), LH and FSH receptors, and in vitro production of cyclic adenosine-3':5'-monophosphate (cAMP), T and some of its steroid precursors were measured in testicular tissue obtained at orchiectomy from seven prostatic cancer patients treated for 6 months with a potent gonadotropin-releasing hormone (GnRH) agonist analog (buserelin, Hoechst, 600 micrograms 3 times a day intranasally). In addition, histologic and morphometric studies were carried out on the testicular tissue. Testicular tissue from age-matched prostatic cancer patients (n = 14), whose first therapy was orchiectomy, served as controls. The peptide treatment decreased intratesticular T by 95% (P less than 0.01) and FSH receptors by 57% (P less than 0.01), but had no effect on LH receptors. The in vitro production of T decreased by 94% (P less than 0.01), but that of cAMP was unaffected. Besides T, the in vitro production of testicular 17-hydroxyprogesterone (17-OHP-4), androstenedione and 5 alpha-dihydrotestosterone (5 alpha-DHT) dropped by 71 to 90% (P less than 0.01 to 0.05) during buserelin treatment, but those of pregnenolone, progesterone and dehydroepiandrosterone (DHEA) were not affected. Histologic studies revealed considerable variation in the seminiferous epithelium of the control group, but spermatogenesis was highly suppressed in nearly all of the buserelin-treated group. The number of Sertoli cells was unaffected, but tubular diameters were reduced (P less than 0.05) by buserelin treatment. Leydig cells appeared dedifferentiated in this group, although their number per testis was not altered. These data indicate that gonadotropin suppression by GnRH agonist most likely affects testicular steroidogenesis by inhibiting 3 beta-hydroxysteroid dehydrogenase and a step(s) prior to pregnenolone formation. The treatment does not impair testicular LH binding or cAMP production, but clearly suppresses FSH receptors. Spermatogenesis in general is suppressed but with considerable variation.

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.

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.