Comparative analysis of human sperm glycocalyx from different freezability ejaculates by lectin microarray and identification of ABA as sperm freezability biomarker.

BACKGROUND: Semen cryopreservation has been widely applied in assisted reproductive technologies and sperm bank, but it causes considerable impairments on sperm quality. It is necessary to find an evaluation indicator for determining the sperm-freezing tolerance. METHODS: The glycocalyx of good freezability ejaculates was compared with poor freezability ejaculates by lectin microarray. The significant different lectins were validated by flow cytometry (FACS). To analyze the relationship between the potential biomarker and the tolerance of sperm to cryopreservation, 60 samples with different recovery rates were collected and detected the lectin-binding intensity by FACS. The receiver operating characteristic (ROC) curve was analyzed to test the capability of the lectin as a potential biomarker for detecting the sperm freezablility. RESULTS: ABA and DSL were found to develop significant differences between them. Further validation showed that ABA was significantly negative correlated with the sperm recovery rates (r = - 0.618, P < 0.000) and could be a potential biomarker for predicting sperm freezability (AUC = 0.733 ± 0.067, 95% CI 0.601 - 0.865, P < 0.01). CONCLUSION: ABA could be a potential biomarker for predicting sperm freezability. It will help to reduce sperm-freezing recovery tests and improve the efficiency of cryopreservation in human sperm bank.

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray.

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.

cDNA cloning and localization of Sp3111 (also called Ms4a14) in the rat testis.

With tetraspanning topology, members of the membrane-spanning four-domain subfamily A (MS4A) may facilitate signaling or ion channel functions in many tissues. In this study, we report the cloning of a full-length cDNA from rat testis, designated Ms4a14 (Sp3111), which encodes the MS4A protein with 1139 amino acid residues. In situ hybridization and immunohistochemical analyses indicate that Ms4a14 is predominantly expressed from round spermatids to spermatozoa at specific stages in the rat testis at both the mRNA and protein level. Immunofluorescence analysis revealed that MS4A14 (SP3111) is located in the acrosome and the midpiece of the flagellum in mature sperm. Previously, we explored and reported the involvement of MS4A14 in reproductive functions, using antibody blockage during IVF and a transgenic RNA interference method in a mouse model. Our results suggested that MS4A14 is involved in fertilization and zygote division. As MS4A14 protein exists in mammals, such as humans, cows, dogs, and rodents, MS4A14 may play a ubiquitous role in mammalian reproduction.

Different effects of androgen on the expression of Fut1, Fut2, Fut4 and Fut9 in male mouse reproductive tract.

The α-(1,2) fucosyltransferases (Fut1 and Fut2) and α-(1,3) fucosyltransferases (Fut4, Fut9) are responsible for the synthesis of Lewis X (LeX) and Lewis Y (LeY) conjugated to glycoproteins. We recently reported that these fucosyltransferases were differentially expressed in the reproductive tract of male mouse. Here, we studied the effect of androgen on fucosyltransferase expression through the use of mouse castration models. We found that Fut1 mRNA and Fut4 mRNA were upregulated, while Fut2 mRNA and Fut9 mRNA were downregulated by androgen in the caput epididymis. However, in the vas deferens and prostate, only Fut4 mRNA and Fut2 mRNA were respectively upregulated following exposure to androgen. In the seminal vesicle, all fucosyltransferases, with the exception of Fut9, were upregulated. We identified the androgen receptor binding sites (ARBSs) of Fut2, Fut4 and Fut9 in the caput epididymis. Luciferase assay for these ARBSs is able to provide an indication as to why Fut4 and Fut9 are differently expressed and regulated by androgen, although they catalyze the same α-(1,3) fucose linkage. Our study showed that androgen could differentially regulate the expression of these fucosyltransferases and provided an insight into the characteristic distribution of each fucosyltransferase responsible for LeX/LeY biosynthesis in the male reproductive tract.

Development of a novel therapy for Lipo-oligosaccharide-induced experimental neuritis: use of peptide glycomimics.

