Abstract
BackgroundMale infertility is a serious social problem in modern society. Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established.MethodsWe choose Kitw/Kitwv mouse as a research model and try to develop a new treatment strategy and “cure” its infertility. Mutant spermatogonial stem cells (SSCs) were isolated from one single unilateral testis of a 14-day-old Kitw/Kitwv mouse and propagated in vitro. The C to T point mutation on Kitwv site of these SSCs was corrected through CRISPR-Cas9-mediated homology-directed repair (HDR) in vitro. Then, the repaired SSCs were screened out, proliferated, and transplanted into the remaining testis, and complete spermatogenesis was established in the recipient testis.ResultsHealthy offsprings with wild type Kit gene or Kitw mutation were obtained through natural mating 4 months after SSC transplantation.ConclusionIn this study, we established an effective new treatment strategy for NOA caused by germ cell gene defects through a combination of SSC isolation, CRISPR-Cas9-mediated gene editing, and SSC transplantation, which brought hope for these NOA patients to restore their natural fertility.
Highlights
Male infertility is a serious social problem in modern society
Male infertility has become an increasingly serious social problem in modern society, and nonobstructive azoospermia (NOA), which accounts for 10% of all infertile men, is an important cause for this [1–3]
Gamete-deficient Nonobstructive azoospermia (NOA) due to germ cells’ genetic mutations, such as RBMY [5], KLHL10 [6], and SYCP3 [7], has been incurable by assisted reproductive technologies and difficult to bear for patients [8]
Summary
Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established. Heterozygous mutant mice with W and WV mutation of this gene, the Kitw/Kitwv mice, whose spermatogonia are considerably reduced or exhausted, have been used as an excellent animal model to develop therapeutic strategies for gamete-deficient patients due to genetic mutations by Yuan et al [9]. They isolated tail-tip fibroblasts from adult Kitw/Kitwv mice and derived embryonic stem cells from these cloned blastocysts obtained by somatic cell nuclear transfer. The produced mutant ESCs’ W site was corrected using TALEN-mediated gene editing and further differentiated into primordial germ cell-like cells in vitro and transplanted into busulfan-treated mouse
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