Frédérique Vidal

Cre expression in primary spermatocytes : A tool for genetic engineering of the germ line

Transgenic mice were generated expressing a testicular Cre recombinase driven by promoter sequences derived from the gene encoding Synaptonemal Complex Protein 1 (Sycp1), expressed at an early stage of the male meiosis (leptotene to zygotene). Recombination at target LoxP sites was examined during germinal differentiation in mice harboring Sycp1‐Cre and a second transgene where LoxP sites flank either the βgeo coding region, the Pgk1 promoter, or a tk‐neo cassette inserted into the Rxrα locus. The LoxP‐flanked transgenes were stably maintained in the somatic tissues of the double transgenic animals, as well as in the progeny of the females. Mice born after mating the double‐transgenic males with normal females showed extensive deletions of the LoxP‐flanked sequences. When the males were hemizygous for the Sycp1‐Cre transgene, the deletions were observed even in the fraction of the offspring which had not inherited the Cre gene, thus demonstrating that expression occurred in the male parent during spermatogenesis. The high efficiency of excision at the LoxP sites makes the Sycp1‐Cre transgenic males suitable for evaluating the role of defined gene functions in the germinal differentiation process. Mol. Reprod. Dev. 51:274–280, 1998.

Germ cells and fatty acids induce translocation of CD36 scavenger receptor to the plasma membrane of Sertoli cells.

The CD36 scavenger receptor is involved in the uptake and transport of fatty acids, as well as the phagocytosis process in macrophages. We show here that the CD36 protein is expressed by Sertoli cells in the seminiferous epithelium, mainly during the stages where phagocytosis takes place. Using a Sertoli-derived cell line, we show that addition of germ cells and residual bodies triggers a re-localization of CD36 from the cytoplasm to the plasma membrane of the cells, while latex beads do not. Moreover, Sertoli cell phagocytosis of germ cells, but not of latex beads, is reduced by the presence of fatty acids in the culture medium. In the testis, CD36 plays a key role in both phagocytosis and lipid recycling, for constant production of mature spermatozoa.

A murine sequence-specific DNA binding protein shows extensive local similarities to the amyloid precursor protein

Microinjection experiments suggested previously that protein binding to the DNA nucleotide sequence GTCACATG, identical to the CDEI element of the yeast centromere, plays an important role in the early development of the mouse. We established from a series of overlapping mouse cDNA clones the sequence of a candidate CDEI-binding protein. Synthesis in Escherichia coli of a fusion protein which binds specifically the CDEI box in vitro confirmed its identification. On the other hand, the translated 511 amino acid sequence shows two regions with high degrees of similarity to the protein precursor (APP) of the beta-protein (amyloid) that accumulates in the brain and blood vessels of Alzheimer patients. A continuous stretch of 195 amino acids includes 133 residues identical to part of the extracellular domain of APP, and 48 of the 70 C-terminal residues of the open reading frame are identical to the APP transmembrane and cytoplasmic domains.

A novel germ line-specific gene of the phosducin-like protein (PhLP) family. A meiotic function conserved from yeast to mice

We identified a new member of the phosducin-like (PhLP) protein family that is predominantly, if not exclusively, expressed in male and female germ cells. In situ analysis on testis sections and analysis of purified spermatogenic cell fractions evidenced a stage-specific expression with high levels of RNA and protein in pachytene spermatocytes and round spermatids. Three mRNA species were detected, which correspond to different polyadenylation sites and vary in abundance during germ cell maturation. Only low levels of RNA were detected in whole ovary extracts, but expression of the protein became detectable within hours after hormonal induction of superovulation. The gene (Mgcphlp) is located on mouse chromosome 5 in the immediate vicinity of the Clock locus. The predicted amino acid sequence shows extensive similarities not only with the known mammalian PhLP proteins but also with the yeast phosducin-like protein Plp2, required for the production and growth of haploid cells. Expression of the murine protein was found to complement the defect of a yeastplp2Δ mutant. We propose that MgcPhLP/Plp2 proteins exert a function in germ cell maturation that is conserved from yeast to mammals.

