Karin Müller

Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles

Abstract PDC-109 is the major protein of bovine seminal plasma. It binds to the bovine sperm surface at ejaculation and modulates sperm capacitation. PDC-109 displays phosphorylcholine- and heparin-binding activities which are thought to account for its sperm surface coating and glycosaminoglycan-induced sperm capacitating activities, respectively. We have characterized the interaction of isolated PDC-109 with membranes of phospholipid vesicles using a biophysical approach. Our results show that PDC-109 interacts not only with the solvent-exposed phosphorylcholine head group but also with the hydrophobic core of liposomes. Binding of PDC-109 to membranes is a very rapid, biphasic process with half times of less than one second. Maximal binding of PDC-109 to small unilamellar vesicles was achieved with a stoichiometric ratio of 10–11 phosphatidylcholine molecules/PDC-109 molecule. Incorporation of phosphatidylethanolamine or phosphatidylserine into phosphatidylcholine vesicles reduced the binding of PDC-109, suggesting that both the density of phosphorylcholine groups and the surface charge determine the interaction of the seminal plasma protein with the surface of the membrane. Electron spin resonance measurements showed that binding of PDC-109 to phosphatidylcholine vesicles caused a rigidification of the membrane. The relevance of the data for describing the role of PDC-109 in the modulation of sperm capacitation is discussed.

Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa

Abstract During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes. Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall. For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only. Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.

Analysis of the lipid composition of bull spermatozoa by MALDI-TOF mass spectrometry—a cautionary note

In this study we demonstrated the combination of MALDI-TOF MS and TLC as a fast and powerful tool to investigate the phospholipid (PL) composition of organic extracts of bull spermatozoa. Since phosphatidylcholine (PC) is the dominant PL species, an adequate resolution of MALDI-TOF spectra for sphingomyelin (SM) or phosphatidylethanolamine (PE) was achieved only after previous PL separation by TLC. We found a poor diversity especially for PE and PC, mainly containing ether-linked fatty acids which were 1-palmityl-2-docosahexaenoyl-PL and the corresponding alkenyl-acyl compound (plasmalogen) 1-palmitenyl-2-docosahexaenoyl-PL. For PC, both lipids were quantified after phospholipase A2 digestion to represent 44.2 and 37.2%, respectively, of the total PC. In contrast, the diacyl-PC content of bull spermatozoa was comparatively low (18.6% of total PC). In the presence of trifluoroacetic acid (TFA), which is routinely added to the MALDI-TOF matrix to improve the signal to noise ratio, a high lysophospholipid (LPL) content was detected in the PL extracts of bull spermatozoa, whereas TLC did not reveal significant amounts of LPL. The TFA mediated hydrolysis of the acid-labile alkenyl-acyl PL to the corresponding LPL was shown to cause this discrepancy. This assumption was verified by analysing the PL composition by MALDI-TOF MS before and after (i) digestion of sperm cell lipids with phospholipase A2 and (ii) exposition of spermatozoa to HCl fumes. We conclude that the analysis of samples containing alkenyl-acyl-PL by MALDI-TOF has to be performed with great caution.

Variance reduction by simultaneous multi-exponential analysis of data sets from different experiments

The analysis of experimental data from the photocycle of bacteriorhodopsin (bR) as sums of exponentials has accumulated a large amount of information on its kinetics which is still controversial. One reason for ambiguous results can be found in the inherent instabilities connected with the fitting of noisy data by sums of exponentials. Nevertheless, there are strategies to optimize the experiments and the data analysis by a proper combination of well known techniques. This paper describes an applicable approach based on the correct weighting of the data, a separation of the linear and the non-linear parameters in the process of the least squares approximation, and a statistical analysis applying the correlation matrix, the determinant of Fishers information matrix, and the variance of the parameters as a measure of the reliability of the results. In addition, the confidence regions for the linear approximation of the non-linear model are compared with confidence regions for the true non-linear model. Evaluation techniques and rules for an optimum experimental design are mainly exemplified by the analysis of numerically generated model data with increasing complexity. The estimation of the number of exponentials significant for the interpretation of a given set of data is demonstrated by using records from eight absorption and photocurrent experiments on the photocycle of bacteriorhodopsin.

