Meiotic Arrest Research Articles

A novel TEX11 mutation induces azoospermia: a case report of infertile brothers and literature review

BackgroundTestis-expressed gene 11 (TEX11) is an X-linked gene and essential for meiotic recombination and chromosomal synapsis. TEX11 deficiency causes meiotic arrest and male infertility, and many TEX11 mutations have been found in azoospermic and infertile men.Case presentationThis study reported one novel TEX11 mutation (2653G → T, in exon 29, GenBank accession number, NM_031276) in two brothers with azoospermia. This mutation was firstly screened out by whole-exome sequencing (WES) and further verified by amplifying and sequencing the specific exon 29. Surprisingly, the same exonic missense mutation (W856C) was observed in two brothers but not in their mother. Histological analysis of testicular biopsy from both brothers revealed meiotic arrest and no post-meiotic round spermatids and mature spermatozoa were observed in the seminiferous tubules. TEX11 expression was observed strongly in spermatogonia and weakly in spermatocytes, but not in Sertoli cells and interstitial cells.ConclusionsWe identified one novel TEX11 mutation in two brothers and summarized the literature regarding TEX11 mutations and male infertility. This study and previous literature indicate that TEX11 mutations are closely associated with male infertility, especially azoospermia, although auxiliary clinical analyses are needed to figure out the causes of male infertility.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption

BackgroundStudies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. ObjectiveWe aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. Experimentaldesign: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. ResultsqRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). ConclusionN-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes.

High level of C-type natriuretic peptide induced by hyperandrogen-mediated anovulation in polycystic ovary syndrome mice.

Polycystic ovary syndrome (PCOS), which is characterized by hyperandrogenism, is a complex endocrinopathy that affects the fertility of 9-18% of reproductive-aged women. However, the exact mechanism of PCOS, especially hyperandrogen-induced anovulation, is largely unknown to date. Physiologically, the natriuretic peptide type C/natriuretic peptide receptor 2 (CNP/NPR2) system is essential for sustaining oocyte meiotic arrest until the preovulatory luteinizing hormone (LH) surge. We therefore hypothesized that the CNP/NPR2 system is also involved in PCOS and contributes to arresting oocyte meiosis and ovulation. Here, based on a dehydroepiandrosterone (DHEA)-induced PCOS-like mouse model, persistent high levels of CNP/NPR2 were detected in anovulation ovaries. Meanwhile, oocytes arrested at thegerminal vesicle stage correlated with persistent high levels of androgen and estrogen. We further showed that ovulation failure in these mice could be a result of elevated Nppc/Npr2 gene transcription that was directly increased by androgen (AR) and estrogen (ER) receptor signaling. Consistent with this, anovulation was alleviated by administration of either exogenous human chorionic gonadotropin (hCG) or inhibitors of AR or ER to reduce the level of CNP/NPR2. Additionally, the CNP/NPR2 expression pattern in the anovulated follicles was, to some extent, consistent with the clinical expression in PCOS patients. Therefore, our study highlights the important role an overactive CNP/NPR2 system caused by hyperandrogenism in preventing oocytes from maturation and ovulation in PCOS mice. Our findings provide insight into potential mechanisms responsible for infertility in women with PCOS.

Genome-wide analysis of gene regulation mechanisms during Drosophila spermatogenesis

