DNA in eucaryotic cells is organized into loop domains, ranging in size from 25 to 100 kb, that are attached at their bases to the structural component of the nucleus termed the nuclear matrix. These DNA loop domains have been shown to be important in the regulation of both DNA replication and RNA transcription. In this study we have compared the structural organization of the DNA loop domains of the 5S rRNA gene cluster in sperm, liver, and brain nuclei in the Syrian golden hamster. The individual loop domains were visualized by fluorescent in situ hybridization to protamine (sperm)- and histone (somatic)-depleted nuclei, termed nuclear matrix halo preparations. We found that in sperm nuclei, the 5S rRNA gene cluster was organized into three small loop domains that were approximately 48 kb each. In both types of somatic cell nuclei examined, the 5S rRNA gene cluster was organized into a single, much larger loop domain that was up to 480 kb in length. The data suggest that at least some of the compaction that sperm DNA undergoes during spermiogenesis is mediated by the nuclear matrix independent of protamine binding. Additionally, this sperm-specific DNA organization may be involved in the specific patterns of DNA replication and transcription of the paternal genome in the embryo.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.

We mapped the positions of three different genes in the flat, hook-shaped hamster sperm nucleus to determine the specificity of sperm DNA positioning. The positions of the 5S rRNA gene cluster, the CAD gene, and the class I 1.6 gene were determined by fluorescent in situ hybridization (FISH) in over 50 hamster sperm nuclei for each gene. We first demonstrated by FISH with mitotic chromosomes that the latter two genes were localized on the same chromosome. Within the sperm nuclei, we found that the precise position was variable for each of the three genes, but that there were two areas of preferred localization that contained 26-31% of the nuclear area and within which 80% of the signals were located. Nuclei were then hybridized to two genes simultaneously, using either two genes located on the same chromosome or two genes located on different chromosomes. We found no preference for orientation of one gene relative to the other for either pair of genes examined. This suggested that the relative arrangements of chromosomes within the sperm nucleus are flexible. These data demonstrate that the topographical arrangements of genes within the hamster sperm nucleus have a limited plasticity allowing for a relatively large range of possible localization.

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