Barend M. Gadella

Dynamics of the mammalian sperm plasma membrane in the process of fertilization.

Sexual reproduction requires the fusion of sperm cell and oocyte during fertilization to produce the diploid zygote. In mammals complex changes in the plasma membrane of the sperm cell are involved in this process. Sperm cells have unusual membranes compared to those of somatic cells. After leaving the testes, sperm cells cease plasma membrane lipid and protein synthesis, and vesicle mediated transport. Biophysical studies reveal that lipids and proteins are organized into lateral regions of the sperm head surface. A delicate reorientation and modification of plasma membrane molecules take place in the female tract when sperm cells are activated by so-called capacitation factors. These surface changes enable the sperm cell to bind to the extra cellular matrix of the egg (zona pellucida, ZP). The ZP primes the sperm cell to initiate the acrosome reaction, which is an exocytotic process that makes available the enzymatic machinery required for sperm penetration through the ZP. After complete penetration the sperm cell meets the plasma membrane of the egg cell (oolemma). A specific set of molecules is involved in a disintegrin-integrin type of anchoring of the two gametes which is completed by fusion of the two gamete plasma membranes. The fertilized egg is activated and zygote formation preludes the development of a new living organism. In this review we focus on the involvement of processes that occur at the sperm plasma membrane in the sequence of events that lead to successful fertilization. For this purpose, dynamics in adhesive and fusion properties, molecular composition and architecture of the sperm plasma membrane, as well as membrane derived signalling are reviewed.

Evaluation of In Vitro Capacitation of Stallion Spermatozoa

Abstract The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca2+-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO2 on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca2+ ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus ∼3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10u200a000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca2+ ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.

A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles

Abstract Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37°C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.

Effects of in vivo prematuration and in vivo final maturation on developmental capacity and quality of pre-implantation embryos

In current in vitro production (IVP) systems, oocytes lack in vivo dominant and preovulatory follicular development, which may compromise pregnancy and viability of calves born. When an oocyte sets off in vivo on the road toward fertilization, it contains numerous transcripts and proteins necessary to survive the first few cell cycles of embryonic development. It is not yet known during which period of development the oocyte builds up the store, possibly primarily during the major growth phase of the oocyte, which is completed at the time a follicle reaches the size of 3 mm. Here, we investigated to what extent the later phases of follicular development, such as prematuration in the dominant follicle before the LH surge and ensuing final maturation in the preovulatory follicle, contribute to oocyte competence and development into viable biastocysts. Recent studies on in vivo vs in vitro oocyte maturation employed oocytes from an identical preovulatory development by applying ovum pick-up (OPU) twice (before and 24 h after the LH surge) in each cow treated for superovulation with a controlled LH surge. The embryo recovery rates at Day 7 of IVC after IVF were similar: 44% (97/219) for in vivo- vs 41% (87/213) for in vitro-matured oocytes, which shows that the natural environment during final maturation is not essential for the mere in vitro development of the prematured oocyte beyond the 8- to 16-cell stage. However, in vivo maturation appeared to contribute to the oocytes quality in a more subtle way, as indicated by a significant increase in the proportion of expanded blastocysts and a more physiological degree of chromosome aberrations of the embryos. In blastocysts derived from in vivo-matured oocytes, 21% of the embryos were mixoploid vs 50% from in vitro-matured oocytes, concomitant with a higher number of cells (96 vs 54 per normal blastocyst). The expression pattern of a set of six developmentally important genes was, however, not significantly altered in blastocysts derived from in vivo-matured oocytes. Certain deviations were observed compared with the levels of entirely in vivo-developed control blastocysts, which suggests that the beneficial effects of in vivo maturation are possibly exerted at initial stages of embryonic development. Prematuration in vivo, occurring in a dominant follicle developing from about 8 mm into the preovulatory follicle, is accompanied by changes in protein synthesis of the cumulus oocyte complex (COC). Presumably, the differentially expressed proteins are involved in equipping the oocyte with further developmental competence. Although we have unraveled some important biochemical and cellular biological features of the oocyte, further research on in vivo processes is essential to improve in vitro embryo production in practice.

Sperm surface proteomics: from protein lists to biological function

Proteomics technologies have matured significantly in recent years and proteomics driven research articles in reproductive biology and medicine are increasingly common. The key challenge is to move from lists of identified proteins to informed understanding of biological function. This review introduces the range of proteomics workflows most commonly used for protein identification before focusing on the mammalian sperm cell at fertilization as an exemplar for proteomic studies. We review the work of others on entire cells but then argue that proper subcellular fractionation and proper solubilization strategies offers critical advantages to achieving increased biological understanding. In relation to understanding initial gamete recognition events at fertilization (capacitation, zona binding and acrosomal exocytosis) it is imperative to study the sperm surface proteome by using purified plasma membrane fractions. Although this task is challenging there are now strategies at our disposal to achieve comprehensive coverage of the proteins at the sperm surface. Within this context it is also important to understand the milieu of the sperm cell during transit from the testis to the oviduct as proteins (or other entities) from the genital tract epithelia and fluids may also affect the composition and organization of proteins on the sperm surface. Finally the arguments presented for studying the cell plasma membrane proteome to understand the role of the cell surface equally apply to all cell types with important roles in reproductive function.

