Pisum Sativum Lectin Research Articles

P-065 HyperSperm, a new sperm preparation method for IVF/ICSI improves sperm capacitation while maintaining a high safety profile

Abstract Study question Does HyperSperm improve sperm capacitation without affecting sperm quality? Summary answer HyperSperm enhances sperm hyperactivated motility, a hallmark of capacitation, without compromising sperm viability, acrosome integrity, or DNA fragmentation. What is known already HyperSperm is a novel semen preparation method for IVF/ICSI based on mimicking effective capacitation that occurs in the female tract. HyperSperm, which consists of 3 media used sequentially to treat sperm before insemination, gave rise to more high-quality blastocysts compared to current standard of care in a pilot split oocyte study in 10 couples. Study design, size, duration This prospective in vitro study was conducted between July 2023 and January 2024 in an academic department. A consecutive cohort of 15 normozoospermic individuals were recruited and included. Each participant provided a semen sample by masturbation after written informed consent was obtained on the day of the collection. Participants/materials, setting, methods Semen samples from normozoospermic individuals (n = 15) were divided into two halves. After density gradient centrifugation, Control half was incubated in modified HTF (Human Tubal Fluid) for 2-4 hours, while the other half underwent HyperSperm treatment. The following parameters were assessed: sperm viability (eosin exclusion); motility and kinematic parameters (CASA); acrosome integrity (fluorescein-labeled Pisum sativum lectin staining); and DNA fragmentation by TUNEL. Differences between groups were evaluated by paired T-test. Significant differences were considered when p < 0.05. Main results and the role of chance Following treatments, sperm viability (90±3% Control vs. 91±3% HyperSperm) and motility (87±2% Control vs. 89±3% HyperSperm) were similar between groups (p > 0.05). Notably, HyperSperm induced a significant hyperactivation increase (9±2% Control vs. 22±3% HyperSperm), along with associated kinematic parameters (i.e., curvilinear velocity and lateral head displacement) (p < 0.05). Furthermore, HyperSperm treatment did not promote motility decrease over time. Finally, there were no differences in the percentage of sperm with intact acrosome (91±2% Control vs. 90±2% HyperSperm) and DNA fragmentation (3±1% Control vs. 3±1% HyperSperm) between groups (p > 0.05). Altogether, HyperSperm has shown to significantly improve sperm capacitation parameters while maintaining a high safety profile in the treated sample. Limitations, reasons for caution The study is limited to normozoospermic samples, and further research should validate these findings across broader male populations undergoing IVF/ICSI. Wider implications of the findings HyperSperm has demonstrated an increase in the production of high-quality blastocysts via IVF, that would reduce barriers to treatment access through higher cumulative success rates and lower treatment repetitions. The observed increase in hyperactivation combined with the safety profile of HyperSperm supports its potential for future adoption in reproductive clinics. Trial registration number not applicable

Candidate Glycoprotein Biomarkers for Canine Visceral Hemangiosarcoma and Validation Using Semi-Quantitative Lectin/Immunohistochemical Assays.

Visceral hemangiosarcoma (HSA) is one of the more frequent cancers in dogs and has a high metastatic rate and poor prognosis, as clinical signs only become apparent in advanced stages of tumor development. In order to improve early and differential diagnostic capabilities and hence, prognosis for dogs with HSA, two types of biomarker are needed: a point-of-care diagnostic biomarker and a prognostic biomarker—preferentially based on samples obtained with minimally invasive methods. In this study, we applied a lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS/MS) workflow through discovery and validation phases to discover serum glycoprotein biomarker candidates for canine HSA. By this approach, we found that Datura stramonium (DSA), wheat germ agglutinin (WGA), Sambucus nigra (SNA), and Pisum sativum (PSA) lectins captured the highest number of validated candidate glycoproteins. Secondly, we independently validated serum LeMBA-MS/MS results by demonstrating the in situ relationship of lectin-binding with tumor cells. Using lectin-histochemistry and immunohistochemistry (IHC) for key proteins on tissues with HSA and semi-quantitation of the signals, we demonstrate that a combination of DSA histochemistry and IHC for complement C7 greatly increases the prospect of a more specific diagnosis of canine HSA.

Aphid Feeding on Plant Lectins Falling Virus Transmission Rates: A Multicase Study.

