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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 4  |  Page : 233-238

Effects of cryopreservation on human sperm glycocalyx


1 Department of Applied Biology, State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East University of Science and Technology, Shanghai 200237, China
2 Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
3 Department of Allergy, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200001, China
4 Department of Reproductive Pharmacology, NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China
5 Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 201100, China
6 Department of Andrology, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China
7 Department of Applied Biology, State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East University of Science and Technology; Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Date of Submission17-Nov-2017
Date of Web Publication7-Feb-2018

Correspondence Address:
Yong-Lian Zhang
Shanghai Institute of Biochemistry and Cell Biology, No. 320 Yueyang Road, Shanghai 200031
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.224914

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  Abstract 


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: CryoSperm™ 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.

Keywords: Cryopreservation Damage; Glycocalyx; Lectin Microarray; Sperm


How to cite this article:
Wu YC, Xin AJ, Lu H, Diao H, Cheng L, Gu YH, Wu B, Tao SC, Li Z, Shi HJ, Zhang YL. Effects of cryopreservation on human sperm glycocalyx. Reprod Dev Med 2017;1:233-8

How to cite this URL:
Wu YC, Xin AJ, Lu H, Diao H, Cheng L, Gu YH, Wu B, Tao SC, Li Z, Shi HJ, Zhang YL. Effects of cryopreservation on human sperm glycocalyx. Reprod Dev Med [serial online] 2017 [cited 2020 Aug 10];1:233-8. Available from: http://www.repdevmed.org/text.asp?2017/1/4/233/224914




  Introduction Top


Sperm cryopreservation has become an important method in assisted reproductive technology and sperm bank. The improvement of the method and stability of recovery rate directly influence the final pregnancy rate.[1],[2],[3] Although cryopreservation is continuously improving, the freezing-thawing process may still cause irreversible damage on the structure and function of sperm.

The surface of mature sperm is covered by a coating of carbohydrate-rich layer that is generated from glycosylated protein and lipid on the membrane surface during sperm passing through epididymis, finally forming a 20–60 nm thick glycocalyx.[4] The sperm glycocalyx is composed of more than 300 different glycoproteins and glycolipids and is the main surface for the interaction of male gametes with the external environment.[4] During the processes of spermiogenesis, spermioteleosis, capacitation and acrosome reaction, the glycoproteins and glycolipids on the sperm surface are rearranged. The subtle change of glycocalyx has great influence on the sperm fertility.[5],[6],[7] Sperm glycocalyx plays important roles in the sperm protection, penetration through cervical mucus, egg recognition, and binding.[8] The maturity and integrity of sperm glycocalyx are closely correlated with sperm fertility.[9]

It has been reported that [5],[10],[11],[12],[13],[14] the motility and activity rates decrease, and DNA integrity, plasma membrane composition, protein composition, and mitochondrial matrix density are damaged in different degrees after freeze-thaw. Besides, the penetrating capability through cervical mucus and eggs are greatly reduced. However, so far, the mechanism of sperm cryodamage is still not clear. Due to the limitation of previous techniques, there are fewer reports on the damage of freeze-thaw on sperm glycocalyx. Through a literature search,[15],[16],[17] only the reports on the changes of poultry sperm glycocalyx after freeze-thaw (correlated with fertility) was found. Nevertheless, there is no report on the changes of human sperm glycocalyx after freeze-thaw. Therefore, the investigation on the cryodamage of sperm glycocalyx can provide important guidance for clinical selection of assisted reproductive technology and optimization of sperm cryopreservation, as well as theoretical basis for male infertility with unknown reason.


  Methods Top


Sample collection

The semen samples were obtained from Human Sperm Bank (Renji Hospital, Shanghai Jiao Tong University). The sperm donors aged between 20 and 35. After 3–5-day suppression of sensual passion, the semen samples were collected by masturbation into sterile containers and liquefied at 37°C in a constant temperature shaker. After semen liquefaction, the semen was analyzed according to the standards of the World Health Organization (Edition 5). The inclusion criteria for the samples were as follows: volume ≥2 mL, sperm concentration ≥15 × 106/mL, and total activity (forward movement + nonforward movement) ≥40%.

