Gabor Huszar

Monitoring of collagen and collagen fragments in chromatography of protein mixtures

Abstract A new method for the detection of collagen and collagen peptides in the presence of other proteins is described. The procedure is based on alkaline hydrolysis of the proteins and monitoring of the free hydroxyproline after chromophore formation. The method is quick and sensitive and the color yield of hydroxyproline is not affected by chromatography solvents. As all steps of the assay take place in a single test tube the method is suitable for batch processing of column fractions. Due to the high sensitivity of the assay it can be used for the analysis of collagen and collagen peptides in extracts of small tissue samples or biopsies.

Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status

OBJECTIVE To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation. DESIGN Comparisons of sperm in semen and in HA-bound sperm fractions. SETTINGS University-based diagnostic and research andrology laboratory. PATIENT(S) Semen samples originated in men being tested for infertility. INTERVENTION(S) The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm). RESULT(S) All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding. CONCLUSION(S) Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone-protamine replacement. Hyaluronic acid-bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects

The testis-expressed chaperone protein, HspA2 (previously creatine kinase M isoform) was established as a measure of human sperm cellular maturity, function and fertility. The presence of HspA2 in the synaptonemal complex is likely to link low HspA2 expression and increased frequency of chromosomal aneuploidies in arrested-maturity spermatozoa. A relationship also exists between HspA2 expression in elongating spermatids and the associated spermatogenetic events, including plasma membrane remodelling and the formation of zona pellucida and hyaluronic acid (HA) binding sites. The HA receptor of mature spermatozoa, when coupled with HA-coated slides and/or Petri dishes, allows visual observation of sperm-HA binding, providing a basis for sperm maturity testing, a major improvement in semen evaluation, and selection of mature spermatozoa for intracytoplasmic sperm injection (ICSI). Thus, in HA-selected spermatozoa the frequency of chromosomal disomy and diploidy is reduced 4- to 6-fold compared with semen sperm fractions. This reduction is similar to the increase in numerical chromosomal aberrations in ICSI children. Combined studies of sperm shape and chromosome probes demonstrated that sperm morphology does not aid selection of haploid spermatozoa. The HA-mediated sperm selection is a novel and efficient technique that may alleviate potential problems related to ICSI fertilization with visually selected spermatozoa.

Putative Creatine Kinase M-Isoform in Human Sperm Is Identifiedas the 70-Kilodalton Heat Shock Protein HspA2

Abstract We previously described a putative creatine kinase M isoform in human sperm that is developmentally regulated and expressed during late spermiogenesis, simultaneous with cytoplasmic extrusion. We have now identified this protein as the testis-expressed 70-kDa heat shock protein chaperone known as HspA2 (the human homologue of mouse Hsp70-2). We have isolated and characterized HspA2 (formerly CK-M) by amino acid sequencing and have localized it by immunocytochemistry to spermatocytes at low levels, to spermatids, and in the tail of mature sperm. The specificity of the CK-M/HspA2 antiserum to HspA2 was demonstrated on immunoblots of one- and two-dimensional SDS-PAGE. In agreement with our earlier biochemical data, immunocytochemistry of testicular tissue indicated that HspA2 is selectively expressed in mature spermatids and in sperm about to be released in the seminiferous tubuli. The identity of HspA2 has been further confirmed by cross-absorption of the mouse HSP70-2 antibody by the HspA2/CK-M fraction, and by identical immunostaining patterns of human testicular tissue using either the anti-CK-M/HspA2 or an anti-mouse Hsp70-2 antisera. During spermiogenesis, both cytoplasmic extrusion and plasma membrane remodeling, which facilitate the formation of the zona pellucida binding site, involve major intrasperm protein transport, which may be chaperoned by HspA2. Accordingly, in immature human sperm, which fail to express HspA2, there is cytoplasmic retention and lack of zona pellucida binding. The present findings provide the biological rationale for the role of the human HspA2 as an objective biochemical marker of sperm function and male fertility, which we have established in earlier clinical studies.