Aphids are insect vectors that have piercing-sucking mouthparts supporting diversified patterns of virus-vector interactions. Aphids primarily retain circulative viruses in the midgut/hindgut, whereas noncirculative viruses tend to be retained in the stylet. Most viruses, and many proteins from animals, have carbohydrate or carbohydrate-binding sites. Lectins vary in their specificity, of which some are able to bind to viral glycoproteins. To assess the potential competition between lectins and viral particles in virus transmission by aphids, this study examined how feeding plant lectins to aphids affects the transmission efficiency of viruses. Sitobion avenae (F, 1794) (Homoptera: Aphididae) aphids fed with Pisum sativum lectin (PSL) transmitted Barley yellow dwarf virus with significantly lower efficiency (four-fold ratio). Pea enation mosaic virus was significantly reduced in Acyrthosiphon pisum Harris (Homoptera: Aphididae) aphids fed with the lectin Concanavalin A. In comparison, the transmission of Potato virus Y was significantly reduced when Myzus persicae Sultzer (Homoptera: Aphididae) aphids were fed with PSL. Thus, lectin could be used as a blocking agent of plant viruses, facilitating an alternative approach for crop protection.

Aqueous two phase based selective extraction of mannose/glucose specific lectin from Indian cultivar of Pisum sativum seed

Pisum sativum lectin (Psl) being a high-value protein has marked its application in the biomedical and therapeutic field. Aqueous two phase extraction (ATPE) was implemented as a selective partitioning technique for the partial purification of Psl from its seeds. PEG/citrate based biodegradable aqueous two phase system (ATPS) was screened and the factors such as the type and concentration of citrate salts, molar mass and concentration of polyethylene glycol (PEG), tie line length (TLL) and additive (NaCl) concentration, pH, crude load and volume ratio were studied for the selective partition of Psl. The Psl was successfully extracted to the top phase in the ATPS formed with 18% PEG 6000/16% sodium citrate at 41.01% TLL, 2% NaCl and pH of 7.5. A volume ratio of 0.76 and a crude load of 20% showed maximum activity yield of 122.12% with the purification factor of 16.26. The subunits of Psl namely α and β were identified with a molecular weight of 6 and 18 kDa respectively during the purity analysis using SDS PAGE and HPLC.

Aqueous two phase partitioning of Pisum sativum lectin in PEG/citrate salt system

Pisum sativum lectin (Psl) is a metalloprotein which is in the center of research interest because of its HIV-1 reverse transcriptase inhibitory activity and mitogenic activity. The application of this lectin in various fields demands the economically feasible and scalable purification strategy other than affinity chromatography. The suitability of aqueous two phase system (ATPS) composed of poly ethylene glycol (PEG) with different salts (sodium citrate, potassium citrate, and ammonium citrate) was evaluated for better partitioning of Psl. The significant factors such as molar mass and concentration of PEG, type and concentration of salts, the effect of tie line length (TLL), ionic strength, and pH were studied to select a suitable system for better partitioning of Psl. ATPS comprising of 18% PEG 6000, 16% sodium citrate, 1% NaCl at the operating condition of pH 8, 40.23% of TLL, and the volume ratio of 1.32 was found to be the best system which gave a maximum partition coefficient and yield of 14.5% and 98.66%, respectively.

Effect of erythrocyte-sperm separation medium on nuclear, acrosomal, and membrane maturity parameters in human sperm.

