AIP Conference Proceedings 2092, 040019 (2019); https://doi.org/10.1063/1.5096752 2092, 040019
© 2019 Author(s).
Sperm Na+,K+-ATPase and dynein ATPase activity: A study of embryo development in in vitro fertilization (IVF)
Cite as: AIP Conference Proceedings 2092, 040019 (2019); https://doi.org/10.1063/1.5096752 Published Online: 09 April 2019
Silvia W. Lestari, Aucky Hinting, Hamdani Lunardi, Debby Aditya, Dessy Noor Miati, and Meidika Dara Rizki
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Sperm Na + ,K + -ATPase and Dynein ATPase Activity: A Study of Embryo Development in In Vitro Fertilization (IVF)
Silvia W. Lestari
1,3,5,a), Aucky Hinting
2, Hamdani Lunardi
2, Debby Aditya
4, Dessy Noor Miati
5, Meidika Dara Rizki
51Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
2Department of Medical Biology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
3Specialist program of Andrology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
4Master program for Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
5Smart IVF Clinic, Anna Hospital, Bekasi, Indonesia
a) Corresponding author E-mail: [email protected]
Abstract. The published data represented that poor semen parameters resulted in the poor embryo development rate following in vitro Fertilization (IVF) - intracytoplasmic sperm injection (ICSI). The disorder of sperm motility involving Na+,K+-ATPase and dynein ATPase enzymes disruption may contribute to the paternal effects on embryo quality and development. This study investigated the activities of Na+,K+-ATPase and dynein ATPase in human sperms of infertile men and the correlation to the ICSI outcome referring to the embryo development. Semen samples were obtained from men performing sperm preparation for the ICSI. The World Health Organization (WHO) 2010 Guidance was used to perform semen analysis. Next, the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The ICSI outcomes including the embryo development grading and scoring were recorded and analyzed. The value of Na+,K+-ATPase and dynein ATPase were 1.847 ± 0.694 and 1.459 ± 0.843 µmol Pi/mg protein/h, while the embryo scoring was 91.83. Using the cut-off value of Na+,K+-ATPase and dynein ATPase, we found that the low level Na+,K+-ATPase and dynein ATPase correlated with the low embryo development quality (p<0.05 and p>0.05, respectively). This study demonstrated that poor activities of Na+,K+-ATPase and dynein ATPase could affect embryo the development following the ICSI.
Keywords: dynein ATPase, embryo development quality, Na+,K+-ATPase
INTRODUCTION
A survey data showed that approximately 48.5 million couples were infertile, wherein the male infertility extent to 40-50%.[1, 2] Male infertility is defined as an inability to inseminate the fertile female couple after one year of regular unprotected intercourse due to an existence of abnormal semen parameters. The abnormal of semen parameters could be in forms of an abnormal of sperm morphology (teratozoospermia), low motility (asthenozoospermia), low sperm count (oligozoospermia), or the combination of each form. A study by Jimenez et al. (2011) demonstrated that an array of biochemical and functional defects may contribute to the paternal failure towards fertilization [3, 4].
The highly regulated sperm ion transport systems residing along its plasma membrane play a pivotal role in the sperm maturation, capacitation and gamete communication. The equilibrium of ion homeostasis is determined by the integral enzyme, such as Na+,K+-ATPase, Ca2+-ATPase and dynein ATPase. The failure of these enzymes contribute to sperm motility defects. The Na+,K+-ATPase is involved in the exchange between the intracellular 3 Na+ and the extracellular 2 K+ in cells, thus maintaining the resting membrane potential and osmotic stability [5]. A previous study reported that the inhibition towards Na+,K+-ATPase by ouabain resulting sperm motility reductions [4, 6].
Besides the homeostasis of sperm ion, sperm motility also depends on the flagellar movement which is generated
contains 1 to 3 heavy chains, along with medium and light chains that associate with microtubules.[7, 8] There are six AAA+ located at the head of dynein, which are arranged in a ring. Four of which (AAA1-AAA4) contain nucleotide binder sites, while two others (AAA5 and AAA6) contain less sites that the entire various cellular activities from these domains associate with ATPase [9]. The previous data reported that the AAA1 is a major site in ATP hydrolysis associated with the flagellum motility. Meanwhile, the AAA2 is also crucial in supporting the role towards ATP- binding pocket, which is fundamental for the sperm movement [10]. Numerous studies stated that there was a declining activity of dynein ATPase and low expression of AAA1 and AAA2 among infertile men [11].
