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

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

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

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

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

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

Silencing markers are retained on pericentric heterochromatin during murine primordial germ cell development

Background: In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins l

Human RAD18 Interacts with Ubiquitylated Chromatin Components and Facilitates RAD9 Recruitment to DNA Double Strand Breaks

RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2

SPO11-Independent DNA Repair Foci and Their Role in Meiotic Silencing

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11YF/YF), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11YF/YFand Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number

Depletion of the Colonic Epithelial Precursor Cell Compartment Upon Conditional Activation of the Hedgehog Pathway

Background & Aims: The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach. Methods: We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction. Results: Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Writ signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium. Conclusions: Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mous