Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.
Abstract: Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After 7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral follicles when compared with SF-IVC7, but both had a lower (P 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P 0.05) among groups. Prior to IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater (P < 0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results herein presented shed light on equine fertility preservation programs using OTC techniques.
Copyright © 2018 Elsevier Inc. All rights reserved.
Publication Date: 2018-12-01 PubMed ID: 30543999DOI: 10.1016/j.theriogenology.2018.11.034Google Scholar: Lookup
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- Journal Article
Summary
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The research investigates the effects of two cryopreservation techniques on equine ovarian tissue, comparing the functionality, integrity, and proliferative ability of the tissue after cryopreservation. It was found that both slow-freezing (SF) and vitrification (VIT) were effective in preserving follicle morphology and ensuring similar tissue functionality after a period of 7 days in in vitro culture.
Objective of the Study
- The study was aimed at examining the effects of slow-freezing (SF) and vitrification (VIT) methods on the equine ovarian tissue after undergoing in vitro culture (IVC) for varying durations.
- Focus was given to the morphological integrity of preantral follicles and their distribution, and to observing cell proliferation and apoptosis factors in the ovarian tissue.
Methodology and Observations
- The number of normal primordial follicles in slow-freezing control, vitrification control, and fresh control groups showed no significant statistical difference.
- After 7 days of IVC, the total percentage of normal preantral follicles in the VIT group was higher compared to the SF group. However, the fresh group outperformed both of these groups.
- There was an insignificant difference in expression levels of EGFR and Ki-67(proliferation markers) amongst fresh, SF and VIT groups both before and after the 7 day culture period.
- Bax (a marker of apoptosis) expression was higher in the VIT group after 7 days of culture compared to the fresh and SF groups, but Bcl-2 levels (another apoptosis marker) were similar across all groups.
- TUNEL signals, which indicate DNA fragmentation, were similar among all groups prior to the IVC. However, the VIT group exhibited a greater number of TUNEL signals after the 7-day culture compared to the fresh group, suggesting more DNA fragmentation.
Conclusion and Implications
- Both SF and VIT were found to be effective in preserving the morphological integrity of follicles compared to the fresh control group. They also offered similar tissue functionality and cell proliferation capability after equine ovarian tissue cryopreservation (OTC) over a 7-day in vitro culture period.
- The research provides critical insights for equine fertility preservation programs using OTC techniques and suggests both SF and VIT can be used reliably for this purpose.
Cite This Article
APA
Gastal GDA, Aguiar FLN, Ishak GM, Cavinder CA, Willard ST, Ryan PL, Feugang JM, Gastal EL.
(2018).
Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.
Theriogenology, 126, 88-94.
https://doi.org/10.1016/j.theriogenology.2018.11.034 Publication
Researcher Affiliations
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, lllinois, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, lllinois, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, lllinois, USA; Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq.
- Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
- Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
- Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
- Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
- Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, lllinois, USA. Electronic address: egastal@siu.edu.
MeSH Terms
- Animals
- Apoptosis
- Cell Proliferation
- Cryopreservation / methods
- Cryopreservation / veterinary
- DNA Fragmentation
- Female
- Fertility Preservation / methods
- Fertility Preservation / veterinary
- Horses / physiology
- Ovary / cytology
- Stress, Physiological
- Tissue Preservation / methods
- Tissue Preservation / veterinary
- Vitrification
Citations
This article has been cited 2 times.- Azevedo AR, Pais AS, Almeida-Santos T, Pires VMR, Pessa P, Marques CC, Nolasco S, Castelo-Branco P, Prates JAM, Lopes-da-Costa L, Laranjo M, Botelho MF, Pereira RMLN, Pimenta JMBGA. Medical Grade Honey as a Promising Treatment to Improve Ovarian Tissue Transplantation. Bioengineering (Basel) 2022 Jul 30;9(8).
- Sacinti KG, Sadat R, Ozkavukcu S, Sonmezer M, Sonmezer M. Ovarian tissue cryopreservation and transplantation as a natural means to delay menopause. Arch Gynecol Obstet 2024 Nov;310(5):2305-2313.
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