Until now, using mouse PSCs, the entire germline cycle can be reconstituted in vitro to form functional gametes, although the efficiency remains limited [17, 18]. The generation of primordial germ cells (PGCs), which can initiate meiosis, is of prime importance for generating haploid gametes [23]. Using ESCs bearing the PGC markers PR/SET domain 1 (Prdm1, also known as Blimp1) and developmental pluripotency–associated 3 (Dppa3, also known Stella), Hayashi et al reported that the combination of bone morphogenetic protein 4, leukemia inhibitory factor (LIF, interleukin 6 family cytokine) and stem cell factor are highly competent for inducing PGC marker expression in epiblast-like cells (EpiLCs); these cells in turn become PGC-like cells (PGCLCs) to facilitate in vitro induction of PGCs (Fig. 3a). This work provides a robust paradigm for the first step for in vitro gametogenesis. Upon transplantation into an environment of appropriate somatic cells in vivo, the induced PGCLCs undergo meiosis and produce functional spermatids and oocytes, which can be subsequently used for generating normal offspring following IVF [24, 25].

Fig. 3 A schematic of ESC derivation and in vitro induced gametogenesis. a In vitro induction of functional gametes from ESCs. EpiLCs and PGCLCs are sequentially induced using well-established female or male ESCs. Next, via aggregation with fetal or neonatal gonadal somatic cells under in vitro conditions, in vitro–derived PGCLCs are successfully converted into primary spermatocytes/oocytes respectively, which are further induced into functional haploid sperm and oocytes. b Derivation and establishment of pluripotent ESC lines from inner cellular mass (ICM) frim in vitro cultured blastocysts Full size image

More recently, in vitro germ cell induction systems have been further optimized to make meiotic differentiation no longer depend on in vivo gonadal niches. Through aggregation with fetal or neonatal gonadal somatic cells under in vitro conditions, in vitro derived PGCLCs are successfully converted into primary spermatocytes/oocytes, respectively, which can be further induced into functional haploid spermatids and oocytes (Fig. 3a). The functionality of these in vitro derived haploid gametes has been confirmed by the production of viable and fertile offspring via intracytoplasmic sperm injection (ICSI) or IVF [17, 18]. It should be noted that blastocysts derived from the in vitro generated gametes can be further used to derive rESCs, which can undergo a new round of in vitro germline induction. Therefore, by integrating in vitro germ cell induction, IVF, and ESC derivation in mouse models, these studies have successfully reconstituted a recurrent life cycle from parental embryos to offspring embryos, without producing offspring animals [17].

The most prominent challenge for establishing in vitro germ cell induction system in farm mammals may be the pluripotent status of PSCs. Pluripotent ESCs are well-established in mice, rhesus monkeys, and humans (Fig. 3b). However, despite the lengthy history of efforts to establish truly undifferentiated ESCs in farm animals, authentic ESC lines that can be proven by stringent germline chimera assay have not been established conclusively in any of these species. Even using the conditions for generating mouse ESCs, such as LIF, BMP4, inhibitors of GSK3 and ERK (2i), derivation of such cell lines has been shown to be chanllenging in nonrodents, especially in domesticated species [26]. Up to date, the majority of the morphologically resembling ESC lines derived from bovine and porcine embryos/fetus, inlcuding those recovered from natural conception, IVF or somatic cell nuclear transfer, fail to contribute to chimeras and exhibite only limited differentiation potential [27, 28]. It should be mentioned here that the putative porcine ESC lines maintained on a basal medium supplemented with FBS plus three growth factors, namely FGF2, LIF, and KITLG, are more capable of forming teratomas [29]. Thus, it is promising that a combination of growth factors may considerably benefit the system for deriving and maintaining dometic ECS lines, as revealed by the fact that the self-renewal capcity of porcine ES-like cells are both LIF-dependent and FGF2-dependent [27]. Similarly, combined use of LIF and FGF2 is also beneficial for maintaining the bovine ES-like cells in an undifferentiated state [30, 31]. These researches, on one hand, have drawn attention to the importance of formulating culture conditions that are consistent with the apparent requirement of factors essential for maintining pluripotency of domestic ESCs. In addition, these data indicates that significant modifications of culture conditions may be needed even for those that had previously proved so successful for mouse and human, since the mechanism for capturing pluripotency may be considerably different between rodent and domestic species. More recently, Bogliotti et al. reported successful derivation of stable primed pluripotent ESCs from bovine blastocysts by using fibroblast growth factor 2 (FGF2) and an inhibitor of the canonical Wnt–β-catenin signaling pathway (IWR1) to optimize culture condition [22]. This work is a breakthrough as it overcomes the challenge of establishing high-quality pluripotent livestock ESCs. Until now, precise mechanisms of how signaling pathways control the pluripotent state and early embryo development remains largely elusive in farm animals, and it appears that the essential pathways are considerably distinct from those of rodent species. Bogliotti’s study, shows that combination of FGF supplementation and WNT signaling inhibition, both of which are critical for capturing bovine pluripotency and important for normal preimplantation embryo development in bovines [32, 33], is critical for capturing bovine pluripotency. This fact highlights that exploring the mechanism underlying pluripotency of domestic embryos, will help identify major obstacles that hamper the establishment of true ESC lines in domestic animals. However, even high-quality ESC lines are established in farm animals, the efficient PGC specification pathway and subsequent aggregation with gonadal somatic cells remains challenging.