BackgroundDuring Drosophila spermatogenesis, testis-specific meiotic arrest complex (tMAC) and testis-specific TBP-associated factors (tTAF) contribute to activation of hundreds of genes required for meiosis and spermiogenesis. Intriguingly, tMAC is paralogous to the broadly expressed complex Myb-MuvB (MMB)/dREAM and Mip40 protein is shared by both complexes. tMAC acts as a gene activator in spermatocytes, while MMB/dREAM was shown to repress gene activity in many cell types.ResultsOur study addresses the intricate interplay between tMAC, tTAF, and MMB/dREAM during spermatogenesis. We used cell type-specific DamID to build the DNA-binding profiles of Cookie monster (tMAC), Cannonball (tTAF), and Mip40 (MMB/dREAM and tMAC) proteins in male germline cells. Incorporating the whole transcriptome analysis, we characterized the regulatory effects of these proteins and identified their gene targets. This analysis revealed that tTAFs complex is involved in activation of achi, vis, and topi meiosis arrest genes, implying that tTAFs may indirectly contribute to the regulation of Achi, Vis, and Topi targets. To understand the relationship between tMAC and MMB/dREAM, we performed Mip40 DamID in tTAF- and tMAC-deficient mutants demonstrating meiosis arrest phenotype. DamID profiles of Mip40 were highly dynamic across the stages of spermatogenesis and demonstrated a strong dependence on tMAC in spermatocytes. Integrative analysis of our data indicated that MMB/dREAM represses genes that are not expressed in spermatogenesis, whereas tMAC recruits Mip40 for subsequent gene activation in spermatocytes.ConclusionsDiscovered interdependencies allow to formulate a renewed model for tMAC and tTAFs action in Drosophila spermatogenesis demonstrating how tissue-specific genes are regulated.

Homozygous Mutations in WEE2 Cause Fertilization Failure and Female Infertility

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted invivo. In clinical practice, some infertility case subjects suffer from recurrent failure of invitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein invitro and in affected individuals' oocytes invivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 invitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts invitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases.

Role of natriuretic peptide receptor 2-mediated signaling in meiotic arrest of zebrafish oocytes and its estrogen regulation through G protein-coupled estrogen receptor (Gper)

Natriuretic peptide type C (NPPC) and its receptor, natriuretic peptide receptor 2 (NPR2), have essential roles in maintaining meiotic arrest of oocytes in several mammalian species. However, it is not known if a similar mechanism exists in non-mammalian vertebrates. Using zebrafish as a model, we show that Nppc is expressed in ovarian follicle cells, whereas Npr2 is mainly detected in oocytes. Treatment of intact and defolliculated oocytes with 100 nM NPPC for 6 h caused a large increase in cGMP concentrations, and a significant decrease in oocyte maturation (OM), an effect that was mimicked by treatment with 8-Br-cGMP. Treatment with E2 and G-1, the specific GPER agonist, also increased cGMP levels. Cyclic AMP levels were also increased by treatments with 8-Br-cGMP, E2 and G1. The estrogen upregulation of cAMP levels was blocked by co-treatment with AG1478, an inhibitor of EGFR activation. Gene expression of npr2, but not nppc, was significantly upregulated in intact oocytes by 6 h treatments with 20 nM E2 and G-1. Both cilostamide, a phosphodiesterase 3 (PDE3) inhibitor, and rolipram, a PDE4 inhibitor, significantly decreased OM of intact and defolliculated oocytes, and enhanced the inhibitory effects of E2 and G-1 on OM. These findings indicate the presence of a Nppc/Npr2/cGMP pathway maintaining meiotic arrest in zebrafish oocytes that is upregulated by estrogen activation of Gper. Collectively, the results suggest that Nppc through Npr2 cooperates with E2 through Gper in upregulation of cGMP levels to inhibit phosphodiesterase activity resulting in maintenance of oocyte meiotic arrest in zebrafish.

Modulation of Prdm9-controlled meiotic chromosome asynapsis overrides hybrid sterility in mice.

Hybrid sterility is one of the reproductive isolation mechanisms leading to speciation. Prdm9, the only known vertebrate hybrid-sterility gene, causes failure of meiotic chromosome synapsis and infertility in male hybrids that are the offspring of two mouse subspecies. Within species, Prdm9 determines the sites of programmed DNA double-strand breaks (DSBs) and meiotic recombination hotspots. To investigate the relation between Prdm9-controlled meiotic arrest and asynapsis, we inserted random stretches of consubspecific homology on several autosomal pairs in sterile hybrids, and analyzed their ability to form synaptonemal complexes and to rescue male fertility. Twenty-seven or more megabases of consubspecific (belonging to the same subspecies) homology fully restored synapsis in a given autosomal pair, and we predicted that two or more DSBs within symmetric hotspots per chromosome are necessary for successful meiosis. We hypothesize that impaired recombination between evolutionarily diverged chromosomes could function as one of the mechanisms of hybrid sterility occurring in various sexually reproducing species.

