Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

Abstract

SUMMARYTesticular tissue cryopreservation offers the hope of preserved future fertility to pre-pubertal boys with cancer before exposition to gonadotoxic treatments. The objective of this study was to compare controlled slow freezing (CSF) with five vitrification techniques for cryopreservation of murine pre-pubertal testicular tissue and to evaluate the best protocol that could provide a successful completion of spermatogenesis after in vitro maturation. Testicular tissue from 24 mice at 6.5 days post-partum (dpp) was used to compare several vitrification protocols with one another, as well as with a CSF protocol. Toxicity test using additional 12 mice was performed for all cryopreservation solutions. Fresh tissue (FT) from six mice was used as a control. Once the optimal vitrification protocol was selected [the modified solid surface vitrification No. 1 (mSSV 1 )], testes from 18 mice were cultured in vitro for 30 days with (i) fresh, (ii) slow-frozen/thawed and (iii) vitrified/warmed tissues. Testes from six mice at 36.5 dpp were used as controls. At day 30 of in vitro culture, germ cells of the seminiferous tubules showed a high ability to proliferate and elongated spermatids were observed after both freezing techniques, confirming the successful completion of in vitro spermatogenesis. However, after mSSV 1 , the morphological alterations and the percentage of pyknotic seminiferous tubules were lower than CSF (4.67 AE 0.53 vs. 10.1 AE 1.12 and 22.7 AE 2.83% vs. 37.3 AE 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV 1 than for CSF (35 AE 3 vs. 9 AE 4 cells), with amounts of secreted testosterone during the culture close to those of FT. The mSSV 1 protocol resulted in success rates better than CSF in maintaining testicular tissue structure, tubular morphology and tissue functions not solely for immediate frozen/thawed tissues but also after a long-term in vitro culture.

In vitrodifferentiation of rat spermatogonia into round spermatids in tissue culture

“…One year later, the same group showed for the first time that it is possible to differentiate spermatogonia in murine pre-pubertal testes into functional sperm in vitro (Sato et al ., 2011). Since then, others have also reported similar data using murine pre-pubertal testis pieces (Dumont et al ., 2015; Arkoun et al ., 2015, 2016). …”

“…However, the efficiency of the organ culture system was low, as only 0.5 ± 1.0% to 4.1 ± 3.0% of the tubules contained round spermatids exhibiting the formation of an acrosomal cap (as identified via PAS). Other recently published studies reported that murine testicular tissue cultured in organ culture resulted in 22 ± 2.8% of the tubules containing round spermatids (Dumont et al ., 2015). If repeated by others, the efficiency of murine germ cell differentiation in vitro into round spermatids would be far higher than the rats as described in this study.…”

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

“…(2011)2.1 M DMSO + 2.7 M EG + 0.5 M sucrose + 20% FBSSSVSpz in allografts, Spz in organotypic cultureBaert et al . (2012), Dumont et al . (2015)2.1 M DMSO + 2.7 M EG + 20% FBS + 0.5 M sucroseVial, LN2Preservation of SSCsGholami et al .…”

“…In rodents, maintenance of tissue integrity and activity in organotypic cultures was reported using slow freezing and vitrification (Travers et al ., 2011; Curaba et al ., 2011b). Complete germ cell differentiation was observed by in vitro maturation of vitrified-thawed TT fragments (Dumont et al ., 2015), as well as after grafting slow-frozen mouse and rat samples and vitrified-thawed mouse samples (Schlatt et al ., 2002; Milazzo et al ., 2010; Baert et al ., 2012; Yildiz et al ., 2013). Importantly, true validation of slow freezing as a means to preserve TT functionality came with the publication from Shinohara et al .…”

“…This procedure includes exposing TT samples to a vitrification solution before placing them on a sterile aluminum boat floating on LN 2 , and transferring the samples into precooled vials followed by plunging them into LN 2 (Abrishami et al ., 2010). Using this approach, the functionality of immature mouse and porcine TT could be maintained during cryopreservation (Baert et al ., 2012; Kaneko et al ., 2013, 2014; Dumont et al ., 2015). Our group studied the feasibility of vitrifying mature human tissue with a protocol proven to preserve the functionality of immature porcine TT upon xenografting (Abrishami et al ., 2010).…”