Effect of immunization of hamsters against recombinant P26h on fertility rates.

Despite the various contraceptive methods available, an effective and inexpensive method remains to be established. Immunocontraception may help to achieve this goal. P26h has been proposed as a candidate for the development of a male contraceptive vaccine. P26h, a hamster sperm protein, interacts with the zona pellucida. Furthermore, in vivo fertilization can be blocked completely by active immunization of male hamsters against P26h. Maltose binding protein (MBP)-P26 shares antigenic determinants with the native P26h present on cauda epididymal spermatozoa. The aim of the present study was to reproduce the immunocontraceptive properties of native P26h by immunizing male hamsters against a recombinant P26h fused with a maltose binding protein (MBP). Active immunization of male hamsters with the MBP-P26h showed that specific anti-P26h circulating IgGs could be generated. Mating of immunized male hamsters with superovulated females resulted in a significant decrease, 20-25%, in the fertilization rate. This result is in agreement with results from in vitro sperm-zona pellucida binding assays. Indeed, the anti-recombinant P26h IgGs showed lower inhibitory properties when compared with anti-native P26h IgG. Despite the high anti-P26h IgG titres generated in hamsters, histological studies showed that active immunization has no pathological sequelae to the reproductive tissues. The potential of P26h as a candidate for a contraceptive vaccine is discussed.

Identification and characterization of P31m, a novel sperm protein in Cynomolgus monkey (Macaca fascicularis).

We have previously described a hamster sperm glycoprotein, P26h, which is implicated in the cascade of events occurring during the interaction between mature spermatozoa and the oocyte's zona pellucida. The P26h is acquired on the acrosomal cap of the spermatozoon during its maturation arising within the epididymis. Lately, using a polyclonal antiserum raised against P26h, a 34 kDa protein, P34H, has been identified on the acrosomal cap of the human spermatozoon. The cloning and sequencing of the cDNA encoding P34H has revealed a 65% similarity between the P34H and P26h amino acid sequences. Considering that P26h shows total immunocontraceptive properties in the hamster, it is of relevant importance to have an animal model phylogenetically closer to the human. Using the Cynomolgus monkey, we searched for a protein autologous to the human P34H. A 31 kDa protein, the P31m, localized on the acrosomal cap of the monkey spermatozoon has been identified by a Western blot analysis and by immunohistochemical techniques using an anti-hamster P26h antiserum. Northern blot analysis showed increasing high levels of the P31m mRNA through the epididymis and at lower levels in the testis. In situ hybridization showed the presence of the P31m mRNA in the principal cells of the epididymis. The cloning and sequencing of the cDNA encoding the P31m showed a high homology of 97% identity between the P31m and P34H nucleotidic sequences. This study clearly demonstrates that the monkey P31m is the homologous protein of the hamster P26h and of the human P34H. Mol. Reprod. Dev. 59: 431-441, 2001.

Expression of the hamster sperm protein P26h during spermatogenesis.

P26h is a hamster sperm protein of 26 kDa that has been previously characterized as a surface protein covering the acrosome acquired during epididymal transit. P26h is involved in sperm-egg interactions. Recently, it has been shown that the P26h transcript is highly expressed in the testis, and the P26h cDNA has been cloned from a hamster testicular cDNA library. Herein we report the production of a fusion protein (maltose binding protein-P26h) with the whole P26h cDNA encoding sequence and the production of a polyclonal antiserum against it. In Western blots, this antiserum recognized both the P26h extracted from cauda epididymal spermatozoa and the MBP-P26h. We also determined the age of appearance of P26h and which germ cell types express P26h mRNA and its translational product. Northern blots and in situ hybridization analysis showed that P26h transcripts appear at 3 wk of age, within the first round of spermatogenesis in the golden hamster. In situ hybridization showed that P26h transcripts are expressed in spermatocytes and round spermatids, whereas immunostaining revealed the presence of P26h in the cytoplasm of round spermatids and elongated spermatids. P26h was undetectable in testicular spermatozoa. Both in situ hybridization and immunostaining showed P26h expression to be dependent of the testicular cell type and the epithelium cycle. The implications for P26h in sperm-egg interaction and the testicular origin of P26h are discussed.

P34H sperm protein is preferentially expressed by the human corpus epididymidis.