Cryoprotectants

Three cryoprotectants were used in this study: CryoSperm™ (Origio, Denmark), Quinn's Advantage ® Sperm Freezing Medium (SAGE BioPharma) and self-made cryoprotectant. The self-made cryoprotectant was prepared as follows: (1) weighted 1.3 g trisodium citrate dehydrate and 1.5 g glucose, and dissolved them in sterile water to make volume 65 mL; (2) added 15 mL glycerin, and mixed well; (3) added 1.3 g glycine, and filtered the solution with 0.45 μm membrane after complete dissolution; and (4) added fresh yelk 20 mL, placed the mixed solution in water bath (56°C) for 40 min, and stirred at times to make yelk completely dissolved. The final pH should be within the range of 6.8–7.2. The cryoprotectant was subpackaged and stored at −80°C.

Sperm cryopreservation and thaw

Cryopreservation

The cryoprotectant was mixed with liquefied semen in the same volume after returning to room temperature. Then, the mixed solution was incubated at 30–35°C for 5 min, and poured into sperm cryovials. Three cryopreservation methods were used in this study: direct fumigation, programmed cooling method, and artificial cooling method.

Direct fumigation

The cryovials were put in the place 5–10 cm higher than liquid nitrogen surface (−175°C) for fumigation, and transferred into liquid nitrogen after 2 h.

Programmed cooling method

The cryovials were put into a controlled rate freezer (Planner Kryo 360-3.3), following the procedure illustrated in [Table 1]. After the cooling was complete, the cryovials were stored in liquid nitrogen.
Table 1: Steps of programmed cooling method

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Artificial cooling method

The cryovials were put into freezing chamber (−20°C) for 30 min, then placed at −80°C for 30 min, put in the neck of the liquid nitrogen container mixed with steam of liquid nitrogen, and finally stored in liquid nitrogen.

Thaw

The cryovials were taken out from liquid nitrogen container, immediately placed into water bath (37°C), and gently shaken until completely thawed. Then, sperm activity was detected. The recovery rate (%) = Sperm motility after cryopreservation/Sperm motility before cryopreservation ×100%.

Preparation for lectin microarray

The lectin microarray was prepared as published literatures described.[18],[19] The concentration of 91 lectins was 2 mg/L, and then mixed with 50% glycerin at a ratio of 1∶1 to make final concentration 1 mg/L. Under humidity, 40% and the samples were applied on the PSH-OP slides with 16 × 18 matrix, and placed on the working stage at 4°C overnight to fix the lectin. On the 2nd day, the lectin slides were sealed in a box and stored at 4°C.

Sperm-lectin binding

The method referred to our published literatures.[19],[20],[21] The samples were washed once with phosphate buffered saline (PBS), fixed with 2% paraformaldehyde/0.2% glutaraldehyde for 30 min, washed with PBS twice, added with 0.5 mL PBS containing 0.2 g/L sodium azide to re-suspend sperm, and stored at 4°C.

The lectin slides were equilibrated at room temperature for 30 min, and then put into Tris Buffered Saline with 0.5% (v/v) Tween-20 (TBST) containing 0.5% tween 20, gently shaken, and blocked for 60 min. Then, the slides were washed with Phosphate Buffered Saline with 0.5% (v/v) Tween-20 (PBST) for 10 min and washed with PBS for 10 min twice. The glass slides with marked matrix arrangement were placed in the back of the slides to indicate the location of lectin spot. Twelve-frame rail was stuck to the dried slides to form 12 blocks.

During the blocking and washing process, the sperm were labeled with fluorescence. The fixed sperm were incubated with propidium iodide (PI: 20 mg/L) at room temperature for 20 min, and collected after centrifugation (2,000 g, 10 min). Then, the sperm were re-suspended with lectin binding buffer (1 × PBS, 50 μmol/L CaCl2, 50 μmol/L MnCl2). Spermatozoa (0.5 × 107) were gently loaded in each block, and incubated at room temperature for 1 h avoiding light. Finally, the binding slides were washed with PBST until the binding profilings could be clearly observed. After the slides were air-dried at room temperature avoiding light, gene chip scanner (GenePix 2000A, GenePix) was used to scan the signal at 532 nm.

