Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit)
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FERTILITY PRESERVATION
Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit) Daniela Bebbere 1
&
Sara Pinna 1 & Stefano Nieddu 1 & Dity Natan 2 & Amir Arav 2 & Sergio Ledda 1
Received: 28 May 2019 / Accepted: 6 August 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract Purpose Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model. Methods Pieces of immature testicular tissue (1 mm3) were inserted into “E.Vit” devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming. Results Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response. Conclusions Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue. Keywords Cryopreservation . Testicular tissue . Gene expression . Prepubertal . In vitro culture
Introduction Gonadotoxic therapies, including cancer treatments, may cause severe damage to gonads and potentially lead to infertility. While semen cryopreservation is recommended to adult patients to preserve ability to reproduce after the treatment, this option is not applicable to prepubertal patients, whose gonads do not yet produce spermatozoa. In this case, cryopreservation of spermatogonial stem cell (SSC) suspension or testicular tissue, which contains mainly SSCs, would offer a
* Daniela Bebbere [email protected] 1
Department of Veterinary Medicine, University of Sassari, Sassari, Italy
2
FertileSAFE Ltd, 11 Haharash St, 7403118 Ness Ziona, Israel
real option for preserving fertility prior to gonadotoxic treatments. Different options to restore fertility can be considered when cured patients desire to
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