In order to better understand the implications of gap junction proteins in spermatogenesis, connexin 43 (Cx43), the most abundant connexin in the testis, was evaluated in testes of wild-type mice and of two mutants with impaired spermatogenesis (ebo/ebo, and jun-d-/-mice). Reverse transcription-polymerase chain reaction (RT-PCR) amplification revealed a constitutive expression of mRNA for Cx43 in both wild-type mice and infertile mutants. In the seminiferous tubules of wild-type mice, indirect immunofluorescence revealed that Cx43 expression was stage-dependent and that the signal was mainly located in the region of Sertoli cell occluding junctions. Colocalization of Cx43 and of the tight-junction-associated protein zonula occludens 1 (ZO-1) was demonstrated in seminiferous tubules by using dual-label immunofluorescence in conjunction with confocal microscopy. The Cx43 staining analyzed by high-resolution confocal microscopy appeared as continuous, anastomozed ribbons and thin dots. The level of Cx43 immunoreactivity was reduced in seminiferous tubules of ebo/ebo and jun-d-/- mutants as compared to the respective wild-type mice. No staining for Cx43 was detected in Sertoli cell-only seminiferous tubules observed sometimes in jun-d-/- mice. The present study represents one of the first in vivo examples of alteration of seminiferous tubule Cx43 in testes with impaired spermatogenesis.

In the present study we report the isolation and characterization of a clonal Sertoli cell line (42GPA9) from sexually mature polyoma virus large T (PyLT) transgenic mice. The cells multiplied indefinitely and expressed large T antigen. The 42GPA9 cell line expressed biochemical features associated with normal Sertoli cells. Transferrin, sulfated glycoprotein-2, and the ligand of c-kit were detected by reverse transcription-polymerase chain reactions and Western blot analyses. Zymographic analysis indicated that the 42GPA9 cell line secreted tissue-type plasminogen activator. These cells also retained FSH receptors as suggested by their specific responsiveness to the gonadotropin (morphological and phagocytic changes, stimulation of cAMP production) and the detection of FSH receptor mRNAs. Another original aspect of the 42GPA9 cell line is its ability to form tight junctions at confluency as demonstrated by electron microscopic study and immunolocalization of the tight junction-associated protein zonula occludens 1. The 42GPA9 cell line, which has retained several important hallmarks of normal Sertoli cells, may prove useful for further studies on Sertoli cell behavior and on Sertoli-germ cell interactions in the mature testis.

We have investigated the male sterility associated with a new recessive mutation of the house mouse: ébouriffé (ebo). All spermatozoa present in the epididymis showed severe malformations, mostly of the tail. Light and electron microscopy showed a drastic decrease of the spermatid population, whereas spermatogonia and spermatocytes seemed moderately affected. This suggests that the mutation affects mostly spermiogenesis. Defects appeared during formation of the acrosome: the acrosomal granule was frequently vacuolated at stages II-III, giving rise first to abnormal acrosomes (stages VI-VII) with dilations and perforations, and then to an abnormal head and acrosome shape (stages IX-XI). However, the most common malformations affected the flagella in elongated spermatids. Sometimes the centriole doublet did not move into the implantation fossa, causing an unattached and isolated flagellum. The major defect occurred in the midpiece region of differentiating spermatozoa: flagellar components were present but highly disorganized, and mitochondria were aggregated in a compact mass. Even though we have no evidence that the ebo gene is a testis structural gene or a regulatory gene that disrupts the complex spermatogenesis process, this mutation may provide an interesting tool for studying the late stages of spermatogenesis. Using an interspecific backcross, we localized the ebo mutation on chromosome 2, with no recombination out of 44 meioses with locus D2Mit32.

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electron-microscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.

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