Anne Gérard

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.

Expression of sex hormone-binding globulin (SHBG) in human granulosa-lutein cells

Sex hormone-binding globulin (SHBG) was classically thought to be a plasma steroid-carrying protein of hepatic origin, but recently, locally produced, membrane-bound SHBG has been shown to influence cell functions in several steroid-responsive tissues. In the ovary, SHBG is known to be present in the follicular fluid, but information about a possible intracellular presence of SHBG in this organ is still very scarce. In this study the presence of SHBG was assessed by immunohistochemistry in human granulosa-lutein cells (GLC) collected by follicle puncture for in vitro fertilization. SHBG was detected in the cytoplasm of GLC before and after in vitro culture for up to 96 h. The presence of full-length SHBG messenger RNA was demonstrated in GLC by reverse transcription-polymerase chain reaction (RT-PCR) in both cultured and uncultured cells. These results demonstrate a local synthesis of SHBG in GLC and raise the question of the physiological significance of these findings in follicular physiology.

Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.

Effects of androgen-binding protein (ABP) on spermatid Tnp1 gene expression in vitro.

In vitro studies were designed to determine whether Sertoli cell-delivered ABP could act on spermatogenetic events, whether such an action could occur via a paracrine or a juxtacrine pathway and whether sex steroids could be involved in this action. ABP delivery to germ cells was achieved using an in vitro model based on recombinant rat ABP-producing mouse Sertoli cells cocultivated with rat spermatids. Using semi-quantitative RT-PCR, the expression of the Tnp 1 gene encoding the Transition Protein 1, involved in the histone to protamine replacement during spermatid nuclear transformation, was analyzed. Our results provide clear evidence that Sertoli cell-derived ABP acts on spermatids by modifying the TP1 mRNA level. This outcome, strictly requiring juxtacrine conditions, is obtained in the absence of sex steroid hormones. To our knowledge this is the first evidence of an effect of ABP itself on male germ cells.

Retinol free and retinol complexed β-lactoglobulin binding sites in bovine germ cells

A high affinity specific binding site for bovine beta-lactoglobulin (BLG) was identified in bovine germ cell plasma membrane enriched fractions. Binding was found to be reversible and pH-dependent with maximum binding occurring at pH 5. The on-rate and off-rate constants were 2.26 +/- 0.8 x 10(5) M(-1) min(-1) (n = 3) and 0.016 +/- 0.004 min(-1) (n = 3), respectively. Scatchard analysis showed a single class of binding sites, with 12.38 +/- 4.62 x 10(12) sites per mg of membrane protein (n = 3) and a dissociation constant (K(D)) estimated at 26.43 +/- 2.68 nM. There was inhibition of iodinated-BLG (variant A) (125I-BLGA) binding to germ cell plasma membrane enriched fractions in the presence of unlabelled BLG variant A, BLG variant B, retinol complexed BLGA and human retinol-binding protein. Inhibition was observed neither with BSA nor with lactoferrin. 125I-BLGA incubated with a Triton X-100 solubilized plasma membrane fraction formed a high molecular mass complex in Superose 12B gel filtration. This receptor complex disappeared in the presence of unlabelled BLGA and in the presence of 10 mM EDTA. The results suggest that germ cell plasma membrane may contain a receptor which is capable of binding either retinol free or retinol complexed BLGA.

Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP)

The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.

High-affinity binding of testosterone in serum from normal developing chick embryos and during the graft-versus-host reaction.

The sera of developing chicken embryos contain high-affinity, low-capacity protein binding sites for testosterone. The affinities remain constant throughout development, with mean values for the association constants of approx. 3.6 X 10(8) M-1 at 25 degrees C, whereas the concentration of sites varies markedly as a function of age: from approx. 2 nmol/g serum proteins in 11-day embryos, it rises to a peak of approx. 5-8 nmol at 14-16 days, then drops to approx. 2.6 nmol at 18 days and only 0.8-1 nmol in adults. Testosterone binding is inhibited by corticosterone, progesterone and dihydrotestosterone, and is little affected by estradiol. The testosterone and corticosterone binding properties of chicken sera show close similarities: parallel ontogenic patterns; constant ratios, throughout development, of the equilibrium binding parameters of the two steroids; mutual binding inhibition. The evidence strongly suggests that the two activities are associated, at least in part, with a common protein carrier(s). In growing embryos which undergo a graft-versus-host reaction, elicited by the graft of adult spleen tissue at 9 days of age, the testosterone and corticosterone binding activities are significantly decreased. This decrease is due to a fall in the number of sites, whereas association constants are not affected. This is the first high-affinity, saturable, testosterone-binding property to be described in an embryonic serum.