Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

Abstract

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230−350 (day 0 = grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230−350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.

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

“…In addition, it is possible to consider the use of a non-permeable high molecular weight compound other than sucrose in the vitrification solution. Indeed, a recent study carried out with the use of trehalose reported the birth of live piglets using spermatozoa retrieved from vitrified/warmed and xenografted pre-pubertal testicular tissue (Kaneko et al, 2013). Moreover, it was observed that the Figure 6 Mouse flagellated spermatozoa after mechanical dilaceration of 36.5 dpp in vivo testicular tissue (A 1-2 ) or explants obtained after an in vitro 30-day-organ culture of FT (B 1-2 ) and after CSF (C 1-2 ) or mSSV 1 (D 1-2 ).…”

“…Transplantation of testicular tissue has generally been used as an approach to complete the maturation of vitrified pre-pubertal testicular tissue in mice (Baert et al, 2012) or piglets (Abrishami et al, 2010;Kaneko et al, 2013). However, a decrease in cell density has been observed in seminiferous tubules of transplanted testicular tissue after grafting that has not been observed after in vitro culture (Poels et al, 2012).…”

“…However, vitrification represents an innovative alternative strategy for pre-pubertal testicular tissue cryopreservation. Indeed, it has been suggested that vitrification may be an effective method to cryopreserve testicular tissue and maintain its ability to initiate (Poels et al, 2013) or even complete (Abrishami et al, 2010;Kaneko et al, 2013;Yokonishi et al, 2014) spermatogenesis after thawing. Vitrification is a physical phenomenon that allows, by an ultrafast cooling velocity, the transformation of a liquid into an amorphous glassy solid (Fahy et al, 1984).…”

“…It is difficult to compare the conclusions of several published data based on testicular tissue vitrification because the protocols were evaluated in a variety of animal models, such as zebrafish (Bono-Mestre et al, 2009), Japanese quails (Liu et al, 2012a(Liu et al, ,b, 2013, mice (Curaba et al, 2011b;Gouk et al, 2011;Hemadi et al, 2011Hemadi et al, , 2014Baert et al, 2012;Gholami et al, 2013a,b;Yokonishi et al, 2014), adult cats (Thuwanut & Chatdarong, 2012), piglets (Zeng et al, 2009;Abrishami et al, 2010;Kaneko et al, 2013), and monkeys (Poels et al, 2012), as well as in pre-pubertal boys (Curaba et al, 2011a;Poels et al, 2013) and adult men (Baert et al, 2013;Tang et al, 2014). Furthermore, the vitrification protocols and techniques used were generally very different, and the methods or parameters chosen to assess the quality of the tissue after thawing were also highly heterogeneous.…”

Male pronucleus formation after ICSI: effect of oocyte cysteine or sperm Triton X-100 treatments

“…The ability of porcine oocytes to adequately develop after in vitro maturation (IVM) and fertilization (IVF), leading to live piglets, has been largely demonstrated by several laboratories (Kikuchi et al 2002;Yoshioka et al 2002Yoshioka et al , 2003Suzuki et al 2004Suzuki et al , 2006Coy et al 2005;Garcia-Rosello et al 2006a;Yong et al 2006;Maehara et al 2012;Kaneko et al 2013). Despite this fact, the efficiency of IVP of blastocysts after intracytoplasmic sperm injection (ICSI) still remains low ).…”