Germ cell transfer into rat, bovine, monkey and human testes.

Germ cell transplantation is a potentially valuable technique offering oncological patients gonadal protection by reinitiating spermatogenesis from stem cells which were reinfused into the seminiferous tubules. In order to achieve an intratubular germ cell transfer, intratubular microinjection, efferent duct injections and rete testis injections were applied on dissected testes of four different species: rat, bull, monkey and man. Ultrasound-guided intratesticular rete testis injection was the best and least invasive injection technique with maximal infusion efficiency for larger testes. Deep infiltration of seminiferous tubules was only achieved in immature or partially regressed testes. This technique was applied in vivo on two cynomolgus monkeys. In the first monkey a deep infusion of injected cells and dye into the lumen of the seminiferous tubules was achieved. In the second, transplanted germ cells were present in the seminiferous epithelium 4 weeks after the transfer. These cells were morphologically identified as B-spermatogonia and located at the base of the seminiferous epithelium. In summary, this paper describes a promising approach for germ cell infusion into large testes. The application of this technique is the first successful attempt of a germ cell transfer in a primate.

The cycle duration of the seminiferous epithelium remains unaltered during GnRH antagonist-induced testicular involution in rats and monkeys.

Although the gonadotropic control of the spermatogenic process is well established, the endocrine regulation of the timing and kinetics of germ cell development has received little attention. We found previously that the administration of a GnRH antagonist (ANT) over a period of 25 days could retard spermatid development and slightly prolong cycle length in intact adult cynomolgus monkeys (Macaca fascicularis). The aim of the present study was to investigate the effects of extended exposure to ANT on the duration of the cycle of the seminiferous epithelium in the monkey. Additionally, the duration of spermatogenesis was studied in the ANT-exposed rat model. In experiment 1, monkeys were given either saline or ANT (n=6/group) and on day 30 all animals received a single injection of 5-bromodeoxyuridine (BrdU) to label S-phase germ cells. Testicular biopsies were taken on days 39, 43, 47 and 51 (end of treatment) for BrdU localization and flow cytometric analysis. ANT treatment suppressed hormone levels, reduced testis size by >70% and severely impaired germ cell production. Despite these alterations, cycle duration remained unchanged at all time-points compared with controls (10.12+/-0.15 days vs 10.16+/- 0.44 days). In experiment 2, adult male Sprague-Dawley rats (n=15/group) received either vehicle (VEH) or ANT for 14 days and received BrdU injection on day 2. Cycle duration was found to be shorter in the ANT-treated group (12.45+/-0.09 days) than in the control group (12.75+/-0.08, P<0.05). As spermatogenic cycle length in this control group was longer than that of our historical controls (range: 12.37-12.53 days), experiment 2 was repeated (n=10/group). In experiment 3, cycle duration was 12.51+/-0.02 for VEH and 12.46+/-0.05 for the ANT-treated group (P>0.05) in both species. We concluded that the duration of the cycle of the seminiferous epithelium in monkeys and rats is independent of gonadotropins but is rather regulated by the spermatogenic tissue itself.

Gonadotrophin-releasing hormone antagonist arrests premeiotic germ cell proliferation but does not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry.

