Allogeneic offspring produced by induction of PD-L1 in spermatogonial stem cells via self-renewal stimulation

The testis is an immune-privileged organ. It is considered that the testis somatic microenvironment is responsible for immune suppression. However, immunological properties of spermatogonial stem cells (SSCs) have remained unknown. Here, we report the birth of allogeneic offspring by enhanced expression of immunosuppressive PD-L1 in SSCs. In vitro supplementation of GDNF and FGF2 increased expression of PD-L1 in SSCs. Cultured SSCs maintained allogeneic spermatogenesis that persisted for >1 year. However, depletion or gene editing of Pd-l1 family genes in SSCs prevented allogeneic spermatogenesis, which suggested that germ cells are responsible for suppression of the allogeneic response. PD-L1 was induced by activation of the MAPK14-BCL6B pathway, which drives self-renewal by reactive oxygen species (ROS) generation. By contrast, reduced ROS or Mapk14 deficiency downregulated PD-L1. Allogeneic offspring were born after SSC transplantation into congenitally infertile and chemically castrated mice. Thus, SSCs have unique immunological properties, which make allogeneic recipients into "surrogate fathers.

Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids

Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 10⁴ donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility

Induction of DNA Methylation by Artificial piRNA Production in Male Germ Cells

SummaryGlobal DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1–3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4–6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7–10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11–16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells

Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA (dsRNA). Among the three types of mammalian ADARs, ADAR1 has long been recognized as an essential enzyme for normal development. The interferon-inducible ADAR1p150 is involved in immune responses to both exogenous and endogenous triggers, whereas the functions of the constitutively expressed ADAR1p110 are variable. Recent findings that ADAR1 is involved in the recognition of self versus non-self dsRNA provide potential explanations for its links to hematopoiesis, type I interferonopathies, and viral infections. Editing in both coding and noncoding sequences results in diseases ranging from cancers to neurological abnormalities. Furthermore, editing of noncoding sequences, like microRNAs, can regulate protein expression, while editing of Alu sequences can affect translational efficiency and editing of proximal sequences. Novel identifications of long noncoding RNA and retrotransposons as editing targets further expand the effects of A-to-I editing. Besides editing, ADAR1 also interacts with other dsRNA-binding proteins in editing-independent manners. Elucidating the disease-specific patterns of editing and/or ADAR1 expression may be useful in making diagnoses and prognoses. In this review, we relate the mechanisms of ADAR1′s actions to its pathological implications, and suggest possible mechanisms for the unexplained associations between ADAR1 and human diseases

Comprehensive DNA Methylation Analysis of Retrotransposons in Male Germ Cells

De novo DNA methylation of retrotransposons is critical for silencing. Here, we use DNA methylation analysis to examine retrotransposons in mouse male germ cells. DNA methylation of long interspersed nuclear elements (LINEs) is dependent on piRNA, and younger LINEs exhibit greater piRNA dependence. In contrast, most long terminal repeat (LTR) retrotransposons produce lower levels of piRNAs and do not show significant piRNA dependence. The relationship between DNA methylation and corresponding piRNA expression of several LTR retrotransposons was reduced in Mili-null cells, but not Miwi2-null cells. These observations raise the possibility of piRNA-dependent DNA methylation without Miwi2. Therefore, it appears that the molecular mechanisms of the gene silencing of retrotransposons are more complicated than previously thought