Physiological State of Bull Sperm Affects Fucose- and Mannose-Binding Properties

Abstract In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to α-l-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean ± SD:167 ± 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 ± 2.7) and in washed sperm with seminal plasma added back (56 ± 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 ± 6.3) and by capacitating washed sperm in medium containing 10 μg/ml heparin (50 ± 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 ± 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 ± 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts.

Reliable protocols were established for investigating asymmetric distributions of 6‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino‐caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane‐damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38°C. After 1 hr of labeling, about 96% of the incorporated C6NBD‐phosphatidylserine, 80% of C6NBD‐phosphatidylethanolamine, 18% of C6NBD‐phosphatidylcholine, and 4% of C6NBD‐sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP‐dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP‐independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions. Mol. Reprod. Dev. 53:108–125, 1999.

Bovine Cumulus Cells Protect Maturing Oocytes from Increased Fatty Acid Levels by Massive Intracellular Lipid Storage

ABSTRACT Metabolic conditions characterized by elevated free fatty acid concentrations in blood and follicular fluid are often associated with impaired female fertility. Especially elevated saturated fatty acid levels can be lipotoxic for several somatic cell types. The aim of this study was to determine the impact of elevated free fatty acid concentrations in follicular fluid on neutral lipids (fatty acids stored in lipid droplets) inside cumulus cells and oocytes and their developmental competence. To this end, cows were exposed to a short-term fasting period during final oocyte maturation. This resulted in elevated, but distinct, free fatty acid concentrations in blood and follicular fluid and a rise in the concentrations of in particular fatty acids with a chain length of 14–18 carbon atoms. Interestingly, elevated free fatty acid concentrations in follicular fluid resulted in a massive increase in the level of neutral lipids in cumulus cells, whereas the level of neutral lipid in oocytes was hardly affected. Furthermore, competence of oocytes to develop to the blastocyst stage after fertilization and culture of cumulus-oocyte-complexes of the experimental and control group was not different. In conclusion these data suggest that short-term elevated free fatty acid concentrations in follicular fluid do not harm oocyte developmental competence. We propose that the involvement of high levels of mobilized oleic acid in follicular fluid in combination with the induced lipid storage in cumulus cells serves to prevent harmful saturated fatty acid exposure to the oocyte.

Mass Spectrometric Detection of Cholesterol Oxidation in Bovine Sperm

We report on the presence and formation of cholesterol oxidation products (oxysterols) in bovine sperm. Although cholesterol is the most abundant molecule in the membrane of mammalian cells and is easily oxidized, this is the first report on cholesterol oxidation in sperm membranes as investigated by state-of-the-art liquid chromatographic and mass spectrometric methods. First, oxysterols are already present in fresh semen samples, showing that lipid peroxidation is part of normal sperm physiology. After chromatographic separation (by high-performance liquid chromatography), the detected oxysterol species were identified with atmospheric pressure chemical ionization mass spectrometry in multiple-reaction-monitoring mode that enabled detection in a broad and linear concentration range (0.05–100 pmol for each oxysterol species detected). Second, exposure of living sperm cells to oxidative stress does not result in the same level and composition of oxysterol species compared with oxidative stress imposed on reconstituted vesicles from protein-free sperm lipid extracts. This suggests that living sperm cells protect themselves against elevated oxysterol formation. Third, sperm capacitation induces the formation of oxysterols, and these formed oxysterols are almost completely depleted from the sperm surface by albumin. Fourth, and most importantly, capacitation after freezing/thawing of sperm fails to induce both the formation of oxysterols and the subsequent albumin-dependent depletion of oxysterols from the sperm surface. The possible physiological relevance of capacitation-dependent oxysterol formation and depletion at the sperm surface as well as the omission of this after freezing/thawing semen is discussed.

Cell biology and functional dynamics of the mammalian sperm surface

Theriogenology has now a 40-year rich history on covering sperm biological aspects with a special emphasis on farm and husbandry animals. The major and most influential of these contributions will be placed into an evolutionary perspective of ongoing and intriguing progresses made in this field. Although many molecular details have been published, it is more the aim of this contribution to provide a guide through the main established aspects and concepts of sperm surface biology and refer only to major molecular players and mechanisms involved in sperm physiology. Those interested in more molecular details and in-depth knowledge can easily access the most relevant literature which is included here for reference purposes. With this approach, a logical and easy to follow buildup can be made of the general picture of sperm surface dynamics and of the ergonomics of sperm physiology and their function in mammalian fertilization. Understanding the ins and outs of sperm surface biology and the dynamics thereof, might challenge future researchers to design novel generation of better sperm-handling procedures. This could be beneficial for assisted reproductive technology and animal breeding industries.