Gene control in germinal differentiation: Rnf6, a transcription regulatory protein in the mouse Sertoli cell

ABSTRACT In mouse Sertoli cells, transcription of the Inha gene encoding the α subunit of inhibin, which acts locally as a tumor suppressor, is down-regulated in tumors and in normal cells during aging. Previous studies suggested that regulation of Inha transcription involves the binding of a protein(s) to a repeat of the GGGGC motif in the promoter. Expression screening identified a cDNA encoding a protein that binds this sequence. Of the RING-H2 family, it is the mouse homologue of a human protein of unknown function, RNF6. The mouse gene, Rnf6, is predominantly expressed in two interacting cell types of the testis, Sertoli cells and pachytene spermatocytes. In Sertoli cells, it colocalizes with the PML and Daxx proteins in punctate nuclear bodies. In transient and stable transfectants, Rnf6 expression from a heterologous promoter increased the expression of reporter genes driven by the Inha promoter. In a Sertoli tumor cell line in which expression of both Inha and Rnf6 was reduced, reexpression of the latter restored the level of Inha while, concomitantly, the cells reverted to normal growth control in culture.

A role of inhibin as a tumor suppressor in Sertoli cells: down-regulation upon aging and repression by a viral oncogene

Inhibin, a member of the TGF-β superfamily, is synthesized in the testis by Sertoli cells and exerts an endocrine regulatory function on pituitary hormone synthesis. A distinct local function has been proposed, negatively controlling cellular growth in the testis (tumor suppressor activity). A critical test for the identification of a tumor suppressor is the reversal of transformed growth properties upon re-expression of the gene in tumor-derived cell lines. Sertoli cell-derived tumoral lines were previously established from tumors that develop in elderly transgenic males which express in the testis the large T antigen of polyoma virus. Both the tumors and the cells in culture exhibited reduced levels of the inhibin α subunit mRNA. Stable transfectants were generated, in which this subunit was expressed from a heterologous promoter. All of them exhibited a strict inhibition of growth at confluency. On the other hand, in addition to an aging-related decrease in inhibin synthesis, the α subunit gene was down regulated in vivo in cells expressing the viral protein. The conjunction of these two factors accounts for the age-related occurrence of testicular cancers in the transgenic model, again pointing to inhibin as a potent cell growth regulator in the seminiferous epithelium.

Ret Finger Protein: An E3 Ubiquitin Ligase Juxtaposed to the XY Body in Meiosis

During prophase I of male meiosis, the sex chromosomes form a compact structure called XY body that associates with the nuclear membrane of pachytene spermatocytes. Ret Finger Protein is a transcriptional repressor, able to interact with both nuclear matrix-associated proteins and double-stranded DNA. We report the precise and unique localization of Ret Finger Protein in pachytene spermatocytes, in which Ret Finger Protein takes place of lamin B1, between the XY body and the inner nuclear membrane. This localization of Ret Finger Protein does not seem to be associated with O-glycosylation or sumoylation. In addition, we demonstrate that Ret Finger Protein contains an E3 ubiquitin ligase activity. These observations lead to an attractive hypothesis in which Ret Finger Protein would be involved in the positioning and the attachment of XY body to the nuclear lamina of pachytene spermatocytes.

Analysis of gene regulation in Sertoli cells by a gene trap approach.

In mammals, the male gamete is the product of complex events, including meiosis, genetic recombination and spermiogenesis. Alterations in some of these regulatory events can affect fertility. The difficulty of reproducing in vitro the necessary requirements for the development of spermatogenesis has limited the progress of our knowledge of the molecular mechanisms which regulate spermatogenesis. Identification of spermatogenic regulatory genes will be useful to develop an in vitro system to test potential pharmacotoxicological agents.