Immunofluorescence and immunogold electron microscopy were employed to examine the assembly of connexins (Cx) 33, 37, and 43 into testis cell gap junctions in mature and postnatal rats. Cx37 was localized by immunofluorescence to the endothelia of blood vessels in both mature and immature testes and was not further characterized. Only Cx43 assembled into Leydig cell gap junctions, but Cx43 also co-assembled with Cx33 in some Sertoli-Sertoli gap junction plaques within and near Sertoli occluding junctions and on adluminal surfaces Assembly of Sertoli gap junctions appeared to be regulated according to the stage of the seminiferous epithelium since Cx33 (and Cx43) immunoreactivity was strong in Sertoli cells from stages II-VII but weak in stages IX-XIV. During postnatal maturation, assembly of Cx33 into gap junctions was regulated independently of Cx43 assembly. Cx43 was present on Sertoli cells of all tubules from postnatal Day 5 through Day 28. In contrast, Cx33 was not apparent on Sertoli cell surfaces until Day 15 and gradually accumulated in all tubules through Day 28. Between postnatal Days 38 and 43, the immunoreactivities of Cx33 and Cx43 became weak in Sertoli cells containing step 9-14 elongated spermatids. Thus, connexin abundance and gap junction composition in Sertoli cells is regulated during testis maturation and the cycle of the seminiferous epithelium.

Sertoli and spermatogenic cells establish germ cell- and epithelial stage-dependent networks of cell-cell communication thought to be important for the initiation and maintenance of spermatogenesis. Since gap junctions assemble between Sertoli cells and between Sertoli and spermatogenic cells, it was hypothesized that multiple, unique routes of cell-cell communication may be established, in part, by the assembly of structurally diverse gap junctions from the connexin (Cx) multigene family. Differences in channel structure may support differences in ion or second messenger permeability between cell types. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that 11 Cx mRNAs were present in total RNA from seminiferous tubules and that 10 of the Cx mRNAs were present in polysomes and presumably translated. RT-PCR analyses also showed that the Cx mRNA population varied between different seminiferous tubule cell types. There were 9 Cx mRNAs in germ cells, 8 in Sertoli cells, and 5 in peritubular cells. One of the Cx mRNAs, Cx-50, was detected only in pachytene spermatocytes and round spermatids. Comparisons of the Cx mRNAs present in tubules at different postnatal ages showed that at least 2 Cxs (Cx-33 and Cx-50) accumulated when leptotene-zygotene stages developed. The multiple Cx genes and proteins produced in spermatogenesis may support the assembly of structurally diverse gap junctions.

DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5-100 micrograms/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5-6 micrograms/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6-100 micrograms/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 micrograms/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by "intermediate-type" protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.

No abstract

Dye coupling experiments were performed to determine whether the gap junctions connecting Sertoli cells with other Sertoli cells and different germ cell stages in rats showed functional variations. Chop loading of adult rat seminiferous tubules was conducted using fluorescent dextran controls and a variety of low-molecular-weight tracers (lucifer yellow, biotin-X-cadaverine, biotin cadaverine, and neurobiotin) to evaluate dye coupling in situ, and scrape loading was used to study dye coupling in Sertoli-germ cell cocultures established using prepuberal rats. Sertoli-Sertoli coupling is relatively short range and nonselective in situ, whereas coupling between Sertoli cells and chains of spermatogonia is strongly selective for the positively charged biotin tracers relative to negatively charged lucifer yellow. Coupling between Sertoli cells and spermatogonia was also asymmetric; lucifer yellow in germ cells never diffused into Sertoli cells, and biotinylated tracers only weakly diffused from spermatogonia to Sertoli cells. Asymmetric coupling would facilitate the concentration in germ cells of molecules diffusing through junctions from Sertoli cells. Dye coupling between Sertoli cells and adluminal germ cells was too weak to detect by fluorescence microscopy, suggesting that the junctional communication between these cells may be functionally different from that between Sertoli and basal germ cells. The results show that there are multiple routes of gap junction communication in rat seminiferous tubules that differ in permeability properties and show alternative gating states. Functional diversity of gap junctions may permit regulated communication among the many interacting Sertoli cells and germ cell stages in the seminiferous epithelium.