Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†.

Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species.

PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT in ovarian granulosa cells.

Controlled maturation of ovarian follicles is necessary for fertility. Follicles are restrained at an immature stage until stimulated by FSH secreted by pituitary gonadotropes. FSH acts on granulosa cells within the immature follicle to inhibit apoptosis, promote proliferation, stimulate production of steroid and protein hormones, and induce ligand receptors and signaling intermediates. The phosphoinositide 3-kinase (PI3K)/AKT (protein kinase B) pathway is a pivotal signaling corridor necessary for transducing the FSH signal. We report that protein kinase A (PKA) mediates the actions of FSH by signaling through multiple targets to activate PI3K/AKT. PKA uses a route that promotes phosphorylation of insulin receptor substrate-1 (IRS-1) on Tyr(989), a canonical binding site for the 85-kDa regulatory subunit of PI3K that allosterically activates the catalytic subunit. PI3K activation leads to activation of AKT through phosphorylation of AKT on Thr(308) and Ser(473). The adaptor growth factor receptor bound protein 2-associated binding protein 2 (GAB2) is present in a preformed complex with PI3K heterodimer and IRS-1, it is an A-kinase anchoring protein that binds the type I regulatory subunit of PKA, and it is phosphorylated by PKA on Ser(159). Overexpression of GAB2 enhances FSH-stimulated AKT phosphorylation. GAB2, thus, seems to coordinate signals from the FSH-stimulated rise in cAMP that leads to activation of PI3K/AKT. The ability of PKA to commandeer IRS-1 and GAB2, adaptors that normally integrate receptor/nonreceptor tyrosine kinase signaling into PI3K/AKT, reveals a previously unrecognized route for PKA to activate a pathway that promotes proliferation, inhibits apoptosis, enhances translation, and initiates differentiation of granulosa cells.

Core binding factor subunit ß plays diverse and essential roles in the male germline.

Much of the foundation for lifelong spermatogenesis is established prior to puberty, and disruptions during this developmental window negatively impact fertility long into adulthood. However, the factors that coordinate prepubertal germline development are incompletely understood. Here, we report that core-binding factor subunit-ß (CBFß) plays critical roles in prepubertal development and the onset of spermatogenesis. Using a mouse conditional knockout (cKO) approach, inactivation of Cbfb in the male germline resulted in rapid degeneration of the germline during the onset of spermatogenesis, impaired overall sperm production, and adult infertility. Utilizing a different Cre driver to generate another Cbfb cKO model, we determined that the function of CBFß in the male germline is likely limited to undifferentiated spermatogonia despite expression in other germ cell types. Within undifferentiated spermatogonia, CBFß regulates proliferation, survival, and overall maintenance of the undifferentiated spermatogonia population. Paradoxically, we discovered that CBFß also distally regulates meiotic progression and spermatid formation but only with Cbfb cKO within undifferentiated spermatogonia. Spatial transcriptomics revealed that CBFß modulates cell cycle checkpoint control genes associated with both proliferation and meiosis. Taken together, our findings demonstrate that core programs established within the prepubertal undifferentiated spermatogonia population are necessary for both germline maintenance and sperm production.