ARRDC5 expression is conserved in mammalian testes and required for normal sperm morphogenesis.

In sexual reproduction, sperm contribute half the genomic material required for creation of offspring yet core molecular mechanisms essential for their formation are undefined. Here, the α-arrestin molecule arrestin-domain containing 5 (ARRDC5) is identified as an essential regulator of mammalian spermatogenesis. Multispecies testicular tissue transcriptome profiling indicates that expression of Arrdc5 is testis enriched, if not specific, in mice, pigs, cattle, and humans. Knockout of Arrdc5 in mice leads to male specific sterility due to production of low numbers of sperm that are immotile and malformed. Spermiogenesis, the final phase of spermatogenesis when round spermatids transform to spermatozoa, is defective in testes of Arrdc5 deficient mice. Also, epididymal sperm in Arrdc5 knockouts are unable to capacitate and fertilize oocytes. These findings establish ARRDC5 as an essential regulator of mammalian spermatogenesis. Considering the role of arrestin molecules as modulators of cellular signaling and ubiquitination, ARRDC5 is a potential male contraceptive target.

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.

Spermatogonial Stem Cell Transplantation: Insights and Outlook for Domestic Animals.

The demand for food will increase to an unprecedented level over the next 30 years owing to human population expansion, thus necessitating an evolution that improves the efficiency of livestock production. Genetic gain to improve production traits of domestic animal populations is most effectively achieved via selective use of gametes from animals deemed to be elite, and this principle has been the basis of selective breeding strategies employed by humans for thousands of years. In modern-day animal agriculture, artificial insemination (AI) has been the staple of selective breeding programs, but it has inherent limitations for applications in beef cattle and pig production systems. In this review, we discuss the potential and current state of development for a concept termed Surrogate Sires as a next-generation breeding tool in livestock production. The scheme capitalizes on the capacity of spermatogonial stem cells to regenerate sperm production after isolation from donor testicular tissue and transfer into the testes of a recipient male that lacks endogenous germline, thereby allowing the surrogate male to produce offspring with the donor haplotype via natural mating. This concept provides an effective selective breeding tool to achieve genetic gain that is conducive for livestock production systems in which AI is difficult to implement.

Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes.

Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle. Using samples from pre-pubertal calves, the CXCR4 protein was detected by immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1, OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell potential.

Fibroblast growth factors activate mitogen-activated protein kinase pathways to promote migration in ovine trophoblast cells.

Fibroblast growth factors (FGFs) 2 and FGF10 are uterine- and conceptus-derived factors that mediate trophoblast activities in cattle and sheep. To extend our understanding of how FGFs may control peri-implantation development in ruminants, we determined whether FGF2 and FGF10 impact trophoblast cell migration. Transwell inserts containing 8 µm pores were used to examine whether FGF2 or FGF10 supplementation increased oTr1 cell migration. Supplementation with 0.5 ng/ml FGF2 or FGF10 did not affect oTr1 cell migration number, but exposure to 5 or 50 ng/ml FGF2 or FGF10 increased (P<0.05) oTr1 cell migration when compared with controls. The involvement of specific MAP kinase (MAPK) cascades in mediating this FGF response was examined by using pharmacological inhibitors of specific MAPKs. Western blot analysis indicated that FGF2 and FGF10 increased phosphorylation status of MAPKs 1, 3, 8, 9, and 14. Exposure to specific inhibitors blocked FGF induction of each MAPK. Exposure to inhibitors before supplementation with FGF2 or FGF10 prevented FGF induction of cell migration, indicating that each of these signaling molecules was required for FGF effects. A final series of studies examined whether FGF2 and FGF10 also mediated the migration of a bovine trophoblast line (CT1 cell). Increases in migration were detected in each cell line by supplementing 5 or 50 ng/ml FGF2 or FGF10 (P<0.05). In summary, FGF2 and FGF10 regulate migratory activity of ovine trophoblast cells through MAPK-dependent pathways. These outcomes provide further evidence that FGFs function as mediators of peri-implantation conceptus development in cattle and sheep.