Gonadotrophin-mediated miRNA expression in testis at onset of puberty in rhesus monkey: predictions on regulation of thyroid hormone activity and DLK1-DIO3 locus.

Molecular mechanisms responsible for the initiation of primate spermatogenesis remain poorly characterized. Previously, 48 h stimulation of the testes of three juvenile rhesus monkeys with pulsatile LH and FSH resulted in down-regulation of a cohort of genes recognized to favor spermatogonia stem cell renewal. This change in genetic landscape occurred in concert with amplification of Sertoli cell proliferation and the commitment of undifferentiated spermatogonia to differentiate. In this report, the non-protein coding small RNA transcriptomes of the same testes were characterized using RNA sequencing: 537 mature micro-RNAs (miRNAs), 322 small nucleolar RNAs (snoRNAs) and 49 small nuclear RNAs (snRNAs) were identified. Pathway analysis of the 20 most highly expressed miRNAs suggested that these transcripts contribute to limiting the proliferation of the primate Sertoli cell during juvenile development. Gonadotrophin treatment resulted in differential expression of 35 miRNAs, 12 snoRNAs and four snRNA transcripts. Ten differentially expressed miRNAs were derived from the imprinted delta-like homolog 1-iodothyronine deiodinase 3 (DLK1-DIO3) locus that is linked to stem cell fate decisions. Four gonadotrophin-regulated expressed miRNAs were predicted to trigger a local increase in thyroid hormone activity within the juvenile testis. The latter finding leads us to predict that, in primates, a gonadotrophin-induced selective increase in testicular thyroid hormone activity, together with the established increase in androgen levels, at the onset of puberty is necessary for the normal timing of Sertoli cell maturation, and therefore initiation of spermatogenesis. Further examination of this hypothesis requires that peripubertal changes in thyroid hormone activity of the testis of a representative higher primate be determined empirically.

The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

STUDY QUESTION: What is the genetic landscape within the testis of the juvenile rhesus monkey (Macaca mulatta) that underlies the decision of undifferentiated spermatogonia to commit to a pathway of differentiation when puberty is induced prematurely by exogenous LH and FSH stimulation? SUMMARY ANSWER: Forty-eight hours of gonadotrophin stimulation of the juvenile monkey testis resulted in the appearance of differentiating B spermatogonia and the emergence of 1362 up-regulated and 225 down-regulated testicular mRNAs encoding a complex network of proteins ranging from enzymes regulating Leydig cell steroidogenesis to membrane receptors, and from juxtacrine and paracrine factors to transcriptional factors governing spermatogonial stem cell fate. WHAT IS KNOWN ALREADY: Our understanding of the cell and molecular biology underlying the fate of undifferentiated spermatogonia is based largely on studies of rodents, particularly of mice, but in the case of primates very little is known. The present study represents the first attempt to comprehensively address this question in a highly evolved primate. STUDY DESIGN, SIZE, DURATION: Global gene expression in the testis from juvenile rhesus monkeys that had been stimulated with recombinant monkey LH and FSH for 48 h (N = 3) or 96 h (N = 4) was compared to that from vehicle treated animals (N = 3). Testicular cell types and testosterone secretion were also monitored. PARTICIPANTS/MATERIALS, SETTING, METHODS: Precocious testicular puberty was initiated in juvenile rhesus monkeys, 14-24 months of age, using a physiologic mode of intermittent stimulation with i.v. recombinant monkey LH and FSH that within 48 h produced 'adult' levels of circulating LH, FSH and testosterone. Mitotic activity was monitored by immunohistochemical assays of 5-bromo-2'-deoxyuridine and 5-ethynyl-2'-deoxyuridine incorporation. Animals were bilaterally castrated and RNA was extracted from the right testis. Global gene expression was determined using RNA-Seq. Differentially expressed genes (DEGs) were identified and evaluated by pathway analysis. mRNAs of particular interest were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. MAIN RESULTS AND THE ROLE OF CHANCE: Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. LARGE SCALE DATA: The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). LIMITATIONS REASONS FOR CAUTION: The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. WIDER IMPLICATIONS OF THE FINDINGS: The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These unexpected results underline the importance of non-human primate models in translating data derived from animal research to the human situation. STUDY FUNDING/COMPETING INTEREST(S): The work described was funded by NIH grant R01 HD072189 to T.M.P. P.A. was supported by an Endocrine Society Summer Research Fellowship Award and CONICET (Argentine Research Council), S.N. by a grant from Vali-e-Asr Reproductive Health Research Center of Tehran University of Medical Sciences (grant #24335-39-92) to Dr Batool Hosseini Rashidi, and M.P.H. by grants from the National Health and Medical Research Council of Australia, and the Victorian State Government's Operational Infrastructure Support Program. The authors have nothing to disclose.