Sperm-lectin binding signal detected by flow cytometry

Spermatozoa (0.5 × 106) were re-suspended with 100 mL PBS before and after freeze-thaw and then added with 100 mg/L FITC-MAA, FITC-PSA, FITC-ABA and FITC-AIA to further incubate at 37°C for 30 min, avoiding light. Then, the spermatozoa were washed with 0.5 ml PBS once, re-suspended with 0.5 ml PBS, and detected using flow cytometry (Facs Calibur). The fluorescence strength was analyzed by WinMID 2.9 (http://scripps.edu/software.html; Scripps Institute, La Jolla, CA, USA).

Data analysis

The signals were extracted from the scan images of lectin slides by GenePix pro 6.0 (Axon-labs, USA). Signal to noise ratio (S/B) was defined as fluorescence signal average intensity/background fluorescence signal average intensity (F532 Mean/B532 Mean). S/B of each lectin spot was normalized and averaged. The data were analyzed by SPSS16.0 software (SPSS Inc., Chicago, IL, USA), and expressed as mean ± standard deviation. To call the positive lectin binding, the cutoff was set as S/B ≥2. The data of sperm-lectin binding before and after freeze-thaw were analyzed by paired t-test.


  Results Top


Influence of cryoprotectants and cooling methods on recovery rate

To investigate which cryoprotectant and cooling method had the smallest damage on sperm, we compared commonly used commercial cryoprotectants, CryoSperm™, Quinn's Advantage ® Sperm Freezing Medium and cryoprotectant made by Renji Hospital. Direct fumigation was used as the cooling method. As shown in [Figure 1]a, the recovery rate of CryoSperm™ is significantly higher than those of other two cryoprotectants. Using this cryoprotectant, we further compared the cooling methods. The result indicated that direct fumigation was significantly better than programmed cooling method and artificial cooling method [Figure 1]b. Thus, we selected CryoSperm™ and direct fumigation to perform following experiments.
Figure 1: The effect of cryoprotective medium (a) and methods (b) on sperm recovery rate.

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Difference in lectin binding profilings before and after cryopreservation

To explore the influences of cryopreservation on sperm glycocalyx, we extracted lectin binding profilings from 12 lectin slides that bound with sperm before and after cryopreservation. The paired t-test indicated that in the 91 lectins, there were 33 lectins in sperm had significant change before and after cryopreservation. Among them, there were 9 lectins with significantly down-regulated binding signal with sperm after cryopreservation [Figure 2]a, and 24 lectins with significantly up-regulated signal [Figure 2]b. Based on the classification of glycan identification type, the types with significantly down-regulated lectin recognition include sialic acid (Sia), GlcNAc, Galb1-4 GlcNAc, GalNAc, fucose (Fuc) and unknown glycan types [Figure 2]a, and those with significantly up-regulated lectin recognition include mannose (Man), GlcNAc, Galb1-3 GlcNAc, Galb1-4 GlcNAc, GalNAc, galactose (Gal), and Fuc.
Figure 2: The significant changes of lection binding profiling of sperm before and after frozen-thawed. (a) 9 lectins were greatly decreased after sperm cryopreservation. (b) 24 lectins were greatly increased after sperm cryopreservation.

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Verification of difference in lectin before and after cryopreservation

To further verify the results, we used flow cytometry to test the lectin MAA that was significantly down regulated, and the lectins PSA, ABA, and AIA that were significantly up regulated in 71 samples after cryopreservation. The results were consistent with those of lectin slides. After cryopreservation, the binding signal of MAA with sperm was significantly down-regulated, and those of PSA, ABA, and AIA were significantly up-regulated [Figure 3].
Figure 3: Validation of the significantly different lectins by FACS before and after frozen-thawed. (a) the binding signal of MAA with sperm was significantly down regulated after frozen-thawed, (b-d) the binding signal of PSA, ABA, and AIA with sperm were significantly up regulated after frozen-thawed. *P<0.05.