Biochemical markers of sperm function: male fertility and sperm selection for ICSI.

The expression of a 70 kDa chaperone protein, HspA2 (formerly called CK-M), has been identified in mature human spermatozoa. The central role of HspA2 has been established, as the expression level of this protein is related to sperm cellular maturity, DNA integrity, chromatin maturity, chromosomal aneuploidy frequency and sperm function, including fertilizing potential. The spermiogenetic events of cytoplasmic extrusion and remodelling of the plasma membrane, which facilitate the formation of zona pellucida binding site(s) in human spermatozoa, are related. Finally, the presence of the hyaluronic acid (HA) receptor on the plasma membrane of mature sperm coupled with the HA-coated slide sperm-binding assay, facilitates the testing of infertile men and the selection of single mature spermatozoa for ICSI. Because mature spermatozoa have no residual cytoplasm, the HA-bound sperm fraction is also enriched in spermatozoa that are normal by the Kruger strict morphology method.

Hyaluronic acid (Sperm Select) improves retention of sperm motility and velocity in normospermic and Oligospermic specimens

The effects of Sperm Select (Pharmacia AB, Uppsala, Sweden), a hyaluronic acid medium, on the motility and membrane integrity properties of sperm were studied. In 15 normospermic specimens after overnight incubation, the motility parameters in the control versus the Sperm Select group were as follows (mean±SEM): motility, 18.8%±2.8% versus 27.4%±2.9%; velocity, 21.5±2.4 versus 27.2±2.2 μ m/s; linearity, 3.8±0.3 versus 4.4±0.2; lateral head displacement, 1.5±0.2 versus 1.9±0.1 μ m; and tail beat/cross frequency, 8.8±1.3 versus 10.8±1.4Hz. The density of motile sperm was 10.8±2.3 versus 18.5±2.5×10 6 sperm/mL. Finally, the velocity coefficient, the multiple of the sperm motility and linear velocity, was 4.6±1.1 versus 8.1±1.4. However, we found no Sperm Select related differences when testing sperm membrane integrity with hypoosmotic swelling and supravital staining. Thus, Sperm Select improves the retention of sperm motility (most prominently velocity) apparently due to a direct action of hyaluronic acid on sperm metabolism or contractility rather than to preservation of sperm membrane integrity. In 20 Oligospermic specimens, Sperm Select caused similar improvements in sperm motility, and the duration of motility could be predicted from the degree of enhancement in sperm velocity after short-term Sperm Select exposure. A modified Sperm Select protocol is described that further increases motile sperm yield without a centrifugation step.

Creatine kinase immunocytochemistry of human sperm-hemizona complexes : selective binding of sperm with mature creatine kinase-staining pattern

Objective To examine the clinical significance of the increased sperm cytoplasmic content that is due to a fault of spermatogenesis, we have further studied the relationship between increased sperm creatine kinase (CK) concentrations and diminished fertilizing potential in men. In the present work, we used CK immunocytochemistry of human sperm-hemizona (HZ) complexes to examine whether the distribution of mature (clear heads), intermediate (sperm heads with light stippling), and immature (heads with heavy stippling or with solid CK staining) spermatozoa bound to the HZ would follow the incidence of these sperm in the samples tested, or if there is a preferential binding by the mature sperm. Design Two pairs of HZ were exposed to washed semen and to their swim-up sperm fractions. The sperm and sperm-HZ complexes were treated with a CK antibody followed by horseradish peroxidase immunostaining, and the sperm were evaluated for maturity. Setting Men presenting for fertility evaluation were studied in two university-based andrology laboratories. Results The binding of the HZ was selective for mature sperm as indicated by the incidence of intermediate and immature sperm in washed semen versus bound to the HZ (intermediate: 20.0% versus 1.4%; immature: 7.6% versus 0.5% [mean ± SEM]) or in swim-up sperm fractions versus the HZ (intermediate: 18.7% versus 3.4%; immature: 2.5% versus 0.2%). The binding was almost exclusive to normal sperm (96.4% to 98.1%) whether the HZ were exposed to washed semen or swim-up fractions in spite of the five to ten times higher incidence of intermediate and immature sperm. Conclusions Mature sperm selectively bind to the zona. We suggest that spermatozoa with immature CK-staining patterns are deficient in the site(s) of oocyte recognition and binding.

Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertilizing potential : selection of sperm for intracytoplasmic sperm injection

Purpose of review The current concepts of sperm biochemical markers and the central role of the HspA2 chaperone protein, a measure of sperm cellular maturity and fertilizing potential, are reviewed. Recent findings Because HspA2 is a component of the synaptonemal complex, low HspA2 levels and increased frequency of chromosomal aneuploidies are related in diminished maturity sperm. We also suggest a relationship between HspA2 expression in elongating spermatids and events of late spermiogenesis, such as cytoplasmic extrusion and plasma membrane remodeling that aid the formation of the zona pellucida binding and hyaluronic acid binding sites. The presence of hyaluronic acid receptor on the plasma membrane of mature sperm, coupled with hyaluronic acid coated glass or plastic surfaces, facilitates testing of sperm function and selection of single mature sperm for intracytoplasmic sperm injection. The frequencies of sperm with chromosomal disomy are reduced approximately fourfold to fivefold in hyaluronic acid selected sperm compared with semen sperm, comparable to the increase in such abnormalities in intracytoplasmic sperm injection offspring. Hyaluronic acid binding also excludes immature sperm with cytoplasmic extrusion, persistent histones, and DNA chain breaks. Summary Hyaluronic acid mediated sperm selection is a novel technique that is comparable to sperm zona pellucida binding. Hyaluronic acid selected sperm will also alleviate the risks related to intracytoplasmic sperm injection fertilization with sperm of diminished maturity that currently cause worldwide concern.

Spermatozoa Bound to Solid State Hyaluronic Acid Show Chromatin Structure With High DNA Chain Integrity: An Acridine Orange Fluorescence Study

During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection.

Sperm maturity and treatment choice of in vitro fertilization (IVF) or intracytoplasmic sperm injection: diminished sperm HspA2 chaperone levels predict IVF failure ☆

OBJECTIVE To reexamine whether low sperm HspA2 ratios (formerly sperm CK-M ratio) are predictive for failure to cause pregnancies by in vitro fertilization (IVF) and to explore whether there are other male or female factors that may be predictive for IVF pregnancy outcome. DESIGN Retrospective, cohort study. SETTING University-based IVF program. PATIENT(S) In 119 IVF cycles, three patient groups were studied: 25 men had a <10% sperm HspA2 ratio and a lack of pregnancies (HS <10% group), 50 men had a >10% sperm HspA2 ratio and no pregnancies (HS >10%NP group), and another 44 couples had a >10% sperm HspA2 ratio but did achieve pregnancies (HS >10%P group). INTERVENTION(S) Sperm HspA2 ratio determinations within 1 year of the IVF cycles and analysis of male and female IVF cycle parameters. MAIN OUTCOME MEASURE(S) Sperm HspA2 ratio determinations within 1 year of the IVF cycles and analysis of male and female IVF cycle parameters. RESULT(S) In the three groups, male and female ages, number and maturation level of oocytes, and morphology of embryos were similar. In the HS < 10% group, mean sperm concentration and motility were close to normal, the fertilization and cleavage rates were lower, and cycles without any oocyte fertilization were higher. These parameters were similar in the two HS > 10% groups. The receiver operating characteristic curve in men with sperm HspA2 ratios of <17% (diminished and borderline sperm maturity) provided a cutoff value of 10.84% HspA2 ratio with a 100% positive predictive value for failure to achieve pregnancy, whether the men were oligospermic or normospermic. CONCLUSION(S) HspA2 ratios of <10% in the diminished sperm maturity range predict the failure to cause pregnancies by IVF. Thus, IVF should be bypassed in favor of ICSI. The occurrence of pregnancy with ICSI depends on the maturity of sperm selected, and it may not be as likely as in other indications for ICSI.