Genetic zygosity of mature ovarian teratomas, struma ovarii, and ovarian carcinoids.

Although ovarian monodermal teratomas, including struma ovarii and carcinoids, are closely associated with mature teratomas, their genetic basis is poorly understood. A series of mature and monodermal ovarian teratomas were analyzed by short tandem repeat genotyping to evaluate their genetic zygosity and its associations. Informative DNA genotyping data were obtained for ten mature teratomas, six struma ovarii, and three carcinoids (one insular, one trabecular, and one mucinous). A homozygous genotype was present in five of the ten (50%) mature teratomas, three of the six (50%) struma ovarii, and one of the three (33%) ovarian carcinoids. There was no significant difference in genetic zygosity between mature and monodermal teratomas. Patients' age was not correlated with the genetic zygosity: the youngest age in the homozygous tumor group of patients was 4years. It is suggested that an oocyte after meiosis I, which has escaped from meiotic arrest, is a significant cause of these tumors. Although one mature teratoma was a rare case with lactating adenoma-like breast tissue, its genetic zygosity was concordant with that of the surrounding teratomatous tissue. In one ovarian carcinoid, the carcinoid and accompanying teratomatous components showed matching zygosity at all but one locus: the carcinoid was heterozygous but teratoma was homozygous at one pericentromeric locus. This suggests that not all carcinoids are secondary neoplasms arising from a fully developed mature teratoma: some are neoplasms deviating from a developing mature teratoma.

Effects of pre-incubation with C-type natriuretic peptide on nuclear maturation, mitochondrial behavior, and developmental competence of sheep oocytes

In vitro produced mammalian embryos suffer from developmental failure, with a large proportion showing embryonic retardation, degradation, or apoptosis. This failure is due, in part, to incomplete oocyte cytoplasmic maturation. C-type natriuretic peptide (CNP) has been reported to act as a meiotic inhibitor. Here we explored the potential effects of CNP pre-treatment sheep oocytes on nuclear maturation, changes in mitochondrial behavior and developmental competence of in vitro fertilized embryos. Sheep cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived ovaries. Nuclear progression was assessed using DAPI chromatin staining, the expression of natriuretic peptide receptor 2 (NPR2) was evaluated by RT-qPCR, active mitochondria localization was assessed with a confocal laser scanning microscopy using MitoTracker Red, and the developmental competence of sheep oocytes subjected to one-step IVM or two-step IVM with or without CNP pretreatment was also investigated. Our results showed that 200 nM CNP could effectively maintain meiotic arrest of sheep COCs in vitro within 4 h. Furthermore, NPR2 mRNA was mainly expressed in cumulus cells. COCs pre-treated with 200 nM CNP for 4 h followed by 24 h IVM showed significantly higher (P < 0.05) cleavage rate and blastocyst rate after in vitro fertilization (IVF), and significantly lower (P < 0.05) proportion of DNA-fragmented nuclei in blastocysts when compared to the conventional 24 h IVM (standard IVM). Non-matured oocytes mainly displayed brilliant circumferential and fine diffuse distribution of mitochondria throughout the cytoplasm. By comparison, 200 nM CNP pre-treated COCs for 4 h led to cytoplasmic mitochondrial granule localization to the peripheral and perinuclear regions. Moreover, oocytes pre-treated with 200 nM CNP for 4 h followed by 24 h IVM, showed mitochondrial organization were similar to those of conventional 24 h matured oocytes in which mitochondria were aggregated more toward the cortical regions of the oocytes, but with larger clumps of stained mitochondria. These results indicate that CNP pre-treatment improves the quality and developmental competence of sheep oocytes and has great potential for facilitating in vitro embryo production.

Oocyte maturation in the toad Rhinella arenarum (Amphibia, Anura): Evidence of cAMP involvement in steroid production and action.