During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction. We have previously described a 34-kDa human epididymal sperm protein, P34H, that has been shown to be involved in sperm-zona pellucida interaction. In the present study, we report the cloning and characterization of the full-length complementary DNA encoding human P34H. The predicted amino acid sequence revealed 65% identity with P26h, the hamster counterpart of the P34H. The deduced P34H amino acid sequence revealed a 71% similarity with a pig lung tetrameric carbonyl reductase, a member of the short chain dehydrogenase/ reductase family proteins. Northern blot analysis revealed that P34H messenger RNA (mRNA) was highly expressed in the human epididymis, principally in the corpus region. A single 912-bp P34H transcript was detected. In situ hybridization experiments showed that the P34H mRNA was predominantly expressed in the proximal and distal sections of the corpus epididymidis. The staining was restricted to the principal cells of the epididymal epithelium. The localization of P34H mRNA was in agreement with the appearance of P34H protein along the male reproductive tract. Western blot analysis revealed that recombinant P34H expressed by a yeast expression system, is antigenically related to the native P34H sperm protein. Based on its pattern of expression and its function in one of the key steps leading to fertilization, P34H can be considered as a marker of epididymal sperm maturation in humans.

Hamster sperm protein, p26h: a member of the short-chain dehydrogenase/reductase superfamily.

For successful fertilization to occur, mammalian spermatozoa must undergo a series of modifications in order to reach and penetrate the oocyte. Some of these modifications occur during passage through the epididymis, the site where spermatozoa acquire their fertilizing ability. We have previously described hamster sperm protein, P26h, which is acquired by spermatozoa during epididymal transit, and have proposed that this protein is involved in sperm-egg binding. In the present study, we report the cloning and characterization of the full-length cDNA encoding hamster P26h. A database search using the predicted hamster P26h amino acid sequence revealed 85% identity with mouse AP27 protein and porcine carbonyl reductase, members of the short-chain dehydrogenase/reductase (SDR) family of proteins. Northern blot analysis revealed a major P26h 1-kilobase transcript in the testis. No signal was detected in other somatic tissues of the hamster. In situ hybridization experiments revealed that the P26h gene was predominantly transcribed in seminiferous tubules of the testis and at a lower level in the corpus epididymidis. The identity of the cloned P26h was confirmed by immunoprecipitating in vitro-translated P26h using polyclonal antiserum raised against purified hamster sperm P26h. Taken together, these results identify P26h as a new member of the SDR family of proteins involved in the processes of mammalian gamete interactions.

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase.

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59,789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC possess an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE.

[Dietary treatment of mild to moderate hypercholesterolemia. Effectiveness of different interventions]. / Traitement diététique de l'hypercholestérolémie légère à modérée. Efficacité de différentes interventions.

OBJECTIVE: To compare the efficacy of brief dietary intervention by family physicians in their daily practice and in group sessions to standard dietetic treatment in mild to moderate hypercholesterolemia. DESIGN: Randomised clinical trial. SETTING: Family practice clinic in a remote community. PARTICIPANTS: Between September 1, 1991 and September 30, 1992, 135 men and women between 20 and 60 years old with mild to moderate hypercholesterolemia were recruited and randomly assigned to three treatment groups to be taught the American Heart Association low fat diet. Each participant had an LDL-C reading higher than the desirable level set by the Canadian Consensus Conference on Cholesterol. INTERVENTIONS: The three treatment groups received different interventions: individual consultations with a family physician in his office (phase I); group sessions with a physician and a dietician (phase II); and individual consultations with a dietician (phase II). Participants were followed for 6 months with visits and blood tests every 2 months. MAIN OUTCOME MEASURES: Reduction in serum levels of total cholesterol, LDL-C, HDL-C, and triglycerides was measured after 2, 4, and 6 months of dietary treatment. Changes in risk factors (smoking, weight, level of physical activity) and patients' cholesterol/saturated fat index were also measured. RESULTS: Ninety-nine subjects completed the 6-month regimen. The mean reduction in serum LDL-C was 0.08 mmol/L (1.8%) in Group I, 0.07 mmol/L (1.6%) in Group II, and 0.28 mmol/L (6.3%) in Group III (P = 0.94). An LDL-C reduction of 10% or more relative to initial level was observed in 27% of participants in Group I and approximately 40% of subjects in the other two groups (P = 0.41). Counseling resulted in a decrease in body weight, smoking, and dietary fat consumption and an increase in physical activity. CONCLUSIONS: Treatment by a dietician achieved better results and should remain the standard. Physicians should focus on the detection and control of other heart disease risk factors.

Development of the ADL Profile.

This article presents a new instrument, the ADL Pcofile, which is designed specifically for the head injured adult. The instrument is based on the model of occupational performance and on Luria's model of cerebral and motor functioning. It divides the concept of activities of daily living (ADL) into 3 dimensions: personal environment, home environment and community environment. It further delineates a number of activities, tasks and operations under each of these dimensions. The article presents a rationale for the instrument's development, a description of the development process and the preliminary validation work, and details of the instrument itself.