Clinical characterization of long-term multiple sclerosis (COLuMbus) patients in Argentina: A cross-sectional non-interventional study.

BACKGROUND: Most Multiple Sclerosis (MS) clinical trials fail to assess the long-term effects of disease-modifying therapies (DMT) or disability. METHODS: COLuMbus was a single-visit, cross-sectional study in Argentina in adult patients with ≥10 years of MS since first diagnosis. The primary endpoint was to determine patient disability using the Expanded Disability Status Scale (EDSS). The secondary endpoints were to evaluate the distribution of diagnoses between relapsing-remitting MS (RRMS) and secondary progressive MS (SPMS), patient demographics, disease history, and the risk of disability progression. The relationship between baseline characteristics and the current disability state and the risk of disability progression was assessed. RESULTS: Out of the 210 patients included, 76.7 % had a diagnosis of RRMS and 23.3 % had been diagnosed with SPMS, with a mean disease duration of 17.9 years and 20.5 years, respectively. The mean delay in the initial MS diagnosis was 2.6 years for the RRMS subgroup and 2.8 years for the SPMS subgroups. At the time of cut-off (28May2020), 90.1 % (RRMS) and 75.5 % (SPMS) of patients were receiving a DMT, with a mean of 1.5 and 2.0 prior DMTs, respectively. The median EDSS scores were 2.5 (RRMS) and 6.5 (SPMS). In the RRMS and SPMS subgroups, 23 % and 95.9 % of patients were at high risk of disability, respectively; the time since first diagnosis showed a significant correlation with the degree of disability. CONCLUSIONS: This is the first local real-world study in patients with long-term MS that highlights the importance of recognizing early disease progression to treat the disease on time and delay disability.

DNA methylation is not involved in specific down-regulation of HSD3B2, NR4A1 and RARB genes in androgen-secreting cells of human adrenal cortex.

We hypothesized that DNA methylation is involved in human adrenal functional zonation. mRNAs expression and methylation pattern of RARB, NR4A1 and HSD3B2 genes in human adrenal tissues (HAT) and in pediatric virilizing adrenocortical tumors (VAT) were analyzed. For analysis of the results samples were divided into 3 age groups according to FeZ involution, pre and post-adrenarche ages. In all HAT, similar RARB mRNA was found including microdissected zona reticularis (ZR) and zona fasciculata, but HSD3B2 and NR4A1 mRNAs were lower in ZR (p < 0.05). NR4A1 and RARB promoters remained unmethylated in HAT and VAT. No adrenal zone-specific differences in NR4A1 methylation were observed. In summary, RARB was not associated with ZR-specific downregulation of HSD3B2 in postnatal human adrenocotical zonation. DNA methylation would not be involved in NR4A1 adrenocortical cell-type specific downregulation. Lack of CpG islands in HSD3B2 suggested that HSD3B2 ZR-specific downregulation would not be directly mediated by DNA methylation.

Androgen Insensitivity Syndrome at Prepuberty: Marked Loss of Spermatogonial Cells at Early Childhood and Presence of Gonocytes up to Puberty.

Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival.