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.

Background: To study the effects of cryopreservation on human sperm glycocalyx. Methods: The lectin binding profilings of sperm after freeze-thaw were compared by lectin microarray. Results: CryoSpermTM and direct fumigation were confirmed to be the optimized cryoprotectant and method by comparing the sperm recovery rate. In 91 lectins, 33 lectins were significantly changed after sperm cryopreservation. Among them, 9 lectins greatly decreased and 24 lectins mainly increased. The binding signals of MAA, PSA, ABA, and AIA were verified by FACS, and the results were consistent with that of lectin microarray. Conclusions: Sperm glycocalyx had significant changes after cryopreservation. The sialic acid, playing an important role in protecting sperm, was greatly lost, which exposed the inner carbohydrates. Thus, the glycocalyx damage due to the cryopreservation might be one of the reasons for low sperm fertility.

Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery.

目的 研究成年山羊精子在附睾成熟过程中与凝集素芯片结合的差异.方法 精子分别取自成年山羊附睾头、体、尾部,经2％多聚甲醛/0.2％戊二醛固定后,然后进行碘化丙啶染色标记,再分别与含有91种凝集素的芯片结合.信号经提取后,利用SPSS16.0和R语言对其进行后续分析和比较.结果 山羊附睾头、体、尾各部分精子与含各类型糖基特异性的凝集素芯片结合分析后发现,与山羊附睾头部精子结合的凝集素有40种,体部精于结合的有48种,尾部精子结合的有42种.来自附睾不同区段的精子与凝集素结合强度也有显著差异,其中,甘露糖特异性的凝集素与体部精子结合最高,复杂结构特异性的凝集素与附睾头、体、尾各部分精子的结合强度呈梯度下降趋势.结论 山羊精子膜表面的糖被,随着精子在附睾中的成熟,也在不断地发生变化的.

DEFB126 rs140685149 mutation was shown to cause sperm dysfunction and subfertility. Indel rs11467497 is another 4-nucleotide frame-shift mutation (151bp upstream of rs140685149) that leads to the premature termination of translation and the expression of peptide truncated at the carboxyl terminus. In the present study, we performed a comprehensive association study to check the contribution of rs140685149 and rs11467497 to male infertility. Our results confirmed the previous findings that there was no association between rs140685149 and sperm motility. In contrast, we found a significant association of another indel rs11467497 with male infertility. Moreover, rs11467497 was shown to be associated with higher number of round cells in the infertile males with low sperm motility. Surprisingly, the two mutations commonly existed in the sperm donors (n=672), suggesting a potential application of the two indels in the screening for eligible sperm donors. Western blotting assays showed the sperms with rs140685149 2-nt deletion tended to have unstable DEFB126 protein in contrast of no DEFB126 protein expressed in the sperms with rs11467497 4-nt deletion, suggesting a more severe consequence caused by rs11467497 mutation. In conclusion, our study presented a significant contribution of another functional frame-shift polymorphism of DEFB126 (rs11467497) to male infertility.

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.