Meiosis constitutes a crucial phase of spermatogenesis since the recombination of genetic information and production of haploid round spermatids need to be achieved. Although it is well established that gonadotrophic hormones are required for completion of the spermatogenic process, little is known about the dynamic and kinetic aspects of development of spermatocytes into spermatids and its endocrine control in the primate. In this study, S-phase germ cells were labelled using 5-bromodeoxyuridine (BrdU) incorporation and were then followed throughout meiosis under normal conditions and following GnRH antagonist (ANT)-induced gonadotrophin withdrawal in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis). Adult animals received either vehicle (VEH, n = 4) or the ANT cetrorelix (n = 5) throughout 25 days. On day 7 all animals received a bolus injection of BrdU. A biopsy was performed after 3 h, one testis was removed 9 days later (day 16 of treatment) and the other testis after 18 days (day 25 of treatment). Serum testosterone and inhibin levels, and testis weight were reduced (P < 0.05) by ANT treatment. BrdU localized to pachytene spermatocytes 9 days after BrdU and to round spermatids 18 days after BrdU in both groups, demonstrating that BrdU-labelled pachytene spermatocytes had undergone meiosis. Flow cytometric analysis revealed that the relative number and number per testis of BrdU-tagged 2C and 4C cells were reduced significantly (P < 0.05) within 16 days of ANT treatment. Numbers of 1C cells were lowered by day 25. The cell ratio for 1C:4C was similar with VEH and ANT (P > 0.05). These findings indicate that ANT reduced the number of cells available for meiosis but did not alter the rate of transition into round spermatids. Unexpectedly, however, the stage-dependent progression of BrdU-tagged round spermatids was significantly (P < 0.05) retarded under ANT as seen from the frequency of tubules containing BrdU-labelled round spermatids. The average duration of spermatogenic cycle was slightly prolonged (9.8 days in the VEH group and 10.8 days in the ANT group (P = 0.09)). Since no atypical germ cell associations could be found, it remains unclear whether this slight prolongation is entirely due to altered spermatid progression or whether earlier phases are affected. We conclude for the nonhuman primate that (1) BrdU-labelling of premeiotic germ cells is suitable for tracing their meiotic transition into postmeiotic cells, (2) unlike in the rat, gonadotrophin suppression initially affects premeiotic cell proliferation and thus the number of cells available for meiosis, (3) the meiotic process continues quantitatively despite gonadotrophin deficiency and (4) prolonged gonadotrophin deficiency might alter the timing of germ cell development.

Estimation of the duration of the cycle of the seminiferous epithelium in the non-human primate Macaca mulatta using the 5-bromodeoxyuridine technique.

A comparatively low yield of germ cells has been reported for the spermatogenic process in primates. Kinetic studies of spermatogenesis and the spermatogenic cycle are needed to investigate this phenomenon but require the application of radioactively labeled compounds or irradiation. We have therefore investigated the suitability of a non-radioactive approach, viz., administration of 5-bromodeoxyuridine, for the determination of the kinetics of the spermatogenic cycle in a non-human primate, the rhesus monkey (Macaca mulatta). Four adult in-season animals received a bolus of 33 mg/kg 5-bromodeoxyuridine, one testis from each monkey was removed 3 h later and the other testis after 10 days and 11 h. Tissue was fixed in Bouin's solution and embedded in Paraplast. 5-Bromodeoxyuridine was localized by immunogold-silver staining with a monoclonal antibody. PAS-hematoxylin counterstaining was used for spermatogenic stage identification. At 3 h, the leptotene and zygotene spermatocytes in stages VII-IX were the most advanced 5-bromodeoxyuridine-positive cells. At 10 days 11 h, the label had advanced and pachytene spermatocytes in stages VI-IX contained 5-bromodeoxyuridine. The duration of the spermatogenic cycle was 10.42+/-0.07 days (range: 10.25-10.62 days). Peritubular cells and interstitial cells were rarely 5-bromodeoxyuridine-positive, and Sertoli cells were consistently negative for 5-bromodeoxyuridine. Importantly, our kinetic data closely resemble those obtained by means of the application of irradiation for this macaque species. We conclude that administration of 5-bromodeoxyuridine represents a non-radioactive reliable approach for studying kinetic aspects of the spermatogenic process in primates.

Expression of mitotic cyclin B1 is not confined to proliferating cells in the rat testis.

