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Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

Transgenic mouse models have contributed considerably to our understanding of the cellular and molecular mechanisms by which androgens control spermatogenesis. Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis. This conclusion is corroborated by similar knockouts in other potential testicular target cells. Mutations resulting in diminished expression of the AR or in alleles with increased length of the CAG repeat mimick specific human forms of disturbed fertility that are not accompanied by defects in male sexual development. Transcriptional profiling studies in mice with cell-selective and general knockouts of the AR, searching for androgen-regulated genes relevant to the control of spermatogenesis, have identified many candidate target genes. However, with the exception of Rhox5, the identified subsets of genes show little overlap. Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre/loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Müllerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte/spermatid development/survival critically depends on androgens.

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, and is characterized by hyperandrogenism, oligo/anovulation, and/or polycystic ovaries. Many women with PCOS also suffer from adverse metabolic phenotypes, including central adiposity, insulin resistance, and glucose intolerance, which can exacerbate reproductive dysfunction. Androgens can act upon androgen receptors (AR), which are expressed in many reproductive and metabolic tissues, and contribute to the pathogenesis of PCOS. AR are highly expressed in the neuroendocrine hypothalamus in areas which regulate the hypothalamic-pituitary-gonadal axis and contribute to the central regulation of metabolism. Many phenotypes of PCOS can be modelled in rodents by administration of the non-aromatizable androgen dihydrotestosterone (DHT) during critical periods of development. Neuronal AR is key in the development of PCOS, as female mice with neuronal AR deletion who are exposed to androgen excess are protected against development of anovulation, polycystic ovaries, and metabolic abnormalities. Yet it is not known which populations of neurons confers this protection. We hypothesize that leptin-receptor (LepR) neurons participate in the pathogenesis of PCOS, as sub-populations of LepR neurons co-express AR in the hypothalamus, and LepR neurons are critical in the central regulation of energy homeostasis, and exert permissive actions on puberty and fertility. We have pre-natally androgenized (PNA) a mouse model of AR deletion specifically in LepR cells (LepRΔAR) and are conducting reproductive and metabolic phenotyping. As previously demonstrated, control PNA females show long periods of acyclicity, whereas LepRΔAR PNA female mice show a similar number of days in each stage of the estrous cycle, number of cycles, and cycle length as vehicle treated LepRΔAR females. Our findings indicate that a subpopulation of AR/LepR cells mediate the effects of prenatal androgen excess on female estrous cycles in a mouse model of PCOS-like phenotype.

The International Network for Young Researchers in Male Fertility （INYRMF; http：//www.youngresearcher.eu） is committed to establish global networks among young （early stage） scientists in testicular research and to promote collaboration and exchange of methodologies and expertise. We provide a more informal way of commu- nication than many other congresses and meetings and promote online discussions on the homepage and during our annual meetings.

Defect-free epitaxial growth of 2D materials is one of the holy grails for a successful integration of van der Waals (vdW) materials in the semiconductor industry. The large-area (quasi-)vdW epitaxy of layered 2D chalcogenides is consequently carefully being researched since these materials hold very promising properties for future nanoelectronic applications. The formation of defects such as stacking faults like 60o twins and consequently 60o grain boundaries is still of major concern for the defect-free epitaxial growth of 2D chalcogenides. Although growth strategies to overcome the occurrence of these defects are currently being considered, more fundamental understanding on the origin of these defects at the initial stages of the growth is highly essential. Therefore this work focuses on the understanding of 60o twin formation in (quasi-)vdW epitaxy of 2D chalcogenides relying on systematic molecular beam epitaxy (MBE) experiments supported by density functional theory (DFT) calculations. The MBE experiments reveal the striking difference in 60o twin formation between WSe2 and Bi2Se3 in both quasi-vdW heteroepitaxy and vdW homoepitaxy, which from our DFT calculations links to the difference in interlayer vdW coupling strength. The stronger interlayer vdW coupling in Bi2Se3 compared to WSe2 results in a striking enhanced control on twin formation and hence shows significantly more promise for defect-free epitaxial integration. This interesting aspect of (quasi-)vdW epitaxy reveals that the strength of interlayer vdW coupling is key for functional 2D materials and opens perspectives for other vdW materials sharing strong interlayer interactions.