Recent etiological studies have revealed that molecular mimicry between the lipo-oligosaccharide (LOS) component of Campylobacter jejuni and gangliosides of peripheral nervous system plays an important role in the pathogenesis of Guillain-Barré syndrome (GBS). Previously, we demonstrated GD3 ganglioside molecular mimicry in a model of GBS in Lewis rats by sensitization with GD3-like LOS (LOS(GD3)) from C. jejuni. Since the neuropathophysiological consequences were due largely to the anti-GD3-like antibodies, we subsequently focused our effort upon eliminating the pathogenic antibodies using several strategies to mimic GD3 in this model. Here, we have validated this strategy by the use of peptide glycomimics based on epitopic mimicry between carbohydrates and peptides. We treated rats by i.p. administration of phage-displayed GD3-like peptides. One GD3-like peptide (P(GD3)-4; RHAYRSMAEWGFLYS) induced in treated rats a remarkable restoration of motor nerve functions, as evidenced by improved histopathology, rotarod performance, and motor nerve conduction velocity. P(GD3)-4 effectively decreased the titer of anti-GD3/anti-LOS(GD3) antibodies and ameliorated peripheral nerve dysfunction in the sera of treated rats. The data suggest that peptide glycomimics of ganglioside may be potential powerful reagents for therapeutic intervention in GBS by neutralizing specific pathogenic anti-ganglioside antibodies.

Reproductive effects of cadmium on sperm function and early embryonic development in vitro.

Cadmium is a major environmental toxicant that is released into the atmosphere, water and soil in the form of cadmium oxide, cadmium chloride, or cadmium sulfide via industrial activities, such as the manufacturing of batteries and pigments, metal smelting and refining and municipal waste incineration. In the present study, we investigated the effects of cadmium exposure on sperm quality parameters, fertilization capacity and early embryonic development. Our study showed that in vitro incubation of human or mouse sperms with cadmium for a long time (up to 24 hours) could significantly decreased sperm motility in a concentration- and time-dependent manner. Exposure to cadmium in the environment for a short term (30 min) did not affect sperm motility but significantly reduced in vitro fertilization rate. We also evaluated the effects of cadmium at concentrations of 0.625 µg/ml, and 1.25 µg/ml on early embryonic development in vitro and observed that the blastocyst formation rate dramatically decreased with increasing cadmium concentration. This finding emphasizes the hazardous effects of cadmium on sperm quality as well as on natural embryo development and raises greater concerns regarding cadmium pollution.

Reproductive effects of two neonicotinoid insecticides on mouse sperm function and early embryonic development in vitro.

Acetamiprid (ACE) and imidacloprid (IMI) are two major members in the family of neonicotinoid pesticides, which are synthesized with a higher selectivity to insects. The present study determined and compared in vitro effects of ACE, IMI and nicotine on mammalian reproduction by using an integrated testing strategy for reproductive toxicology, which covered sperm quality, sperm penetration into oocytes and preimplantation embryonic development. Direct chemical exposure (500 µM or 5 mM) on spermatozoa during capacitation was performed, and in vitro fertilization (IVF) process, zygotes and 2-cell embryos were respectively incubated with chemical-supplemented medium until blastocyst formation to evaluate the reproductive toxicity of these chemicals and monitor the stages mainly affected. Generally, treatment of 500 µM or 5 mM chemicals for 30 min did not change sperm motility and DNA integrity significantly but the fertilization ability in in vitro fertilization (IVF) process, indicating that IVF process could detect and distinguish subtle effect of spermatozoa exposed to different chemicals. Culture experiment in the presence of chemicals in medium showed that fertilization process and zygotes are adversely affected by direct exposure of chemicals (P<0.05), in an order of nicotine>IMI>ACE, whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). These findings unveiled the hazardous effects of neonicotinoid pesticides exposure on mammalian sperm fertilization ability as well as embryonic development, raising the concerns that neonicotinoid pesticides may pose reproductive risks on human reproductive health, especially in professional populations.

The effects of Di-(2-ethylhexyl)-phthalate exposure on fertilization and embryonic development in vitro and testicular genomic mutation in vivo.

The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 µg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 µg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ - at least in testes, are of great concern to human male reproductive health.