The purpose of this study is to investigatewhether erythrocyte-sperm separation medium (ESSM) has effects on human sperm motility, morphology, viability, membrane maturity, acrosome integrity, and nuclear attributes before and after cryopreservation. Semen samples from normozoospermic (n = 36) and oligozoospermic (n = 9) patients were analyzed. Samples from the same patient were divided into three aliquots: group 1 and group 2 were resuspended in sperm washing media and ESSM, respectively. Group 3 was resuspended in ESSM with blood sample to mimic the extensive number of erythrocytes in the testicular sperm extraction (TESE) material. All groups were evaluated for sperm concentration, motility, Kruger/Tygerberg strict morphology, viability by eosin-nigrosin staining, membrane maturity by hyaluronic acid-binding assay (HBA), acrosomal integrity by Pisum sativum lectin staining, chromatin maturity by aniline blue staining, and DNA integrity by TUNEL assay before and after cryopreservation. No significant difference was determined between ESSM-treated and ESSM-untreated sperm samples for the sperm parameters tested (p > 0.05). After cryopreservation, total sperm motility and viability decreased regardless of ESSM used. The percentages of sperm with Tygerberg normal morphology, intact acrosome, and HA-bound sperm were found to be lower in oligozoospermic samples before cryopreservation in each group. However, no statistically significant differences were found between oligozoospermic and normozoospermic samples when all groups were compared. Thus, ESSM treatment did not cause a significant change on sperm motility, normal morphology, viability, HA-binding capacity, chromatin maturity, and DNA fragmentation. ESSM can enhance the efficiency of sperm retrieval protocol and can also decrease the time required to collect spermatozoa while not affecting sperm morphogenetic properties.

Effect of Akti-2 on sperm motility, capacitation and acrosome reaction in a mouse model

The aim of the present study was to investigate the effect of the Akt inhibitor, Akti-2, on the sperm motility and acrosome reaction in mice. Mature sperms from the adult mice, aged 8 weeks, were co-incubated with Akti-2 for ~30 min at 37°C in 5% CO2, and the sperm viability was assessed by eosin-nigrosin staining. The sperm total and progressive motility were analyzed by computer-aided sperm analysis. In addition, the acrosome reaction of sperms was detected by the acid phosphatase assay, Coomassie Brilliant Blue staining and fluorescein-isothiocyanate conjugated pisum sativum lectin staining, respectively. Compared with the control (dimethyl sulfoxide), Akti-2 had no effect on sperm viability, but it suppressed the total and progressive motility significantly. Furthermore, the capacitation-associated protein tyrosine phosphorylation and the acrosome reaction induced by calcium ionophore A23187 could be suppressed by Akti-2. These experiments confirmed that Akti-2 significantly impaired the sperm functions, including motility, capacitation and acrosome reaction, and provide the proof for its potential in male reproductive toxicity.

Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates

Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

Cell microarrays for the screening of factors that allow the enrichment of bovine testicular cells

Cell microarrays can serve as high-throughput platforms for the screening of a diverse range of biologically active factors and biomaterials that can induce desired cellular responses such as attachment, proliferation, or differentiation. Here, we demonstrate that surface-engineered microarrays can be used for the screening and identification of factors that allow the enrichment and isolation of rare cells from tissue-derived heterogeneous cell populations. In particular, we have focused on the enrichment of bovine testicular cells including type A spermatogonia and Sertoli cells. Microarray slides were coated with a copolymer synthesized from poly(ethylene glycol) methacrylate and glycidyl methacrylate to enable both the prevention of cell attachment between printed spots and the covalent anchoring of various factors such as antibodies, lectins, growth factors, extracellular matrix proteins, and synthetic macromolecules on printed spots. Microarrays were incubated with mixed cell populations from freshly isolated bovine testicular tissue. Overall, cell attachment was evaluated using CellTracker staining, whereas differential attachment of testicular cells was determined by immunohistochemistry staining with Plzf and vimentin antibodies as markers for type A spermatogonia and Sertoli cells, respectively. The results indicate that various surface immobilized factors, but in particular Dolichos biflorus lectin, allowed the enrichment of Plzf positive cells. Furthermore, Pisum sativum lectin, concanavalin A, collagen type IV, and vitronectin were identified as suitable negative selection factors. To our best knowledge, this work is the first to demonstrate the utility of surface engineered cell-based microarrays for the identification of factors that allow the selective capture of rare cells from tissue isolated heterogeneous mixtures.

Preincubation in peritoneal fluid decreases the follicular fluid-induced acrosomal reactivity of human spermatozoa.

The aim of this study was to determine the effects of preincubation in peritoneal fluid on the follicular fluid-induced acrosomal reactivity of human spermatozoa in vitro. Thirty women participating in our IVF-ET program were given a GnRH-analogue, highly purified FSH and hCG in order to induce superovulation. Peritoneal and follicular fluids were aspirated during pick-up laparoscopy, centrifuged, filtered and frozen until use. An aliquot of swim-up suspension from normospermic semen specimens (n = 30) was incubated with peritoneal fluid or HAM-F10 for 30-180 min, and follicular fluid (in volumetric proportion approximately 50/50 with peritoneal fluid) was subsequently added. The percentage of acrosomally-reacted spermatozoa was assessed using the FITC-conjugated Pisum sativum lectin before and after incubation in peritoneal fluid or control medium, as well as after follicular fluid addition. Peritoneal fluid was not able to stimulate acrosomal reactivity; further, preincubation in peritoneal fluid decreased, but not abolished, the follicular fluid-induced acrosomal reactivity. A longer pre-incubation in peritoneal fluid was associated with a lower percentage of reacted spermatozoa in response to the addition of follicular fluid. In conclusion, our data suggest that peritoneal fluid acts maintaining spermatozoa in an unreacted status in the upper female genital tract. After mixing with follicular fluid, a phenomenon that is likely to occur at ovulation, peritoneal fluid reduces, but does not abolish, the stimulating effect of follicular fluid on acrosomal reactivity.

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction.

Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml-1 of pentoxifylline at 37 degrees C, 5% CO2 for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 x 10(6) cells ml-1. One hundred microlitres of each sperm suspension was then deposited under oil and 30-40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.

The effects of natural and hybrid lectins on the legume-rhizobium interactions

The effects of hybrid lectins--full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)--on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.

Inverse PCR-based site-directed mutagenesis of nucleotide sequences coding for carbohydrate-binding fragments of legume lectins

A new method of site-directed mutagenesis was developed to allow manipulation with extended plasmid-cloned gene fragments irrespective of their position and the presence of restriction sites. The method was used to obtain chimeric constructs encoding a Pisum sativum lectin with the native carbohydrate-binding region replaced by its counterpart from other legumes. The method can be used in plasmid construction to clone a coding gene fragment under the control of a promoter in a certain reading frame.

Expression of the legume symbiotic lectin genes psl and gs52 promotes rhizobial colonization of roots in rice

Transgenic rice ( Oryza sativa L. cv. Murasaki) carrying genes encoding pea ( Pisum sativum) lectin (PSL) or wild-soybean ( Glycine soja) lectin-nucleotide phosphohydrolase (GS52) were inoculated with Rhizobium leguminosarum bv. v iciae or Bradyrhizobium japonicum USDA110, respectively, as well as with Rhizobium sp. NGR234, and root colonization was assessed in comparison to comparably inoculated control plants. The data showed that expression of PSL and GS52 significantly promoted rhizobial colonization of root epidermal cells including root hairs in rice. In addition, in the case of R. leguminosarum bv. v iciae and B. japonicum USDA110 colonization of the psl and gs52 transgenic rice plants, respectively, the bacterial cells were found to preferentially home towards and aggregate maximally at the root hair tip regions rather than on the root hair “stalks”. The above data suggest that the lectins PSL and GS52, which participate in rhizobial recognition by root epidermal cells in pea and soybean, respectively, are also able to facilitate rhizobial attachment and colonization of the epidermal cells in rice roots. Moreover, aggregation of R. leguminosarum bv. v iciae and B. japonicum USDA110 cells preferentially at root hair tip regions suggest that similar to legumes, the PSL and GS52 lectins are targeted to the root hair tips in transgenic rice, enabling higher bacterial attachment/colonization at the tip region. Rhizobial colonization at root hair tips in the psl and gs52 rice plants frequently led to the localized dissolution of the cell wall creating perforations at the tip region. It is likely that the presence of lectins, such as PSL and GS52 leads to structural modifications in cell wall organization of the root hair/epidermal cells, making them prone to localized dissolution by the hydrolytic activity of compatible rhizobia to permit invasion of the root cells.

Lectinocytochemical detection of apoptotic murine leukemia L1210 cells.

Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about its changes during apoptosis. We investigated whether specific changes in the expression of plasma membrane glycoproteins take place during apoptosis and whether these changes could be used for a quantitative estimation of apoptosis. Lectin cytochemical study of normal and apoptotic murine leukemia cells of the L1210 line was done. Horseradish peroxidase-labeled Laburnum anagyroides bark agglutinin, Phaseolus vulgaris agglutinin, Pisum sativum lectin (PSL), Ricinus communis agglutinin (RCA-120; 120 kDa), Solanum tuberosum agglutinin, Triticum vulgaris lectin (wheat germ agglutinin), Viscum album agglutinin, Canavalia ensiformis lectin (concanavalin A), and Helix pomatia lectin were used to compare specific glycoprotein expressions in normal and apoptotic murine leukemia cells of the L1210 line sensitive (L1210) and resistant (L1210R) to apoptosis induction by cisplatin. The data demonstrated significantly increased binding of alpha-D-mannose-specific PSL lectin (P<0.01) and beta-D-galactose-specific RCA-120 lectin (P<0.001) by the apoptotic cells of the L1210 and L1210R lines in comparison with the intact cells. That binding was shown to be specific because it was blocked by the corresponding inhibitory sugars. Lectins specific to alpha-D-mannose (PSL) and beta-D-galactose (RCA-120) can be used to distinguish between native and apoptotic murine leukemia L1210 cells and to quantitatively estimate apoptosis in a population of these cells.

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. Setting 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.

Decreased sperm function of patients with myotonic muscular dystrophy.

Myotonic muscular dystrophy (MMD) is a genetic disease caused by a defective enzyme, myotoninkinase. Male patients with MMD are reported to have reduced fertility. The purpose of this work was to study sperm capacitation and acrosome reaction in the ejaculates of sterile males with MMD and of healthy males (control group). The expression of the specific D-mannose receptors was explored by microscopic examination and by flow cytometry analysis. In addition, the binding patterns of Pisum sativum (PSA) lectin to acrosome content and outer acrosomal membrane in the spermatozoa of each group were analysed. Both the capacitation and the acrosome reaction in the spermatozoa of the MMD group were deficient and these findings strongly suggest that these anomalies may account for the sterility of these patients.

Molecular cloning of the bark and seed lectins from the Japanese pagoda tree (Sophora japonica).

cDNA clones encoding the bark and seed lectins from Sophora japonica were isolated and their sequences analyzed. Screening of a cDNA library constructed from polyA RNA isolated from the bark resulted in the isolation of three different lectin cDNA clones. The first clone encodes the GalNAc-specific bark lectin which was originally described by Hankins et al. [9] whereas the other clones encode the two isoforms of the mannose/glucose-specific lectin reported by Ueno et al. [34]. Molecular cloning of the seed lectin genes revealed that Sophora seeds contain only a GalNAc-specific lectin which is highly homologous to though not identical with the GalNAc-specific lectin from the bark. All lectin polypeptides are translated from mRNAs of ca. 1.3 kb encoding a precursor carrying a signal peptide. In the case of the mannose/glucose-specific bark lectins this precursor is post-translationally processed in two smaller peptides. Alignment of the deduced amino acid sequences of the different clones revealed striking sequence similarities between the mannose/glucose-binding and the GalNAc-specific lectins. Furthermore, there was a high degree of sequence homology with other legume lectins which allowed molecular modelling of the Sophora lectins using the coordinates of the Pisum sativum, Lathyrus ochrus and Erythrina corallodendron lectins.

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Ram spermatozoa were treated with fluorescein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIC) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC. Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved samples.

Sugar-binding activity of pea (Pisum sativum) lectin is essential for heterologous infection of transgenic white clover hairy roots by Rhizobium leguminosarum biovar viciae.

Legume lectin stimulates infection of roots in the symbiosis between leguminous plants and bacteria of the genus Rhizobium. Introduction of the Pisum sativum lectin gene (psl) into white clover hairy roots enables heterologous infection and nodulation by the pea symbiont R. leguminosarum biovar viciae (R.l. viciae). Legume lectins contain a specific sugar-binding site. Here, we show that inoculation of white clover hairy roots co-transformed with a psl mutant encoding a non-sugar-binding lectin (PSL N125D) with R.l. viciae yielded only background pseudo-nodule formation, in contrast to the situation after transformation with wild type psl or with a psl mutant encoding sugar-binding PSL (PSL A126V). For every construct tested, nodulation by the homologous symbiont R.l. trifolii was normal. These results strongly suggest that (1) sugar-binding activity of PSL is necessary for infection of white clover hairy roots by R.l. viciae, and (2) the rhizobial ligand of host lectin is a sugar residue rather than a lipid.