Preservation of Primordial Follicles from Lions by Slow Freezing and Xenotransplantation of Ovarian Cortex into an Immunodeficient Mouse

Abstract

ContentsAssisted reproductive technology (ART) is considered an important tool in the conservation of endangered species, but often the most limiting factor of ART is the availability of mature oocytes. The aim of the present study was to investigate the feasibility of preserving female germ cells from ovaries of female lions (Panthera leo). Good quality cumulus-oocyte complexes (COCs) were isolated and subjected to in vitro maturation (IVM). In addition, ovarian cortex was obtained and cut into pieces for culture and cryopreservation by slow freezing. The survival of ovarian follicles was assessed by histology. Frozen-thawed samples of ovarian cortex samples were xenotransplanted under the skin of ovariectomized immunodeficient mouse for 28 days. Overall, 178 intact COCs were obtained from 13 lions, but only 28.1% were matured in vitro indicating insufficient IVM conditions. In contrast, almost all follicles within the ovarian cortex survived culture when the original sample was from a young healthy lion collected immediately after euthanasia. Within the xenotransplants, the number of primordial follicles decreased after 28 days by 20%, but the relation between primordial and growing follicles changed in favour of follicular growth. Female gamete rescue from valuable felids may be performed by slow freeze cryopreservation of ovarian cortex. Although the IVM protocol for lions is not yet optimized, mature oocytes may be obtained after longterm xenotransplantation and IVM and could potentially represent one way of salvage of endangered felid species in the future.

Ovarian and testicular xenografting: state of art and perspectives in domestic and wild mammals

“…Se por um lado, o xenotransplante serve para confirmar a eficiência de técnicas de congelação e vitrificação, estas, uma vez validadas, para uma espécie, são plenamente aplicáveis na reprodução assistida em animais de interesse ecológico (Abassi et al, 2011, Wiedemann et al, 2012, Campos-Júnior et al, 2014, econômico (Senbon et al, 2003), bem como para humanos (Oktay et al, 1998).…”

Preservation of female fertility in humans and animal species

“…Evidence from a number of species indicate that xenografting of cryopreserved ovarian tissue into mice with severe combined immunodeficiency may provide a temporary solution to this problem. Indeed, xenografting has been shown to support the activation of primordial follicle growth and antral follicle production in cryopreserved ovarian tissues from a range of rodent and domestic species including mouse (Snow et al, 2002), cat (Bosch et al, 2004), and cow as well as a variety of wild animals including tammar wallaby (Mattiske et al, 2002), common wombat (Cleary et al, 2003), African elephant, (Gunasena et al, 1998) and lion (Wiedemann et al, 2012). The combined approach of ovarian tissue banking followed by xenografting may in the future be used in conjunction with follicle culture technologies to produce full sized, MII oocytes for in vitro fertilisation and embryo transfer into a suitable recipient animal.…”

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model

“…These options also are particularly interesting in endangered species conservation to bank ovarian tissue containing an abundant amount of preantral and primordial follicles for individuals undergoing surgical ovarian removal or dying unexpectedly [2][3][4]. In this type of studies, the domestic cat represents an excellent biomedical model for nondomestic cats and human [3,5,6].…”

“…These options also are particularly interesting in endangered species conservation to bank ovarian tissue containing an abundant amount of preantral and primordial follicles for individuals undergoing surgical ovarian removal or dying unexpectedly [2][3][4]. In this type of studies, the domestic cat represents an excellent biomedical model for nondomestic cats and human [3,5,6]. In association with the freezing techniques, culture of the ovarian tissue in vitro is a fundamental approach to reanimate the tissue and grow more oocytes to an advanced stage [5,7,8].…”

“…In association with the freezing techniques, culture of the ovarian tissue in vitro is a fundamental approach to reanimate the tissue and grow more oocytes to an advanced stage [5,7,8]. However, there may be several hours between the ovarian tissue collection and the cryopreservation process itself which compromises (through ischemia and oxidative stress) the viability and developmental competence of the germ cells within the gonads [3,9,10]. During this critical period, cellular respiration in mitochondria is still occurring despite the ischemic environment [10][11][12].…”