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  Discussion Top


Sperm glycocalyx lies on the outer surface of plasma membrane, composed of polysaccharide and protein and lipid in the plasma membrane. Most of the reports on the cryodamage of sperm plasma membrane mainly focus on the changes of composition and structure of protein and lipid of plasma membrane. However, there was no report on the damage of glycocalyx on the plasma membrane. Sperm glycocalyx plays very important roles in the activity rate, permeability through cervical mucus, egg recognition, and binding. The integrity of sperm glycocalyx directly influences the fertility.[4],[9],[22] Lectin can specifically recognize and bind to glycan, which is a main tool in glycobiology. In this study, lectin slide was used to compare the glycocalyx change before and after cryopreservation, and flow cytometry was used to verify the results in large-sample size. We firstly reported the cyrodamage of sperm glycocalyx.

Because lectin specifically recognizes glycan, the decrease in sperm binding signal represents decreased glycan on the sperm surface and vice versa. Our study indicated that after freeze-thaw, the sperm binding signals of MAA, MAL II, and SNA-I specifically recognizing Sia significantly decreased, suggesting the Sia of glycocalyx was damaged and lost after freeze-thaw. In general, Sia lies at the end of glycoprotein chain, wrapped in the outermost layer of sperm glycocalyx, and is also the main contributor for the negative charge on the sperm surface.[23],[24] If the freeze damages the sperm glycocalyx, Sia should be firstly affected, which is in line with our study. The Sia from glycocalyx can protect sperm from immunological surveillance of female reproductive tract, which is conducive to the survival of sperm in the reproductive tract.[25],[26] The decrease in Sia on the sperm surface will increase the probability of immunoreaction from the female, so the sperm could not arrive in oviduct. On the other hand, the numerous negative charges from Sia can repulse sperm from each other rather than agglutination, which can protect sperm. The decrease in Sia will cause reduced negative charge on the sperm surface, leading to agglutination, which is against forward movement of sperm. It may be one of the reasons why the fertility rate of frozen sperm is lower than the fresh sperm.

As we discussed above, generally, Sia modifies the end of glycocalyx, playing the role of the protection cover. Besides, our results also proved that the content of Sia on the surface of frozen sperm significantly decreased. The subterminal glycan covered by Sia will be exposed due to lose of Sia, which further causes increase in the lectin signal recognizing these subterminal glycan. Glycoproteins are divided into N-linked glycosylation and O-linked glycosylation, based on glycosylation modification site in the peptide chain. N-linked glycosylation has three types: high-mannose type only with mannose residues, complex type without other mannose residues except manninotriose core, and hybrid type combining with high-mannose type and complex type. Our study has indicated that sperm glycocalyx possesses many oligosaccharide structures of high-mannose type and complex type. After cryopreservation, the signal of mannose significantly increased. O-linked glycosylation structure is simpler than N-linked glycosylation, but the link types are various. First, GalNAc (generally at least one, or up to more than ten) binds with serine/threonine, and then glycans (including Gal and GlcNAC) are added to modify into Sia, or sometimes, Fuc. However, the whole oligosaccharide does not contain man modification. The results have indicated that sperm glycocalyx possesses large amounts of O-type oligosaccharide. After cryopreservation, the GalNAc and Gal in the oligosaccharide significantly increased.

Above all, after cryopreservation, lots of changes occur in the sperm glycocalyx. Lots of Sia that could protect sperm is lost, and the glycans inside oligosaccharide are also exposed. This study can provide experimental and theoretical basis for selection of cryoprotectants and cryopreservation protocols, and also indicate the influence of cryopreservation on sperm fertility from the view of sperm glycocalyx.

Financial support and sponsorship

We thank the financial support from National Natural Science Foundation of China (81401252) and MerckSerono China Research Fund for Fertility Experts.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]


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