Spermatogenesis is a precisely controlled and timed process comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, and the maturation and differentiation of haploid spermatids. Cell proliferation is controlled by genes involved in the regulation of the cell cycle. Among the principal regulatory proteins are cyclins, which are categorized according to their appearance during the cell cycle. B-type cyclins are mitotic cyclins and function at the G2/M transition of the cell cycle. We have investigated the expression and regulation of cyclin B1 during rat spermatogenesis. Rat cyclin B1 was isolated from a testis cDNA library and further used as a probe to detect mRNA expression. Northern blot hybridization of testis mRNA revealed the presence of a single 1.7-kilobase transcript. In situ hybridization showed stage-specific expression during spermatogenesis with highest expression found in late pachytene spermatocytes and early round spermatids. This pattern was confirmed in fractions of isolated germ cells. Immunocytochemistry displayed highest protein levels in round spermatids. Depletion of gonadotropins did not change the quantitative and qualitative expression pattern of cyclin B1. Therefore, the signals triggering the onset of cyclin B1 expression seem not to originate from the pituitary-gonadal endocrine axis and might therefore be paracrine factors originating within the germinal epithelium. Our observations suggest that cyclin B1 plays a hitherto unknown role in spermatid maturation in addition to its known function in dividing cells.

The duration of the cycle of the seminiferous epithelium is altered by administration of 2,5-hexanedione in the adult Sprague-Dawley rat.

The duration of the cycle of the seminiferous epithelium is believed to be under genetic control rather than being influenced by other factors. However, the frequencies of certain spermatogenic stages--reflecting their relative durations--can be altered under various conditions including treatment with the n-hexane metabolite, 2,5-hexanedione (HD). To investigate whether HD administration alters the duration of the spermatogenic process, adult Sprague-Dawley rats were exposed to vehicle (n = 20) or 1% HD dissolved in the drinking water (n = 40) throughout a period of 29 days. On day 17, 100 mg/kg of 5-bromodeoxyuridine (BrdU) was given intraperitoneally; 3 hours later one testis was removed, and the remaining testis was excised 12 days later. BrdU was localized in Bouin's-fixed, Paraplast-embedded testes using immunogold-silver-staining, and sections were counterstained with periodic acid--Schiff and hematoxylin to permit identification of the spermatogenic stages. HD treatment reduced weights of body, testis, and epididymis (P < 0.05) but had no effects on serum levels of follicle-stimulating hormone or testosterone. The seminiferous epithelium was vacuolized, and spermatogenesis was affected to various degrees. HD significantly reduced the frequency of stage VII tubules (P < 0.01) and increased the frequency of stage IX and stage X tubules (P < 0.05). Furthermore, HD reduced the stage-dependent progression of BrdU-labeled germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Duration of the cycle of the seminiferous epithelium, estimated by the 5-bromodeoxyuridine technique, in laboratory and feral rats.

The stability of the duration of the cycle of the seminiferous epithelium was determined by investigating incorporation of 5-bromodeoxyuridine into S-phase germ cells of normal and hemicastrated standard laboratory rats (Sprague-Dawley) and feral Brown/Norway rats (Rattus norvegicus). Feral rats were trapped on farms in the surroundings of Münster. The duration of the cycle of the seminiferous epithelium, determined at intervals of 12 days (3 h versus 12 days 3 h after 5-bromodeoxyuridine injection), was remarkably constant and similar in intact laboratory rats (12.49 +/- 0.05 days, n = 13, mean +/- SEM) and feral rats (12.44 +/- 0.06 days, n = 8). In hemicastrated laboratory and feral rats the duration of the cycle was similar to that in intact animals, indicating that hemicastration did not influence the kinetics of the seminiferous epithelium cycle. However, the coefficients of variation of the estimated duration of the cycle of the seminiferous epithelium were at least three times lower in hemicastrated rats (one testis from the same animal serving as reference point) compared with that of intact rats (the reference point based on the average staining frequency at 3 h). Overall, no significant differences between laboratory and feral rats could be observed with regard to testis weight and serum concentrations of FSH and testosterone. The number of cells per testis, determined by flow cytometry, was similar in laboratory and feral rats, except for a slight but significant difference in the haploid:tetraploid cell ratio (6.3 +/- 0.2 versus 7.5 +/- 0.3, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)