In this work, we describe the participation of the adenylate cyclase/3'-5'-cyclic adenonsine monophosphate (cAMP) pathway in the seasonal follicular secretion of progesterone (P4 ) and testosterone (T), and its relationship with the maturation of Rhinella arenarum oocytes. Under gonadotropin stimulation, P4 secretion was the dominant steroid produced during the reproductive period, resulting in 100% germinal vesicle breakdown (GVBD) in oocytes in vitro; in contrast, T and estradiol (E2 ) secretion increased (∼16 nM/20 follicles and ∼80 pM/20 follicles, respectively) during the non-reproductive period, but only yielded 50% GVBD. Treatment of the follicles with dibutyryl-cAMP or forskolin induced a significant increase in T secretion during both periods, but P4 secretion did not significantly change and GVBD did not occur. These results suggest that high cAMP levels in the oocyte maintain meiotic arrest and prevent the induction effect of follicular steroids. An increase in cAMP levels in denuded oocytes, however, negatively regulated T-induced maturation since treatment with increasing db-cAMP or forskolin inhibited their maturation. Therefore, we hypothesize that an elevation in T during the non-reproductive period favors its aromatization to E2 , leading to follicle growth. During the reproductive period, P4 production might promote oocyte maturation when environmental conditions are favorable for reproduction. Together, the results indicate that steroidogenesis is seasonal and depends on gonadotropic activity in R. arenarum.

The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis.

Esco1 has been reported to function as a cohesion establishment factor that mediates chromosome cohesion and segregation in mitotic cells. However, its exact roles in meiosis have not been clearly defined. Here, we document that Esco1 is expressed and localized to both the nucleus and cytoplasm during mouse oocyte meiotic maturation. Depletion of Esco1 by siRNA microinjection causes the meiotic progression arrest with a severe spindle abnormality and chromosome misalignment, which is coupled with a higher incidence of the erroneous kinetochore–microtubule attachments and activation of spindle assembly checkpoint. In addition, depletion of Esco1 leads to the impaired microtubule stability shown by the weakened resistance ability to the microtubule depolymerizing drug nocodazole and the decreased level of acetylated α-tubulin. Conversely, overexpression of Esco1 causes hyperacetylation of α-tubulin and spindle defects. Moreover, we find that Esco1 binds to α-tubulin and is required for its acetylation. The reduced acetylation level of α-tubulin in Esco1-depleted oocytes can be restored by the ectopic expression of exogenous wild-type Esco1 but not enzymatically dead Esco1-G768D. Purified wild-type Esco1 instead of mutant Esco1-G768D acetylates the synthesized peptide of α-tubulin in vitro. Collectively, our data assign a novel function to Esco1 as a microtubule regulator during oocyte meiotic maturation beyond its conventional role in chromosome cohesion.

Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis

Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling.

DMC1 mutation that causes human non-obstructive azoospermia and premature ovarian insufficiency identified by whole-exome sequencing

BackgroundThe genetic causes of the majority of male and female infertility caused by human non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) with meiotic arrest are unknown.ObjectiveTo identify the genetic...

MORC2B is essential for meiotic progression and fertility.

The microrchidia (MORC) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing. Here, we report that mouse Morc2b encodes a functional germ cell-specific member of the MORC protein family. Morc2b arose specifically in the rodent lineage through retrotransposition of Morc2a during evolution. Inactivation of Morc2b leads to meiotic arrest and sterility in both sexes. Morc2b-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis. Loss of MORC2B causes mis-regulated expression of meiosis-specific genes. Furthermore, we find that MORC2B interacts with MORC2A, its sequence paralogue. Our results demonstrate that Morc2b, a relatively recent gene, has evolved an essential role in meiosis and fertility.

Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis

Telomeres integrity is indispensable for chromosomal stability by preventing chromosome erosion and end-to-end fusions. During meiosis, telomeres attach to the inner nuclear envelope and cluster into a highly crowded microenvironment at the bouquet stage, which requires specific mechanisms to protect the telomeres from fusion. Here, we demonstrate that germ cell-specific knockout of a shelterin complex subunit, Trf1, results in arrest of spermatocytes at two different stages. The obliterated telomere-nuclear envelope attachment in Trf1-deficient spermatocytes impairs homologue synapsis and recombination, resulting in a pachytene-like arrest, while the meiotic division arrest might stem from chromosome end-to-end fusion due to the failure of recruiting meiosis specific telomere associated proteins. Further investigations uncovered that TRF1 could directly interact with Speedy A, and Speedy A might work as a scaffold protein to further recruit Cdk2, thus protecting telomeres from fusion at this stage. Together, our results reveal a novel mechanism of TRF1, Speedy A, and Cdk2 in protecting telomere from fusion in a highly crowded microenvironment during meiosis.

