Extrathymic AIRE-Expressing Cells: A Historical Perspective.

Since its discovery, Aire has been the topic of numerous studies in its role as a transcriptional regulator in the thymus where it promotes the "promiscuous" expression of a large repertoire of tissue-restricted antigens (TRAs) that are normally expressed only in the immune periphery. This process occurs in specialized medullary thymic epithelial cells (mTECs) and mediates the elimination of self-reactive T cells or promotes their conversion to the Foxp3+ regulatory T cell lineage, both of which are required for the prevention of autoimmunity. In recent years, there has been increasing interest in the role of extrathymic Aire expression in peripheral organs. The focus has primarily been on the identification of the cellular source(s) and mechanism(s) by which extrathymic AIRE affects tolerance-related or other physiological processes. A cadre of OMICs tools including single cell RNA sequencing and novel transgenic models to trace Aire expression to perform lineage tracing experiments have shed light on a phenomenon that is more complex than previously thought. In this chapter, we provide a deeper analysis of how extrathymic Aire research has developed and progressed, how cellular sources were identified, and how the function of AIRE was determined. Current data suggests that extrathymic AIRE fulfills a function that differs from what has been observed in the thymus and strongly argues that its main purpose is to regulate transcriptional programs in a cell content-dependent manner. Surprisingly, there is data that also suggests a non-transcriptional role of extrathymic AIRE in the cytoplasm. We have arrived at a potential turning point that will take the field from the classical understanding of AIRE as a transcription factor in control of TRA expression to its role in immunological and non-immunological processes in the periphery.

Envisioning a role for nuclear actin in prophase I spermatocytes.

Actin is a multi-functional protein that is involved in numerous cellular processes including cytoskeleton regulation, cell migration, and cellular integrity. In these processes, actin's role in respect to its structure, complex mechanical, and protein-binding properties has been studied primarily in the cytoplasmic and cellular membrane compartments. However, its role in somatic cell nuclei has recently become evident where it participates in transcription, chromatin remodeling, and DNA damage repair. What remains enigmatic is the involvement of nuclear actin in physiological processes that lead to the generation of germ cells, in general, and primary spermatocytes, in particular. Here, we will discuss the possible role and nuclear localization of actin during meiotic prophase I and its interaction with chromatin remodeling complexes, the latter being essential for the control of pairing of homologous chromosomes, cross-over formation, and recombination. It is our hope that this perspective article will extend the scope of actin's nuclear function in germ cells undergoing meiotic division.

AIRE in Male Fertility: A New Hypothesis.

Male infertility affects approximately 14% of all European men, of which ~44% are characterized as idiopathic. There is an urgency to identify the factors that affect male fertility. One such factor, Autoimmune Regulator (AIRE), a protein found in the thymus, has been studied in the context of central tolerance functioning as a nuclear transcription modulator, responsible for the expression of tissue-restricted antigens in specialized thymic cells that prevent autoimmunity. While its expression in the testes remains enigmatic, we recently observed that sterility in mice correlates with the absence of Aire in the testes, regardless of the deficient expression in medullary thymic epithelial cells or cells of the hematopoietic system. By assessing the Aire transcript levels, we discovered that Sertoli cells are the exclusive source of Aire in the testes, where it most likely plays a non-immune role, suggesting an unknown mechanism by which testicular Aire regulates fertility. Here, we discuss these results in the context of previous reports which have suggested that infertility observed in Aire deficient mice is of an autoimmune aetiology. We present an alternative point of view for the role of Aire in testes in respect to fertility altering the perspective of how Aire's function in the testes is currently perceived.

Two complementary approaches for efficient isolation of Sertoli cells for transcriptomic analysis.

Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature "nurturing" state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.

Focal Adhesion Protein Vinculin Is Required for Proper Meiotic Progression during Mouse Spermatogenesis.

The focal adhesion protein Vinculin (VCL) is ascribed to various cytoplasmic functions; however, its nuclear role has so far been ambiguous. We observed that VCL localizes to the nuclei of mouse primary spermatocytes undergoing first meiotic division. Specifically, VCL localizes along the meiosis-specific structure synaptonemal complex (SC) during prophase I and the centromeric regions, where it remains until metaphase I. To study the role of VCL in meiotic division, we prepared a conditional knock-out mouse (VCLcKO). We found that the VCLcKO male mice were semi-fertile, with a decreased number of offspring compared to wild-type animals. This study of events in late prophase I indicated premature splitting of homologous chromosomes, accompanied by an untimely loss of SCP1. This caused erroneous kinetochore formation, followed by failure of the meiotic spindle assembly and metaphase I arrest. To assess the mechanism of VCL involvement in meiosis, we searched for its possible interacting partners. A mass spectrometry approach identified several putative interactors which belong to the ubiquitin-proteasome pathway (UPS). The depletion of VLC leads to the dysregulation of a key subunit of the proteasome complex in the meiotic nuclei and an altered nuclear SUMOylation level. Taken together, we show for the first time the presence of VCL in the nucleus of spermatocytes and its involvement in proper meiotic progress. It also suggests the direction for future studies regarding the role of VCL in spermatogenesis through regulation of UPS.

FBXO38 Ubiquitin Ligase Controls Sertoli Cell Maturation.

The ubiquitin ligase SCFFBXO38 controls centromeric chromatin by promoting the degradation of the ZXDB protein. To determine the importance of this pathway during development, Fbxo38-deficient mice were generated. The loss of FBXO38 resulted in growth retardation affecting several organs, including the male reproductive system. A detailed analysis of the mutant testes revealed pathological changes in the seminiferous tubules, accompanied by a significant decrease in sperm production and reduced fertility. In adult testes, FBXO38 was specifically expressed in Sertoli cells, a somatic population essential for spermatogenesis initiation and progression. Sertoli cells lacking FBXO38 exhibited stabilized ZXDB protein and upregulated centromeric chromatin. Furthermore, the gene expression profile revealed that the absence of FBXO38 led to a defect in Sertoli cell maturation, specifically characterized by dysregulation in genes controlling retinoic acid metabolism and intercellular communication. Consequently, we documented significant changes in their ability to initiate spermatogonial differentiation. In conclusion, we show that FBXO38 acts as a Sertoli cell maturation factor, affecting the Sertoli cell transcription program, centromere integrity, and, subsequently, the ability to control spermatogenesis.