Chaipipat Suparat, Prukudom Sukumal, Sritabtim Kornkanok, Kuwana Takashi, Piyasanti Yanika, Sinsiri Rungthiwa, Piantham Chayada, Sangkalerd Sornchai, Boonsanong Sompong, Pitiwong Klinsak, Pidthong Apisit, Wanghongsa Sawai, Siripattarapravat Kannika
Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom, Thailand; Center of Excellence on Agricultural Biotechnology:(AG-BIO/PERDO-CHE), Bangkok, Thailand.
Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom, Thailand; Center of Excellence on Agricultural Biotechnology:(AG-BIO/PERDO-CHE), Bangkok, Thailand; Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
Theriogenology. 2021 Apr 15;165:59-68. doi: 10.1016/j.theriogenology.2021.02.010. Epub 2021 Feb 18.
Interspecific germline chimerism mediated by transplantation of primordial germ cells (PGCs) of wild species to domestic hosts promises the conservation of wild birds. Cryopreservation of avian eggs and embryos is impracticable, and currently only frozen PGCs enable conservation of both the male and female descendants. Purebred offspring have been obtained from germline chimeras of wild avian species, proving the feasibility of such technology. In vitro propagation has been optimized for avian PGCs of domestic species; however, evidence is rather limited for successful isolation as well as long-term culture from a single embryo of wild species. With accelerating biodiversity loss, we have committed to preserving current genetic resources by freezing PGCs isolated from individual embryos in addition to their genetic material. We have devised a reliable protocol for the isolation and proliferation of PGCs from wild fowls in the family Phasianidae that are conserved in captive breeding (red junglefowl, bar-tailed pheasant, kalij pheasant, Siamese fireback pheasant, and silver pheasant). We obtained individual isolates of cultured circulating PGCs (49.7%, 79/155) as well as tissue PGCs (92.9%, 144/155). The specific co-culture conditions of autologous embryonic cells, without additional growth factors, facilitated the proliferation of so-called tissue PGCs (the remaining PGCs in embryonic tissue following blood aspiration). Only circulating PGCs left in blood vessels and of PGCs migrating to developing gonads have been previously reported. However, the present study is the first to report on the harvest of ectopic PGCs. The defined conditions sustained continuous proliferation of tissue PGCs for at least six months and maintained PGC identity following cryopreservation. Cultured tissue PGCs of these wild species were extensively characterized for their expression of the germ cell-specific proteins, chicken vasa homolog (CVH) and deleted in azoospermia-like (DAZL), as well as the ability to colonize chicken embryonic gonads. The novel protocol is practical for generating enough PGCs for cryopreservation, transplantation, and additionally, it enables isolation of PGCs from both blood circulation and embryonic tissue simultaneously. For conservation purposes, this approach is potentially applicable more widely to other non-domestic birds than those in the family Phasianidae that were investigated in the present study.
将野生物种的原始生殖细胞（PGC）移植到家养宿主中介导的种间生殖系嵌合体有望实现野生鸟类的保护。禽类卵和胚胎的冷冻保存是不可行的，目前只有冷冻的PGC能够保存雄性和雌性后代。已经从野生鸟类物种的生殖系嵌合体中获得了纯种后代，证明了这种技术的可行性。已针对家养物种的禽类PGC优化了体外繁殖；然而，从单个野生物种胚胎成功分离以及长期培养的证据相当有限。随着生物多样性的加速丧失，我们致力于通过冷冻从单个胚胎中分离的PGC及其遗传物质来保存当前的遗传资源。我们设计了一种可靠的方案，用于从圈养繁殖中保存的雉科野生禽类（红原鸡、斑尾雉、黑鹇、暹罗火背鹇和白鹇）中分离和增殖PGC。我们获得了培养的循环PGC（49.7%，79/155）以及组织PGC（92.9%，144/155）的单个分离物。自体胚胎细胞的特定共培养条件，无需额外的生长因子，促进了所谓组织PGC（采血后胚胎组织中剩余的PGC）的增殖。此前仅报道过血管中残留的循环PGC以及迁移到发育中的性腺的PGC。然而，本研究首次报道了异位PGC的收获。确定的条件使组织PGC持续增殖至少六个月，并在冷冻保存后保持PGC特性。对这些野生物种培养的组织PGC进行了广泛表征，检测了其生殖细胞特异性蛋白鸡vasa同源物（CVH）和无精子症样缺失蛋白（DAZL）的表达，以及定殖于鸡胚胎性腺的能力。该新方案对于产生足够用于冷冻保存、移植的PGC是可行的，此外，它还能同时从血液循环和胚胎组织中分离PGC。出于保护目的，这种方法可能比本研究中所调查的雉科鸟类更广泛地适用于其他非家养鸟类。