Dynamics, ultrastructure and gene expression of human in vitro organized testis cells from testicular sperm extraction biopsies.

Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3-4 weeks. Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2-3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1-3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68+/CD163-/IL6+ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. N/A. Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.

Historical and Clinical Perspectives on Chromosomal Translocations.

Chromosomal translocations, rearrangements involving the exchange of segments between chromosomes, were documented in humans in 1959. The first accurately reported clinical phenotype resulting from a translocation was that of Down syndrome. In a small percentage of Down syndrome cases, an extra 21q is provided by a Robertsonian translocation chromosome, either occurring de novo or inherited from a phenotypically normal parent with the translocation chromosome and a balanced genome of 45 chromosomes. Balanced translocations, including both Robertsonian and reciprocal translocations, are typically benign, but meiosis in germ cells with balanced translocations may result in meiotic arrest and subsequent infertility, or in unbalanced gametes, with attendant risks of miscarriage and unbalanced progeny. Most reciprocal translocations are unique. A few to several percent of translocations disrupt haploinsufficient genes or their regulatory regions and result in clinical phenotypes. Balanced translocations from patients with clinical phenotypes have been valuable in mapping disease genes and in illuminating cis-regulatory regions. Mapping of discordant mate pairs from long-insert, low-pass genome sequencing now permits efficient and cost-effective discovery and nucleotide-level resolution of rearrangement breakpoints, information that is absolutely necessary for interpreting the etiology of clinical phenotypes in patients with rearrangements. Pathogenic translocations and other balanced chromosomal rearrangements constitute a class of typically highly penetrant mutation that is cryptic to both clinical microarray and exome sequencing. A significant proportion of rearrangements include additional complexity that is not visible by conventional karyotype analysis. Some proportion of patients with negative findings on exome/genome sequencing and clinical microarray will be found to have etiologic balanced rearrangements only discoverable by genome sequencing with analysis pipelines optimized to recover rearrangement breakpoints.

Distinct prophase arrest mechanisms in human male meiosis.

ABSTRACTTo prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX. Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

Heat stress impairs gap junction communication and cumulus function of bovine oocytes.

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5’ adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.

The switch from cAMP-independent to cAMP-dependent arrest of meiotic prophase is associated with coordinated GPR3 and CDK1 expression in mouse oocytes

Mammalian oocytes are arrested in meiotic prophase from around the time of birth until just before ovulation. Following an extended period of growth, they are stimulated to mature to the metaphase II stage by a preovulatory luteinizing hormone (LH) surge that occurs with each reproductive cycle. Small, growing oocytes are not competent to mature into fertilizable eggs because they do not possess adequate amounts of cell cycle regulatory proteins, particularly cyclin-dependent kinase 1 (CDK1). As oocytes grow, they synthesize CDK1 and acquire the ability to mature. After oocytes achieve meiotic competence, meiotic arrest at the prophase stage is dependent on high levels of cAMP that are generated in the oocyte under the control of the constitutively active Gs-coupled receptor, GPR3. In this study, we examined the switch between GPR3-independent and GPR3-dependent meiotic arrest. We found that the ability of oocytes to mature, as well as oocyte CDK1 levels, were dependent on follicle size, but CDK1 expression in oocytes from preantral follicles was not acutely altered by the activity of follicle stimulating hormone (FSH). Gpr3 was expressed and active in incompetent oocytes within early stage follicles, well before cAMP is required to maintain meiotic arrest. Oocytes from Gpr3-/- mice were less competent to mature than oocytes from Gpr3+/+ mice, as assessed by the time course of germinal vesicle breakdown. Correspondingly, Gpr3-/- oocytes contained significantly lower CDK1 levels than their Gpr3+/+ counterparts that were at the same stage of follicle development. These results demonstrate that GPR3 potentiates meiotic competence, most likely by raising cAMP.