Franck Boué

Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the eggs zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High‐salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose‐dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol‐anchored protein and that prostasomes may be implicated in this process. Mol. Reprod. Dev. 52:225–233, 1999.

In Vivo Regulation of Rat Epididymal Proteins by Retinoids: Analysis by Two—Dimensional Electrophoresis

The role of retinoids in the regulation of epididymal fluid protein expression was investigated. We compared the patterns of two-dimensional electrophoretic gels of proteins from luminal fluids, cytosols and spermatozoa (from control rats only) of control, retinoid-depleted, retinoid-depleted retinoic acid-complemented and retinoid-depleted testosterone-supplemented rats. This study compared the luminal fluid patterns from the 4 diets and observed 13 proteins whose expression was dependent on nutritional status. Eight were either absent or very weakly expressed in retinoid-depleted animals only, while their presence was obvious in control rats and in the retinoid-deficient retinoic acid- and testosterone-complemented groups. The expression of 8 proteins was greatly enhanced in retinoid-depleted testosterone-supplemented fluids as compared to control fluids. Five of the regulated proteins seemed to be captured by spermatozoa as they were observed in sperm protein patterns of control rats. These results clearly show that the synthesis of several epididymal proteins is influenced by retinoids. Since testosterone-supplemented animals on retinoid-free diet elicited the same response as retinol and retinoic acid ones, testosterone is likely to be the mediator of retinoid action on epididymal protein synthesis. Nevertheless, the observation of one protein whose expression is stimulated by retinoic acid only and is totally independent of testosterone also favors the direct influence of this retinoid.

Identification of post-vasectomy sperm auto-antigens in fox (Vulpes vulpes) by two-dimensional gel electrophoresis and Western blotting.

The aim of this work was to identify antigenic surface proteins on fox spermatozoa. Six foxes were inguitinally vasectomised, and the time course of antibody response in the sera was studied. Five out of the six foxes reacted to vasectomy with a production of antisperm antibodies. The number of bands recognised by Western blot was maximal 120-150 days after the vasectomy, at the end of the reproductive season. On the whole, 30 bands were recognised between 9 and 150 kDa. The pattern of recognised proteins varied from one fox to another. The humoral response was studied in one fox 2 years after the vasectomy, before, during and after the breeding season. The same proteins were recognised, but the intensity of staining was increased during the testis regression. Using FITC-labelling on sperm smears with fox sera, antigens were localised at or near the sperm surface, either on the acrosome or both on the acrosome and on the flagellum. Using two-dimensional gel electrophoresis and Western blotting, we identified eight areas containing major antigens, recognised by at least two sera. The molecular weights (kDa) and isoelectric points of the repeated antigens were, respectively [150, 6.6-6.0]; [105-98, 6.0-5.5]; [97, 4.6-4.3]; [95, 5.0]; [85-80, 5.4-5.1]; [42, 5.0-4.8]; [17-15, 6.5-5.9]; and [17-15, 5.5-4.8]. The results of this study can be used to characterise more precisely fox sperm auto-antigens by microsequencing the selected proteins.

Identification of a New, Testis-Specific Sperm Antigen Localized on the Principal Piece of the Spermatozoa Tail in the Fox (Vulpes vulpes)

Abstract Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH2-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RIIα domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.