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Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying

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Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration.


Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR.


CRNPs effectively transported trehalose into COCs within 4 h of co-incubation at 38.5 °C followed by a cold-triggered release at 4 °C for 15 min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1 at dehydration levels reaching up to 63% of water loss.


Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

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Data are available upon request to the corresponding author.


  1. Devi L, Goel S. Fertility preservation through gonadal cryopreservation. Reprod Med Biol. 2016;15(4):235–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Comizzoli P, He X, Lee PC. Long-term preservation of germ cells and gonadal tissues at ambient temperatures. Reprod Fertil. 2022;3(2):R42–50.

    Article  PubMed Central  Google Scholar 

  3. Comizzoli P, Amelkina O, Lee PC. Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation. Mol Reprod Dev. 2022;89(12):565–78.

    Article  CAS  PubMed  Google Scholar 

  4. Saragusty J, Loi P. Exploring dry storage as an alternative biobanking strategy inspired by Nature. Theriogenology. 2019;126:17–27.

    Article  PubMed  Google Scholar 

  5. Graves-Herring JE, Wildt DE, Comizzoli P. Retention of structure and function of the cat germinal vesicle after air-drying and storage at suprazero temperature. Biol Reprod. 2013;88(6):139.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Dang-Nguyen TQ, Nguyen HT, Nguyen MT, Somfai T, Noguchi J, Kaneko H, et al. Maturation ability after transfer of freeze-dried germinal vesicles from porcine oocytes. Anim Sci J. 2018;89(9):1253–60.

    Article  CAS  PubMed  Google Scholar 

  7. Wakayama S, et al. Healthy cloned offspring derived from freeze-dried somatic cells. Nat Commun. 2022;13(1):3666.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hibshman JD, Clegg JS, Goldstein B. Mechanisms of desiccation tolerance: themes and variations in brine shrimp, roundworms, and tardigrades. Front Physiol. 2020;11: 592016.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Patrick JL, Elliott GD, Comizzoli P. Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model. Theriogenology. 2017;103:36–43.

    Article  CAS  PubMed  Google Scholar 

  10. Elliott GD, et al. Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model. Biopreserv Biobank. 2015;13(3):164–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang Y, et al. Cold-responsive nanoparticle enables intracellular delivery and rapid release of trehalose for organic-solvent-free cryopreservation. Nano Lett. 2019;19(12):9051–61.

    Article  CAS  PubMed  Google Scholar 

  12. Lee PC, Comizzoli P. Desiccation and supra-zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation. Mol Reprod Dev. 2019;86(12):1822–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Amelkina O, et al. Apoptosis-related factors in the luteal phase of the domestic cat and their involvement in the persistence of Corpora lutea in lynx. PLoS One. 2015;10(11): e0143414.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Hachen A, Jewgenow K, Braun BC. Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus). Theriogenology. 2012;77(3):539–49.

    Article  CAS  PubMed  Google Scholar 

  15. Siemieniuch MJ, et al. Steroidogenic capacity of the placenta as a supplemental source of progesterone during pregnancy in domestic cats. Reprod Biol Endocrinol. 2012;10:89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Braun BC, Zschockelt L, Dehnhard M, Jewgenow K. Progesterone and estradiol in cat placenta--biosynthesis and tissue concentration. J Steroid Biochem Mol Biol. 2012;132(3–5):295–302.

    Article  CAS  PubMed  Google Scholar 

  17. Wood TC, Wildt DE. Effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro. J Reprod Fertil. 1997;110(2):355–60.

    Article  CAS  PubMed  Google Scholar 

  18. Grupen CG, Fung M, Armstrong DT. Effects of milrinone and butyrolactone-I on porcine oocyte meiotic progression and developmental competence. Reprod Fertil Dev. 2006;18(3):309–17.

    Article  CAS  PubMed  Google Scholar 

  19. Shah S, et al. Fluorescence properties of doxorubicin in PBS buffer and PVA films. J Photochem Photobiol B. 2017;170:65–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lee PC, et al. Desiccated cat spermatozoa retain DNA integrity and developmental potential after prolonged storage and shipping at non-cryogenic temperatures. J Assist Reprod Genet. 2021.

  21. Vandesompele J, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):ESEARCH0034.

    Article  Google Scholar 

  22. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9): e45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Comizzoli P, Wildt DE, Pukazhenthi BS. In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes. Reprod Domest Anim. 2009;44(Suppl 2):269–74.

    Article  PubMed  Google Scholar 

  24. Comizzoli P, Wildt DE, Pukazhenthi BS. Impact of anisosmotic conditions on structural and functional integrity of cumulus-oocyte complexes at the germinal vesicle stage in the domestic cat. Mol Reprod Dev. 2008;75(2):345–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Goncalves DR, et al. Cellular uptake of polymeric nanoparticles by bovine cumulus-oocyte complexes and their effect on in vitro developmental competence. Eur J Pharm Biopharm. 2021;158:143–55.

    Article  CAS  PubMed  Google Scholar 

  26. Kim HJ, et al. TRITC-loaded PLGA nanoparticles as drug delivery carriers in mouse oocytes and embryos. ACS Appl Mater Interfaces. 2021;13(5):5975–88.

    Article  CAS  PubMed  Google Scholar 

  27. Huang C, Wu D, Khan FA, Wang Y, Xu J, Luo C, et al. Zinc oxide nanoparticle causes toxicity to the development of mouse oocyte and early embryo. Toxicol Lett. 2022;358:48–58.

    Article  CAS  PubMed  Google Scholar 

  28. Sribna VO, Voznesenska TY, Blashkiv TV. The influence of zero-valent iron nanoparticles on oocytes and surrounding follicular cells in mice. Appl Nanosci. 2019;9(6):1395–403.