The sophisticated assisted reproductive technology brings enormous benefits toward male infertility to have offspring. The IVF-ICSI was successfully conducted in 62.5% of the cycle to achieve pregnancies. Nevertheless, approximately only 34.1% were able to undergo delivery among the infertile couples, indicating the low successful number of IVF-ICSI [12]. Generally, the quality of sperm performance, including sperm motility, plays an important role in the success of ovum fertilization. Nevertheless, there are only a few reports evaluating the correlation of the activity of Na+,K+-ATPase, and dynein -ATPase towards the embryo development quality in the IVF-ICSI. In this study, we evaluated the activity of Na+,K+-ATPase, and dynein-ATPase using a colorimetric method and determining the both cut-off value. As indicated, the activities of Na+,K+-ATPase and dynein ATPase may be used to evaluate and predict the embryo development quality following the IVF-ICSI.
METHOD
Semen Collection and Analysis
In this study, the 15 ejaculated sperm samples were obtained from infertile men involved in the IVF-ICSI program, in Smart IVF Clinic, Anna Hospital (Bekasi, Indonesia). The semen samples were collected by masturbation after 3- 5 d of abstinence. Subsequently, the samples were stored in semen containers for complete liquefying for approximately 30 min at room temperature. The semen analysis was performed according to WHO 2010 Guideline, which included sperm concentration, motility, morphology, viability and membrane integrity [13]. The obtained aliquots were used for Na+,K+-ATPase and dynein ATPase activity assays. The research protocol and ethical clearance was approved by the Ethics Committee of Faculty of Medicine, Universitas Indonesia, and the informed consent was obtained from all patients.
Isolation of sperm plasma membrane
The modified Olson method was conducted to isolate the sperm plasma membranes [14, 15]. The semen samples were re-suspended in 9 mL of an Olson A solution (0.05 M Tris-HCl; 0.1 M NaCl, pH 7.4), followed by a centrifugation at 1000 xg for 10 min at 4 °C. The supernatant was discarded and the Olson B solution (10 mM Tris- HCl; 0.5 mM EDTA, pH 7.4) was added to re-suspend the pellet, followed by a centrifugation at 1000 xg for 10 min at 4 °C. The obtained supernatant was homogenized by the Ultra Turrax homogenizer. Thus, the resulting suspension was layered by the sucrose gradient solution (15% sucrose with 25 mmol/l Tris-HCl (pH 7.4) and 50% sucrose with 25 mmol/l Tris-HCl in pH 7.4). Another serial centrifugation was performed at 17,000 xg for 30 min and the pellet was re-suspended by the Olson B solution, followed by a centrifugation. The obtained pellet was used for Na+, K+- ATPase activity analysis.
Isolation of sperm axoneme
The adjustment of the Olson method was implemented to separate the axoneme fraction of sperms. [4, 16] The Olson A solution (0.05 M Tris-HCl; 0.1 M NaCl, pH 7.4) of 9 ml was mixed the semen. Next, the suspension was centrifuged at 1000 xg for 10 minutes at 4°C. Subsequently, the supernatant was discarded, then the pellet was mixed with the Olson B solution (10 mMTris-HCl; 0.5 mM EDTA, pH 7.4) and centrifuged at 1000 xg for 10 minutes at 4°C. Next, the obtained supernatant was homogenized with the Ultra Turrax homogenizer dan performed gradually sucrose solution (15% sucrose with 25 mmol/l Tris-HCl (pH 7.4) and 50% sucrose with 25 mmol/l Tris-HCl in pH 7.4) in layers. After that, the suspension was centrifuged at 17,000 xg for 30 minutes and the obtained pellet was mixed again with the Olson B solution. To remove the membrane component on the axoneme fraction, 2% sodium dodecyl sulfate (SDS) and 1% bovine serum albumin (BSA) was added and then incubated at 37 °C for 20 minutes.
The Bio-Rad micro-method of Bradford method was performed to measure the protein presence of axoneme fraction.
[17] After that, this suspension was assayed for dynein ATPase analysis.