Except the promising studies in ESCs, iPSCs also provide a practical alternative for successful in vitro germ cell induction. By continuous formulation and optimization of reprogramming factors and medium conditions, primed- or naive-type iPSCs have been successfully derived from porcine and bovine embryonic fibroblast cells or other cell types [34,35,36,37]. Using porcine iPSCs as progenitor cells, our group has successfully induced porcine iPSCs to the PGCLCs. Further, xenotransplantation of the PGCLCs into seminiferous tubules of infertile immunodeficient mice can result in immunohistochemically identifiable germ cells [38]. Moreover, with the extensive studies over the past decades that investigate the origins and mechanisms underlying PGC and germ line specification/differentiation in domestic animal, a series of key growth factors (e.g. SCF, LIF, FGF2, BMP4) [39,40,41,42] and signaling pathways (Activin/Nodal signaling, redox/apoptotic signaling) [42, 43] have been identified to be implicated in maintaining the survival and self-renewal of domestic PGCs. All these findings will benefit the high-efficient system of domestic PGC induction. Interestinly, a more recent study, using in vitro model of germ cell induction, showed conserved principles of epiblast development for PGC fate among porcine and model animals, although the mechanisms underlying pluripotency networks and early post-implantation development are thought to be divergent among species [44]. In addition, studies highlighting the origins of domestic germline-potential stem cells, provide alternate source of domestic PGSs. Aside for those from developing fetal gonad, stem cells derived from adult bovine and porcine ovaries [45, 46] or fetal porcine skin [47, 48] also exhibit the intrinsic ability to differentiate into PGCLCs or even oocyte-like cells (OLCs). However, these germline-potential stem cells are not preferred in our proposed breeding system, because developmentally advanced stem cells will prolong the breeding cycle since differentiated fetal or adult somatic cells are needed. Considering the big challenge of establishing high-quality ESC lines in domestic animals, iPSCs or germline-potential stem cells, may be feasible alternates for connecting transgenerational breeding cycles. Furthermore, Hayashi’s work also offers a valuable reference for formatting and purifying PGCLCs from ESCs without relevant transgenic markers from domestic animals. Specifially, they identified SSEA1 (stage-specific embryonic antigen) and Integrin β3 as essential surface markers for achieving PGCLC isolation and purification [24]. A more recent study further indicated that epithelial cell adhesion molecule (EpCAM) and integrin α6 are efficient in distinguishing PGCLC following human iPS induction [49]. These advances, together with the studies of germ cell biology in porcine and bovine, provide more substantial basis for eventually achieving in vitro germ cell induction in domestic animals.

From the feasibility perspective, a relative low-frequent but noticeable de novo generation of single-nucleotide variants (SNVs) can be elicited in the proposed breeding system, along with the derivation culture and passage of ESCs, especially by the induced reprogramming of iPSCs [50, 51]. For example, dozens to several hundred de novo SNVs can be detected between generations in ESCs or somatic cells and the mutation rate (approximately 10− 9 to 10− 8 at global genome level) is more frequent than that from in vivo germline differentiation (approximately 10− 10 to 10− 9 at global genome level which varies largely based on species, cell types, and culture or induction methods). Although de novo mutations induced by the manipulation of pluripotent cells have minimal contributions to the reference sites of genome selection, the biological significance and potential application as well as the risk of de novo mutations should be re-evaluated based on offspring phenotypes.