    Article  CAS  Google Scholar 

  29. Lin YH, et al. The effects of graphene quantum dots on the maturation of mouse oocytes and development of offspring. J Cell Physiol. 2019;234(8):13820–31.

    Article  CAS  PubMed  Google Scholar 

  30. Wang R, et al. Potential adverse effects of nanoparticles on the reproductive system. Int J Nanomedicine. 2018;13:8487–506.

    Article  CAS  PubMed Central  Google Scholar 

  31. Lei R, et al. Effects of fullerenol nanoparticles on rat oocyte meiosis resumption. Int J Mol Sci. 2018;19(3).

  32. Das J, Choi YJ, Song H, Kim JH. Potential toxicity of engineered nanoparticles in mammalian germ cells and developing embryos: treatment strategies and anticipated applications of nanoparticles in gene delivery. Hum Reprod Update. 2016;22(5):588–619.

    Article  CAS  PubMed  Google Scholar 

  33. Kim YS, et al. PLGA nanoparticles with multiple modes are a biologically safe nanocarrier for mammalian development and their offspring. Biomaterials. 2018;183:43–53.

    Article  CAS  PubMed  Google Scholar 

  34. Adekiya TA, et al. In vivo evaluation of praziquantel-loaded solid lipid nanoparticles against S. mansoni infection in preclinical murine models. Int J Mol Sci. 2022;23(16):1.

    Article  Google Scholar 

  35. Naziris N, et al. Thermoresponsive chimeric nanocarriers as drug delivery systems. Colloids Surf B Biointerfaces. 2021;208: 112141.

    Article  CAS  PubMed  Google Scholar 

  36. Turathum B, Gao EM, Chian RC. The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization. Cells. 2021;10(9).

  37. Lopez A, et al. DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development. Porcine Health Manag. 2021;7(1):56.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Suttirojpattana T, Somfai T, Matoba S, Nagai T, Parnpai R, Geshi M. The effect of temperature during liquid storage of in vitro-matured bovine oocytes on subsequent embryo development. Theriogenology. 2016;85(3):509-18.e1.

    Article  CAS  PubMed  Google Scholar 

  39. Han D, Zhao BT, Liu Y, Li JJ, Wu YG, Lan GC, et al. Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes. Mol Reprod Dev. 2008;75(5):838–46.

  40. Martino A, Pollard JW, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev. 1996;45(4):503–12.

    Article  CAS  PubMed  Google Scholar 

  41. Iryani MTM, et al. Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana. Cell Stress Chaperones. 2020;25(6):1099–103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Alterio T, et al. Heat shock proteins in encysted and anhydrobiotic eutardigrades. J Limnol. 2012;71(1).

  43. Menezo Y, et al. Methylation: an ineluctable biochemical and physiological process essential to the transmission of life. Int J Mol Sci. 2020;21(23).

  44. Huan Y, et al. A novel role for DNA methyltransferase 1 in regulating oocyte cytoplasmic maturation in pigs. PLoS One. 2015;10(5): e0127512.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Mitchell LE. Maternal effect genes: update and review of evidence for a link with birth defects. HGG Adv. 2022;3(1): 100067.

    CAS  PubMed  Google Scholar 

  46. Turathum B, et al. Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Reprod Biol Endocrinol. 2010;8:70.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Huang J, et al. Dynamic changes in the global transcriptome of bovine germinal vesicle oocytes after vitrification followed by in vitro maturation. Reprod Fertil Dev. 2018;30(10):1298–313.

    Article  CAS  PubMed  Google Scholar 

  48. Ruiz-Conca M, et al. Apoptosis and glucocorticoid-related genes mRNA expression is modulated by coenzyme Q10 supplementation during in vitro maturation and vitrification of bovine oocytes and cumulus cells. Theriogenology. 2022;192:62–72.

    Article  CAS  PubMed  Google Scholar 

  49. Somfai T, et al. Vitrification of porcine cumulus-oocyte complexes at the germinal vesicle stage does not trigger apoptosis in oocytes and early embryos, but activates anti-apoptotic Bcl-XL gene expression beyond 4-cell stage. J Reprod Dev. 2020;66(2):115–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ebrahimi B, et al. In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes. J Assist Reprod Genet. 2010;27(5):239–46.

    Article  PubMed Central  Google Scholar 

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We thank Dr. Keiko Antoku (Last Chance Animal Rescue) and Dr. Joy Lewis (Spay Now Animal Surgery Clinic), and their staff for providing domestic cat testes and ovaries.


This work was supported by the Office of the Director, National Institutes of Health, grant/award number: R01OD023139. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations



PL: conceptualization, project administration, methodology, investigation, formal analysis, visualization, and writing—original draft preparation; SS: methodology and writing—methodology; OA: investigation, analysis, writing—methodology, and review; HS: methodology and writing—review; XH: resources and writing—review; PC: conceptualization, funding acquisition, resources, supervision, and writing—review and editing.

Corresponding author

Correspondence to Pierre Comizzoli.

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Ethical disclosure

The Animal Care and Use Committee from the Smithsonian’s National Zoo and Conservation Biology Institute granted a waiver of the animal care and use approval for that study because testes and ovaries were collected at local veterinary clinics as byproducts from owner-requested routine neutering and spaying.

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The authors declare no competing interests.

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Suppl. Fig. 1

High resolution image (TIF 12966 kb)


Kinetics of water content during microwave drying. A 40 µl drop of 0.3 M trehalose was microwave dried for 0 to 40 min (5 min interval). Water contents were expressed as gH2O/gDW (left Y axis) and percentage of water weight (right Y axis). Values are mean ± SEM. (PNG 87 kb)

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Lee, PC., Stewart, S., Amelkina, O. et al. Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying. J Assist Reprod Genet 40, 1817–1828 (2023).

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