Na
+, K
+-ATPase activity assay
The Na+, K+-ATPase activity was assessed by using Kitao and Vignini method and measured by incubating 1 mL of isolated sperm plasma membrane in a medium containing 5 mmol/l MgCl2, 140 mmol/l NaCl, and 14 mmol/l KCl in 40 mmol/l Tris-HCl with pH 7.7 at 37°C. The ATPase reaction was inducted by the addition 3 mmol/L of Na2ATP and terminated by 1 mL of 15% trichloroacetic acid 20 minutes later. The reaction was followed by the centrifugation at 1100 xg for 10 minutes and the released inorganic phosphate (Pi) was determined by the colorimetric method based on the KH2PO4 standard [18]. The Na+,K+-ATPase activity was stated as the difference in Pi contents both in the presence and absence of 10 mM ouabain in the reaction.
Dynein activity assay
A medium containing 5 mmol/l MgCl2, Trisbase, the addition of 10 mM ouabain, 1uM thapsigargin and 1mM vanadate was added to the 1 mL of isolated sperm axoneme to measure dynein ATPase activity. The ATPase reaction was performed by the supplementation of 3 mmol/L of Na2ATP and stopped by 1 mL of 15% trichloroacetic acid 20 minutes later. The released inorganic phosphate (Pi) from a centrifugation at 1100 xg for 10 minutes was defined by the colorimetric method based on the KH2PO4 standard. The difference in Pi contents both in the presence and absence of 1 mM vanadate in the reaction was determined as dynein ATPase activity.
ICSI procedure and Embryo culture
Right before the ICSI, the obtained sperm was added to a micro droplet, which consisted of 10%
Polyvinylpyrrolidone (PVP) (Medicult, Origio) in an ICSI dish. Besides the PVP drops, there were some micro droplets comprising a GMOPs medium (Vitrolife, Sweden) to put the fresh oocytes which utilized for the ICSI. Next, all of the micro droplets were overlaid by liquid paraffin (Medicult, Origio). During ICSI procedures, each of oocytes was injected by a single viable and motile sperm, which apparently has a normal morphology. The sperm was immobilized by stroking the injecting pipette on to the sperm tail. The oocyte with a polar body was protected and placed at the 6 or 12 o’clock position onto the holding pipette. The sperm was aspirated, followed by the penetration into the ooplasm at the 3 o’clock position. Furthermore, the injection pipette was released gently from the oocyte. The injected oocytes were placed into drops in the culture dish consisting of ISM1 culture medium (Medicult, Origio) and stored at 37 °C in a triple-gas (5% O2, 6% CO2, and 5% N2) incubator for 3 days.
Embryo development grading and scoring
The morphological grading of embryo development and cleavage were focused on 2-cell and 8-cell stages, based on the embryo grading by Veeck (1998)[19] (Table 1) and the morphological embryo scoring by van Loendersloot (2014) = 103 + (2 x early cleavage) + {-3 x (∑ blastomeres D3 – 8)} + (-5 x morphology score on D3) + (-11 x morula on D3).[20] The embryo development was observed under the reverse phase light microscope (Leica IRB, Japan) and inverted microscope (Olympus IX81 SF-3, Japan). The fertilized oocytes were assessed at 16-18 hours after the ICSI, marked by the appearance of two equally sized pro-nuclei, while the cleaved embryos were evaluated on day 3 (68 – 72 hours after insemination) after the ICSI.
TABLE 1. Scoring criteria for cleaved embryo [19]
Measurement Grade Description
Cleavage stage morphology
Grade 1 Embryo with blastomeres of equal size, no cytoplasmic fragments Grade 2 Embryo with blastomeres of equal size, minor cytoplasmic fragments
Grade 3 Embryo with blastomeres of distinctly of unequal size, none or few cytoplasmic fragments Grade 4 Embryo with blastomeres of equal or unequal size, significant cytoplasmic fragments Grade 5 Embryo with few blastomeres of any size, severe or complete fragments
Statistical Analysis
The data were analyzed using a Statistical Package for Social Science (SPSS) version 22 software for Windows.
The correlation between Na+,K+-ATPase, and dynein -ATPase and the quality of embryo development was analyzed using the Spearman’s test. The p-values less than 0.05 was regarded as significant.
