A novel approach to differentiate rat embryonic stem cells in vitro reveals a role for RNF12 in activation of X chromosome inactivation

textabstractX chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse

Evidence that the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in human serum may not be secreted by the pituitary gland

Recent studies have revealed that TRH-like immunoreactivity (TRH-LI) in human serum is predominantly pGlu-Glu-ProNH2 (< EEP-NH2), a peptide previously found in, among others tissues, the pituitary gland of various mammalian species. In the rat pituitary, < EEP-NH2 is present in gonadotrophs and its pituitary content is regulated by gonadal steroids and gonadotrophin-releasing hormone (GnRH). Hence, we reasoned that < EEP-NH2 in human serum may also arise, at least in part, from the pituitary, and that its secretion may correlate with that of gonadotrophins. Therefore, blood was simultaneously sampled from both inferior petrosal sinuses, which are major sites of the venous drainage of the pituitary gland, and a peripheral vein from seven patients with suspected adrenocorticotrophin-secreting pituitary tumours. In addition, in six postmenopausal and six cyclic women, peripheral vein blood was collected at 10-min intervals for 6 h, then a standard 100 micrograms GnRH test was performed. In the sera, TRH-LI was estimated by RIA with antiserum 4319, which binds most tripeptides that share the N- and C-terminal amino acids with TRH (pGlu-His-ProNH2). In addition, LH and FSH were measured in these sera b

Repair of exogenous DNA double-strand breaks promotes chromosome synapsis in SPO11-mutant mouse meiocytes, and is altered in the absence of HORMAD1

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11YF/YF mice, 140–160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11YF/YF compared to Spo11+/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

TRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because serum was kept for at least 2 h at room temperature, which causes degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is not caused by these peptides. On high-performance liquid chromatography, serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced in, among others, the prostate. Urine of normals and control patients also contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with similar levels in males and females. TRH represented only 2% of urinary TRH-LI, and anion-exchange chromatography and high-performance liquid chromatography revealed that most TRH-LI in urine was < EEP-NH2. In patients with carcinoid tumors, increased urinary TRH-LI levels were noted (range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with urinary creatinine, and the urinary clearance rate of TRH-LI was similar to the glomerular filtration rate. In addition, serum TRH-LI was increased in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum < EEP-NH2 is cleared by glomerular filtration wit

Renal clearance of the thyrotropin-releasing hormone-like peptide pyroglutamyl-glutamyl-prolineamide in humans

TRH-like peptides have been identified that differ from TRH (pGlu-His-ProNH2) in the middle amino acid. We have estimated TRH-like immunoreactivity (TRH-LI) in human serum and urine by RIA with TRH-specific antiserum 8880 or with antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2. TRH was undetectable in serum (< 25 pg/mL), but TRH-LI was detected with antiserum 4319 in serum of 27 normal subjects, 21 control patients, and 12 patients with carcinoid tumors (range 17-45, 5-79, and 18-16,600 pg/mL, respectively). Because serum was kept for at least 2 h at room temperature, which causes degradation of TRH, pGlu-Phe-ProNH2, and pGlu-Tyr-ProNH2, serum TRH-LI is not caused by these peptides. On high-performance liquid chromatography, serum TRH-LI coeluted with pGlu-Glu-ProNH2 (< EEP-NH2), a peptide produced in, among others, the prostate. Urine of normals and control patients also contained TRH-LI (range 1.14-4.97 and 0.24-5.51 ng/mL, respectively), with similar levels in males and females. TRH represented only 2% of urinary TRH-LI, and anion-exchange chromatography and high-performance liquid chromatography revealed that most TRH-LI in urine was < EEP-NH2. In patients with carcinoid tumors, increased urinary TRH-LI levels were noted (range 1.35-962.4 ng/mL). Urinary TRH-LI correlated positively with urinary creatinine, and the urinary clearance rate of TRH-LI was similar to the glomerular filtration rate. In addition, serum TRH-LI was increased in 17 hemodialysis patients (43-373 pg/mL). This suggests that serum < EEP-NH2 is cleared by glomerular filtration with little tubular resorption. The possible role of the prostate as a source of urinary TRH-LI was evaluated in 11 men with prostate cancer, showing a 25% decrease in urinary TRH-LI excretion after prostatectomy (0.19 +/- 0.02 vs. 0.15 +/- 0.01 ng/mumol creatinine, mean +/- SEM). However, TRH-LI was similar in spontaneously voided urine and in urine obtained through a nephrostomy cannula from 16 patients with unilateral urinary tract obstruction (0.15 +/- 0.01 vs. 0.14 +/- 0.01 ng/mumol creatinine). These data indicate that: 1) TRH-LI in human serum represents largely < EEP-NH2, which is cleared by renal excretion; 2) part of urinary < EEP-NH2 is derived from prostatic secretion into the blood and not directly into urine; and 3) urinary < EEP-NH2 can be used as marker for carcinoid tumors

