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W. J. Barcellona and M. L. Meistrich

Summary. Mouse testicular cells were examined ultrastructurally to determine whether the cells are damaged during the preparation of single-cell suspensions or during cell separation. The testicular cells were dissociated from seminiferous tubules by trypsinization and were fixed immediately; fixed after being held in suspension for 4 h at 4°C; or fixed after being separated into enriched fractions by sedimentation velocity either at unit gravity or by centrifugal elutriation. In general, the ultrastructural integrity of the cells, compared with that of corresponding testicular cells fixed in situ, was maintained during the dissociation and separation procedures. Ultrastructural abnormalities were most frequently produced in Sertoli cells and were occasionally observed in the acrosomes and nuclei of round spermatids. The cytoplasmic matrix of the midpiece of mature elongated spermatids or spermatozoa and the acrosomes of these cells were often disrupted. It is suggested that the dissociation procedures were responsible for most of the observed alterations of ultrastructural integrity.

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M. E. A. B. van Beek and M. L. Meistrich

Summary. Rats maintained on a diet deficient in retinol and retinoic acid were given a diet containing retinoic acid for 21–29 days after the start of weight loss. The testes of four of these rats were studied. Spermatogonia of all types were observed, though in lower numbers than in controls, and their mitotic activity was normal. Normal preleptotene spermatocytes were encountered, but no normal spermatocytes in further stages of development were seen. Pale cells that appeared to be in prophase were observed. It was concluded that, in retinol-deficient rats maintained on retinoic acid, the spermatogonial population is qualitatively normal, but quantitatively subnormal, while spermatocyte development is qualitatively and quantitatively abnormal. No evidence of spermatogonial arrest or any other form of synchronization was found in testes of these rats, but when the remaining rats were injected with retinol, the seminiferous epithelium did show stage synchronization at 36 and 128 days after the injection.

Keywords: spermatogenesis; retinol; rat

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D. E. Walters, R. G. Edwards and M. L. Meistrich

Summary. All the available data relating to pregnancy and multiple implantations from Bourn Hall Clinic are used to assess the accuracy of two statistical models of implantation. The simple binomial model was found to be incapable of describing both the pregnancy rates and the incidence of multiple implantations, whereas a two-parameter model, incorporating both patient and embryo variability, provided a good fit to the data. The estimates obtained for the two parameters in the second model suggested that 36% of the embryos could implant, and that 43% of patients were capable of sustaining implantation. The Maximum Likelihood method of constructing 95% confidence limits on the estimates of the parameter values is demonstrated.

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F. M. F. van Dissel-Emiliani, D. G. de Rooji and M. L. Meistrich

Summary. A method for obtaining enriched populations of gonocytes from rat embryos at 18 days p.c. has been developed. Single cell suspensions with high cell yield and good viability of the cells were obtained by a collagenase/trypsin digestion of the testes. Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity. Populations of 600 000 gonocytes (70–75% purity), sedimenting at about 12 mm/h, could be obtained from 30–35 fetal rats within 8 h after killing. Purities were determined by Nomarski microscopy and verified in fixed preparations and by Coulter volume measurements.

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M. L. Meistrich, T. J. Hughes, T. Ambus and W. R. Bruce

Summary. Male BC3F1 hybrid mice were injected with 10 μg oestradiol benzoate/day alone or in conjunction with 80 μg testosterone propionate/day. Oestrogen alone decreased the weights of the accessory sex organs, but oestrogen + testosterone resulted in an increase. Assessment of spermatogenesis by a variety of methods revealed little effect of the hormones. Testicular weight and the histological appearance of the seminiferous epithelium was not altered appreciably, although Leydig cell atrophy was apparent when testosterone was given. The kinetics of spermatogenesis, studied by cell separation techniques and biochemical features of spermatogenesis, including protamine biosynthesis, lactate dehydrogenase levels and the formation of the highly resistant, condensed sperm nucleus, were also unaffected by the hormonal treatments. However, a reduction in the number of intact testicular spermatozoa in mechanically prepared cell suspensions was obtained. It is concluded that, in these animals, spermatogenesis is relatively insensitive to the administration of oestrogen.

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C A Rezende-Melo, A L Caldeira-Brant, A L Drumond-Bock, G M Buchold, G Shetty, F R C L Almeida, M M Matzuk, K Hara, S Yoshida, M L Meistrich and H Chiarini-Garcia

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14−/ mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.