RESULT AND DISCUSSION
The semen profile of infertile man patients is presented in Table 2. The finding of this study demonstrated that all sperm parameters in the semen analysis were shown in normal values. Moreover, this study also showed the biochemical parameters of sperms which were Na+, K+-ATPase and dynein ATPase activity 1.847 ± 0.694 and 3.010
± 0.390 µmol Pi/mg protein/h, respectively. (Table 3) This finding indicated that sperm analyses could not ensure the quality of sperms.
TABLE 2. Semen analysis Parameter
Volume (ml) 3
Ph 7.2
Concentration (million.ml-) 22.5
Motility (%) 43
Morphology (%) 12
Viability (%) 63
Membrane integrity (%) 61
Values are expressed as the mean ± SEM
TABLE 3. Activity of Na+,K+-ATPase and dynein ATPase ATPase activity (⎧M Pi/mg protein/h)
Na+,K+-ATPase 1.847 ± 0.694
Dynein ATPase 3.010 ± 0.390
Values are expressed as the mean ± SEM
In addition, our findings about the morphological grading of embryo and the scoring were demonstrated in Table 4. In Table 4, there were more of Grade 1 embryos compared to other grades.
TABLE 4. Embryo development quality
Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
Figure of embryo
Scoring embryo 91.83
The cut-off values for a good Na+, K+-ATPase and dynein ATPase activity were determined by a statistical analysis of normozoopermic samples, resulting in 13.851±1.884 and 2.725±0.545 µmol Pi/mg protein/h. [11, 21] We further categorized the Na+, K+-ATPase and dynein ATPase activity as low based on the cut-off values, and measured the correlation of each towards embryo development quality. We analyzed the correlation between Na+, K+-ATPase and dynein ATPase activity and the embryo development rate. A different result was obtained when we developed subset analyses using the cut-off values of Na+, K+-ATPase and dynein ATPase to correlate both variables to an embryo development quality. There was a moderate and significant correlation between the low activity of Na+, K+-ATPase activity and the embryo development (r=0.521; p=0.019). In contrast, there was low and insignificant correlation between the low activity of dynein ATPase activity and the embryo development (r=0.316; p=0.116) (Figure 1)
FIGURE 1. Correlation analysis between (A) Na+,K+-ATPase activity and embryo development (r=0.521; p=0.019) and (B) Dynein ATPase activity and embryo development (r=0.316; p=0.116)
This finding presented that low activity of Na+, K+-ATPase and dynein ATPase could influence the development of embryo after the ICSI, but Na+, K+-ATPase influenced more, compared to dynein ATPase. In infertile men, the pathological condition may alter the Na+, K+-ATPase and dynein ATPase activities, thus generating the impairment of sperm structure and function. Therefore, prior being used in the IVF-ICSI, sperm underwent preparation resulted in the higher percentage and the velocity of motile sperms than the whole semen. These revealed that the high presence of good sperms guaranteeing the normal function of Na+, K+-ATPase and dynein ATPase activities, generating the better motile sperms by maintaining its ion homeostasis.
Apparently, sperms with normal morphology and motility was selected during the ICSI. However, the quality of sperms remained unknown, whether the quality is good or improper. Sperm defects, such as fragmented DNA and the impairment of Na+, K+-ATPase and dynein ATPase, affected the embryo development resulting in poor the IVF outcome.[16, 22] Measuring the Na+, K+-ATPase and dynein ATPase could be used as a prospective model for infertile man management, particularly in the idiopathic infertility. The findings in our research presented that the low activity of Na+, K+-ATPase and dynein ATPase correlated to the poor development of an embryo after the ICSI. The approach of measuring Na+, K+-ATPase and dynein ATPase activities might be utilized as an alternative reference for the evaluation of sperm quality considering its correlation towards the embryo development. Surely, further studies should be conducted to investigate other molecular phenomena on sperms that might interfere the quality of embryos.
CONCLUSION
This study showed that the Na+, K+-ATPase and dynein ATPase could affect the embryo development after the ICSI. The Na+, K+-ATPase and dynein ATPase activities might be used as an alternative of sperm screening before the ICSI.
ACKNOWLEDGMENT
The authors would like to express gratitude to Hibah Publikasi Terindeks Internasional untuk Tugas Akhir Mahasiswa (PITTA), Direktorat Riset dan Pengabdian Masyarakat (DRPM), Universitas Indonesia, 2018 for supporting this study.
