Marvin L. Meistrich

Chapter 2 Separation of Spermatogenic Cells and Nuclei from Rodent Testes

Publisher Summary This chapter describes and evaluates separation methods employing two physical parameters— density and sedimentation rate. The chapter also discusses the use of ultrasound or enzymes to destroy cells and nuclei selectively. The techniques, as currently being used in the laboratory are outlined. Velocity sedimentation, equilibrium density centrifugation, and ultrasonic disruption are used to obtain enrichment of all classes of cells and nuclei from adult rodent testes. Several types of nuclei, including pachytene spermatocytes, round spermatids, early elongated spermatids, and late elongated spermatids can be obtained in purities by using the appropriate combinations of methods. The important factors and principles involved in the development of the separation methods are emphasized. The chapter reviews the methods developed in other laboratories and compares the results obtained with the different procedures. Using the cell separation methods, spermatogenesis as a model of cell differentiation can be exploited and the sequence of macromolecular events involved in sperm development can be mapped. The availability of purified populations of specific testicular cell types will permit the elucidation of other important events of spermatogenesis, such as genetic recombination, meiosis, spermatid morphogenesis, and nuclear inactivation and condensation.

Nuclear protein transitions in rat testis spermatids.

Abstract Dramatic transitions occur in the nuclear proteins during spermiogenesis in rats. In order to determine more precisely when these transitions occur, we have employed centrifugal elutriation, velocity sedimentation at unit gravity, centrifugation in metrizamide gradients, and sonication to obtain relatively homogeneous populations of testis cells and nuclei. The results indicate that histones are present in step 1–8 spermatid nuclei but are not detectable after step 12. Nuclear proteins designated TP and TP2 are not detectable in step 1–8 spermatids but are present and actively synthesized in step 13–15 spermatids. These two proteins are turned over within 5 days after synthesis. A spermatidal basic nuclear protein, designated TP3, and the sperm basic nuclear protein, S1, are present in step 16–19 spermatids. Biochemical characterization of TP2 and TP3 are presented.

Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes.

The presence and biosynthesis of the testis-specific isozyme of lactate dehydrogenase (LDH-X) in cells at various stages of spermatogenesis have been examined. Enrichment of testicular cells in various stages of spermatogenesis has been achieved by two methods: (1) cell separation by velocity sedimentation in the Elutriator rotor and (2) γ irradiation of testes to eliminate specific classes of testicular cells. Separation of cells from immature mice indicated that cells prior to the midpachytene stage contain no LDH-X. Measurement of LDH-X levels in cells separated from adult mice and in testicular homogenates prepared at various times after irradiation indicated that the highest level of LDH-X per cell (normalized for DNA content) was in spermatids. Synthesis of LDH-X was determined, after in vivo injection of [3H]valine, by measurement of the radioactivity in LDH-X precipitated with specific antiserum. After irradiation, the rate of LDH-X synthesis remained constant, despite the loss of early primary spermatocytes. In separated cells, the rate of LDH-X synthesis was highest in late pachytene spermatocytes, lower in round spermatids, and even lower, but still significant, in elongated spermatids. Therefore, the synthesis of LDH-X begins at a specific point during spermatogenesis, the midpachytene stage of spermatocyte development, and continues throughout spermatid differentiation.

Changes in sperm nuclei during spermiogenesis and epididymal maturation

Abstract Mouse and rat spermatid nuclei pass abruptly through developmental stages characterized by increased resistance to disruption by various agents. The mouse spermatid nucleus becomes resistant to sonication at step 12, resistant to digestion by trypsin-DNase at step 15 and resistant to lysis by SDS between the testis and the caput epididymis. These alterations are correlated with changes in the basic nuclear proteins.

Synchronization of mouse L-P59 cells by centrifugal elutriation separation.

Abstract Cell separation by centrifugal elutriation was employed to obtain synchronized cells from asynchronous populations of mouse L-P59 fibroblast cells. The fractions most enriched in specific phases of the cell cycle contained about 90% G1-phase cells, 70% S phase cells and 60% G2 + M phase cells, respectively, as determined by pulse cytophotometry and autoradiography. When replated, cells from separated fractions divided synchronously in culture. The method is rapid and large numbers of cells (3 × 10 8 ) can be processed in a single run with no loss of viability. This method should be applicable to most cell lines which can be obtained in single-cell suspension.

Partial characterization of a new basic nuclear protein from rat testis elongated spermatids

Abstract A basic protein designated TP2 has been isolated from rat testis elongated spermatids. This new protein contains basic and acidic amino acids in relative amounts similar to those in histone F2al but is unusually rich in serine and proline. Techniques which were developed for preparing relatively homogeneous populations of spermatid nuclei were used to demonstrate that TP2 is most abundant and most actively synthesized in spermatids representing steps 12 through 15 of spermiogenesis.

Flow cytometry of human spermatozoa

SummaryMethods are given for the preparation and staining of human spermatozoa for flow cytometric DNA measurements. Using agents for the reductive cleavage of disulfide crosslinks and suitable proteolytic enzymes and effective decondensation of the sperm chromatin and a DNA-proportional uptake of fluorochromes is achieved. Thus reliable and precise measurements of the relative DNA content of human spermatozoa are possible and the two subpopulations of haploid spermatozoa can be distinguished according to the difference in their DNA content.

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Abstract Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by 3 H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.

Flow cytometry of DNA in mouse sperm and testis nuclei

Abstract Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation fo DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine-Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei. The additional peaks, located at 3.0 and 3.7 times the fluorescence intensity of round spermatid nuclei correspond to leptotene and zygotene spermatocytes and to late pachytene spermatocytes, respectively. These peaks contained clumps of nuclei. The homogeneity of the nuclei sorted from the peaks, as well as the relative sizes of the peaks, was enhanced when the nuclei were prepared from cells enriched in specific stages of development. The relative fluorescence intensities of the various testis nuclei were characteristic and repeatedly found but were not stoichiometric with the DNA content of the nuclei.

“Cytogenetic” studies of spermatids of mice carrying Cattanach's translocation by flow cytometry

The DNA content of spermatids of mice carrying Cattanachs translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X-and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2±0.1% of the total haploid genome as compared to 3.4±0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanachs translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.