The ubiquitin-conjugating enzyme HR6B is required for maintenance of X chromosome silencing in mouse spermatocytes and spermatids

<p>Abstract</p> <p>Background</p> <p>The ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids.</p> <p>Results</p> <p>HR6B is enriched on the XY body and on centromeric regions in pachytene spermatocytes. Global gene expression analyses revealed that spermatid-specific single- and multicopy X-linked genes are prematurely expressed in <it>Hr6b </it>knockout spermatocytes. Very few other differences in gene expression were observed in these cells, except for upregulation of major satellite repeat transcription. In contrast, in <it>Hr6b </it>knockout spermatids, 7298 genes were differentially expressed; 65% of these genes was downregulated, but we observed a global upregulation of gene transcription from the X chromosome. In wild type spermatids, approximately 20% of the single-copy X-linked genes reach an average expression level that is similar to the average expression from autosomes.</p> <p>Conclusions</p> <p>Spermatids maintain an enrichment of repressive chromatin marks on the X chromosome, originating from meiotic prophase, but this does not interfere with transcription of the single-copy X-linked genes that are reactivated or specifically activated in spermatids. HR6B represses major satellite repeat transcription in spermatocytes, and functions in the maintenance of X chromosome silencing in spermatocytes and spermatids. It is discussed that these functions involve modification of chromatin structure, possibly including H2B ubiquitylation.</p

Genomes of Ellobius species provide insight into the evolutionary dynamics of mammalian sex chromosomes

The X and Y sex chromosomes of placental mammals show hallmarks of a tumultuous evolutionary past. The X Chromosome has a rich and conserved gene content, while the Y Chromosome has lost most of its genes. In the Transcaucasian mole vole Ellobius lutescens, the Y Chromosome including Sry has been lost, and both females and males have a 17,X diploid karyotype. Similarly, the closely related Ellobius talpinus, has a 54,XX karyotype in both females and males. Here, we report the sequencing and assembly of the E. lutescens and E. talpinus genomes. The results indicate that the loss of the Y Chromosome in E. lutescens and E. talpinus occurred in two independent events. Four functional homologs of mouse Y-Chromosomal genes were detected in both female and male E. lutescens, of which three were also detected in the E. talpinus genome. One of these is Eif2s3y, known as the only Y-derived gene that is crucial for successful male meiosis. Female and male E. lutescens can carry one and the same X Chromosome with a largely conserved gene content, including all genes known to function in X Chromosome inactivation. The availability of the genomes of these mole vole species provides unique models to study the dynamics of sex chromosome evolution

Meiotic arrest occurs most frequently at metaphase and is often incomplete in azoospermic men

Objective: To establish which meiotic checkpoints are activated in males with severe spermatogenic impairment to improve phenotypic characterization of meiotic defects. Design: Retrospective observational study. Setting: University medical center research laboratory and andrology clinic. Patient(s): Forty-eight patients with confirmed spermatogenic impairment (Johnsen scores 3–6) and 15 controls (Johnsen score 10). Intervention(s): None. Main Outcome Measure(s): Quantitative assessment of immunofluorescent analyses of specific markers to determine meiotic entry, chromosome pairing, progression of DNA double-strand break repair, crossover formation, formation of meiotic metaphases, metaphase arrest, and spermatid formation, resulting in a novel classification of human meiotic arrest types. Result(s): Complete metaphase arrest was observed most frequently (27%), and the patients with the highest frequency of apoptotic metaphases also displayed a reduction in crossover number. Incomplete metaphase arrest was observed in 17% of the patients. Only four patients (8%) displayed a failure to complete meiotic chromosome pairin