REFERENCES
1. Mascarenhas M, Flaxman S, Boerma T, Vanderpoel S, Stevens G. National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. PloS Med. 2012;9;e10011356.
2. Kamel R. Managemnet of the infertile couple: an evidence-based protocol. Reprod Biol Endocrinol. 2010;8(1):21.
3. Aitken R, Best F, Richardson D, Djahanbakhch O, Templeton A, Lees M. An analysis of semen quality and sperm function in cases of oligozoospermia. Fertil Steril. 1982;38(6):705-11.
4. Jimenez T, Sanchez G, Blanco G. Activity of the Na,K-ATPase alpha4 isoform is regulated during sperm capacitation to support sperm motility. J Androl. 2012;33(5):1047-57.
5. Lestari S, Miati D, Soeharso P, Sugiyanto R, Pujianto D. SpermNa+, K+-ATPase α4 and plasma membrane Ca2+- ATPase (PMCA) 4 regulation in asthenozoospermia. Systems Biology in Reproductive Medicine. 2017.
6. Da Costa R, Botana D, Pinero S, Proverbio F, Marin R. Cadmium inhibits motility, activities of plasma membrane Ca(2+)-ATPase and axonemal dynein-ATPase of human spermatozoa. Andrologia. 2016;48(4):464-9.
7. Porter M, Johnson K. Dynein structure and function. Annu Rev Cell Biol. 1989;5(1):119-51.
8. Fang S, Baker H. Male infertility and adult polycystic kidney disease are associated with necrospermia. Fertil Steril. 2003;79:643-4.
9. Koonce M, Smaso M. Overexpression of cytoplasmic dyneis's globular head causes a collapse of the interphase microtubule network in Dictyostelium. Mol Biol Cell. 1996;7(6):935-48.
10. Kardon J, Vale R. Regulators of the cytoplasmic dynein motor. Nat Rev Mol Cell Biol. 2009;10(12):854-65.
11. Lestari S, Larasati M, Mansur I, Soelaeman M, Rahmat F, Azzahra F, et al. Sperm dynein AAA1 and AAA2 expression in human sperm: a regulation in sperm preparation. Biomedical & Pharmacology Journal.
2018;11(1):77-84.
12. Asmarinah, Syauqy A, Umar L, Lestari S, Mansyur E, Hestiantoro A, et al. Sperm chromatin maturity and integrity correlated to zygote development in ICSI program. Systems Biology in Reproductive Medicine. 2016.
13. WHO. WHO laboratory manual for the examination and processing of human sperm. In: Organization WH, editor.
5 ed. Geneva, Switzerland: WHO Press; 2010.
14. Olson G, Winfrey V, Garbers D, Noland T. Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa. Biol Reprod 1985;33(3):761-79.
15. Vignini A, Buldreghini E, Nanetti L, Amoroso S, Boscaro M, Ricciardo-Lamonica G. Free thiols in human spermatozoa: are Na+/K+-ATPase, Ca2+-ATPase activities involved in sperm motility through peroxynitrite formation? . Reprod Biomed Online. 2009;18(1):132-40.
16. Desai N, Hafez F, Sabanegh E, Goldfarb J. Paternal effect on genomic activation, clinical pregnancy and live birth rate after ICSI with cryopreserved epididymal versus testicular spermatozoa. Reprod Biol Endocrinol. 2009;7:142.
17. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1-2):248-54.
18. Fiske C, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem. 1925;66(2):375-400.
19. Veeck L. Oocyte assessment and biological performance. Ann NY Acad. 1988;541(1):259-74.
20. van Loendersloot L, van Wely M, van der Veen F, Bossuyt P, Repping S. Selection of embryos for transfer in IVF:
ranking embryos based on their implantation potential using morphological scoring. Reprod Biomed Online.
2014;29(2):222-30.
21. Lestari SW, Miati DN, Seoharso P, Sugiyanto R, Pujianto DA. Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia. Syst Biol Reprod Med. 2017;63(5):294-302.
22. Sedo C, Bilinski M, Lorenzi D, Uriondo H, Noblia F, Longobucco V, et al. Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects. JBRA Assist Reprod. 2017;21(4):343-50