Interaction of mammalian seminal plasma protein PDC-109 with cholesterol: implications for a putative CRAC domain.

Seminal plasma proteins of the fibronectin type II (Fn2) family modulate mammalian spermatogenesis by triggering the release of the lipids phosphatidylcholine and cholesterol from sperm cells. Whereas the specific interaction of these proteins with phosphatidylcholine is well-understood, their selectivity for cholesterol is unknown. To characterize the interaction between the bovine Fn2 protein PDC-109 and cholesterol, we have investigated the effect of PDC-109 on the dynamics of fluorescent cholesterol analogues in lipid vesicles by time-resolved fluorescence anisotropy. The data show that PDC-109 decreases the rotational mobility of cholesterol within the membrane and that the extent of this impact depends on the cholesterol structure, indicating a specific influence of PDC-109 on cholesterol. We propose that the cholesterol recognition/interaction amino acid consensus (CRAC) regions of PDC-109 are involved in the interaction with cholesterol.

Characteristic oxidation products of choline plasmalogens are detectable in cattle and roe deer spermatozoa by MALDI-TOF mass spectrometry.

Plasmalogens (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphocholines and -phosphoethanolamines) are important constituents of spermatozoa membranes and possess significant antioxidative properties. This particularly holds as plasmalogens from spermatozoa also possess a very high content of highly unsaturated fatty acyl residues (especially 22:6). The organic spermatozoa extracts of two different ruminants (cattle and roe deer) were analyzed for their contents of characteristic choline plasmalogen oxidation products by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry. It will be shown that 1-hydroxy-2-docosahexaenoyl-sn-glycero-3-phosphocholine (LPC 22:6) and formyl-LPC 22:6 are reliable measures of lipid oxidation of spermatozoa and allow, accordingly, conclusions about the storage conditions. All data on spermatozoa were also confirmed by the investigation of the oxidation behavior of selected reference compounds. It will be shown that, equally if plasmalogens or diacyl PC species are used, oxidation takes place primarily at the double bond next to the glycerol backbone. These data were additionally confirmed by recording the corresponding post source decay (PSD) fragment ion spectra.

Lysophospholipids: potential markers of diseases and infertility?

The in vivo concentration of lysophospholipids (LPL) such as lysophosphatidylcholine (LPC) increases under different pathological conditions and, thus, LPL attract nowadays considerable diagnostic and pharmacological interest. LPL are particularly interesting because they possess pro- and anti-inflammatory properties and can be generated by two completely different pathways: either by the influence of (a) phospholipases and (b) different reactive oxygen species (ROS) that are generated in significant amounts under inflammatory conditions. This review provides a summary of the mechanisms by which LPL can be generated under in vitro and in vivo conditions. The focus will be on lysophosphatidylcholine (LPC) because this LPL is most abundant among all LPL and was, thus, most intensively studied so far. Additionally, biochemical, chromatographic and spectroscopic methods of LPL and LPC determinations will be discussed. Finally, the effects of LPL as signaling molecules and their roles in different pathologies such as infertility, cancer, atherosclerosis or inflammatory diseases are discussed. Special emphasis will be on the role of LPL in reproduction failures related to poor semen quality and, in that context, the potential role of LPC as a disease-indicative molecule.

Polysialic Acid Is Present in Mammalian Semen as a Post-translational Modification of the Neural Cell Adhesion Molecule NCAM and the Polysialyltransferase ST8SiaII

Background: Polysialylated glycoproteins play an import role during numerous biological processes. Results: Polysialylated ST8SiaII and NCAM are components of mammalian semen and are partially associated with spermatozoa. Conclusion: Polysialic acid represents a further glyco-motif in mammalian ejaculates, which is known to influence the immune system. Significance: Administration of polysialic acid during insemination might be useful to increase the number of spermatozoa escaping the female immune system. Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85–98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.