Identification of human sperm surface glycoproteins recognized by autoantisera from immune infertile men, women, and vasectomized men

To identify the surface antigens of human sperm recognized by antisera from immune infertility patients and vasectomized men, we labeled sperm surface proteins with 125I- and used patient antisera for immunoprecipitation. Sera were studied from 27 infertile males, 18 infertile females, and 4 vasectomized males, each possessing anti-sperm antibodies detected by immunobead binding. Sera from different infertile males, different infertile females, and vasectomized males were remarkably similar in their surface antigen recognition. The different sera specifically immunoprecipitated the same small group of 125I-labeled surface proteins, which included polypeptides in the region 90 kDa, 40-45 kDa, and 26 kDa. Treatment with N-glycanase showed that the proteins of 90 kDa, 40-45 kDa, and 26 kDa were glycoproteins with N-linked carbohydrate. The immunoprecipitated 125I-labeled proteins and the total extract of 125I-labeled surface proteins were compared on two-dimensional (2D) gels. The results show the 90 kDa polypeptide is a major sperm surface component, whereas 40-45 kDa and 26 kDa polypeptides are minor components. The 2D gel comparison also indicates that 90 kDa, 40-45 kDa, and 26 kDa are a small subset of the total ensemble of sperm surface proteins. Clinical data suggest antibodies to these few proteins interfere with sperm function.     

Isolation and Purification of Human Serum Alpha-1 Acid Glycoprotein

Abstract

Alpha-1 acid glycoprotein (or orosmucoid) was obtained in a pure state from normal human serum by ion exchange chromatography followed by curtain electrophoresis and a final ion exchange chromatography step. Pure α₁ acid glycoprotein (α₁A) has a sedimentation coefficient of 3.1 S and a diffusion coefficient of 5.2 × 10^?7cm² sec^?1, which yields a molecular weight of 44,680 Daltons and an asymmetry factor of 14.6. The αA prepared in the manner here described appears less denatured than the same protein isolated by the Cohn fractionation method.^1,2 ' Alpha-1 A acts as a depressant of phagocytosis³ and is one of the constituents of Mowbray's serum fraction,″which induces a prolongation of skin homografts.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

Autoradiography of progesterone and model compound entry and distribution in Xenopus laevis oocytes

Xenopus laevis oocytes resume meiosis in response to progesterone. The initial interaction involves surface binding to numerous low-affinity receptor proteins. The mechanism of entry and functions of intracellular steroid are unknown. Because the latter are important for understanding progesterone-induced maturation, a dry-mount autoradiographic technique for analyzing entry and intracellular distribution of radiolabeled steroids was developed and tested. The distinguishing feature of this cryo-technique is sample preparation directly in incubation media using uncross-linked polyacrylamide for inert support. The external ligand functions as an internal standard, so quantitation is by simple ratio (bound/free). The entry kinetics and subcellular binding patterns in large oocytes were studied using this method at nM levels of radiolabeled steroids and model compounds. Progesterone, estradiol, corticosterone, and 1,25-dihydroxycholecalciferol all showed rapid entry (P approximately 10(-6) cm/sec). Entry rates were not saturable with unlabeled steroid. Intracellular patterns of these steroids were highly specific and negatively associated with yolk protein and lipid. Intracellular binding in animal hemisphere ooplasm was 10x that of the yolk-rich vegetative ooplasm. In contrast, dexamethasone, ponasterone-A, and ecdysone displayed entry rates 20-60x slower than progesterone with little compartmentalization. Glycerol, glucose, and leucine entered over 10x slower than progesterone. Cholesterol and Ca++ had entry rates below detection. Evidence for mediated entry of progesterone included the rapid saturation of a cortical compartment equivalent in magnitude to reported receptor numbers. The kinetics and specificity of cortical uptake were consistent with low-affinity, high capacity protein binding. Intracellular binding was seen to correlate with rhodamine 123 patterns, suggesting involvement of mitochondrial or other microtubule-associated structures in steroid responses. Mitochondrial binding is consistent with the limited steroid metabolism seen in oocytes. Since several maturation events are consistent with respiratory uncoupling, reported by others for steroids and isolated organelles, and since mitochondria contain nearly all of the oocyte DNA, a role for these organelles in steroid-induced oocyte maturation was proposed.