Synaptonemal Complex Protein Research Articles

The role of tyrosine phosphatase Shp2 in spermatogonial differentiation and spermatocyte meiosis.

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11–13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.

A molecular model for self-assembly of the synaptonemal complex protein SYCE3

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates homologous chromosome synapsis during meiosis. This zipper-like structure assembles in a continuous manner between homologous chromosome axes, enforcing a 100-nm separation along their entire length and providing the necessary three-dimensional framework for cross-over formation. The mammalian SC comprises eight components—synaptonemal complex protein 1–3 (SYCP1–3), synaptonemal complex central element protein 1–3 (SYCE1–3), testis-expressed 12 (TEX12), and six6 opposite strand transcript 1 (SIX6OS1)—arranged in transverse and longitudinal structures. These largely α-helical, coiled-coil proteins undergo heterotypic interactions, coupled with recursive self-assembly of SYCP1, SYCE2–TEX12, and SYCP2–SYCP3, to achieve the vast supramolecular SC structure. Here, we report a novel self-assembly mechanism of the SC central element component SYCE3, identified through multi-angle light scattering and small-angle X-ray scattering (SAXS) experiments. These analyses revealed that SYCE3 adopts a dimeric four-helical bundle structure that acts as the building block for concentration-dependent self-assembly into a series of discrete higher-order oligomers. We observed that this is achieved through staggered lateral interactions between self-assembly surfaces of SYCE3 dimers and through end-on interactions that likely occur through intermolecular domain swapping between dimer folds. These mechanisms are combined to achieve potentially limitless SYCE3 assembly, particularly favoring formation of dodecamers of three laterally associated end-on tetramers. Our findings extend the family of self-assembling proteins within the SC and reveal additional means for structural stabilization of the SC central element.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

SCRE serves as a unique synaptonemal complex fastener and is essential for progression of meiosis prophase I in mice.

Meiosis is a specialized cell division for producing haploid gametes from diploid germ cells. During meiosis, synaptonemal complex (SC) mediates the alignment of homologs and plays essential roles in homologous recombination and therefore in promoting accurate chromosome segregation. In this study, we have identified a novel protein SCRE (synaptonemal complex reinforcing element) as a key molecule in maintaining the integrity of SC during meiosis prophase I in mice. Deletion of Scre (synaptonemal complex reinforcing element) caused germ cell death in both male and female mice, resulting in infertility. Our mechanistic studies showed that the synapses and SCs in Scre knockout mice were unstable due to the lack of the SC reinforcing function of SCRE, which is sparsely localized as discrete foci along the central elements in normal synaptic homologous chromosomes. The lack of Scre leads to meiosis collapse at the late zygotene stage. We further showed that SCRE interacts with synaptonemal complex protein 1 (SYCP1) and synaptonemal complex central element 3 (SYCE3). We conclude that the function of SCRE is to reinforce the integrity of the central elements, thereby stabilizing the SC and ensuring meiotic cell cycle progression. Our study identified SCRE as a novel SC fastener protein that is distinct from other known SC proteins.

Osteocacin favours the expression of synaptonemal complex protein 3 in azoospermic mouse model

Objective Infertility is the inability to conceive after regular unprotected sex for more than 1 year without the use of contraceptive.Azoospermia is defined as an absence of fertile sperm in seminal fluid. Men with azoospermia will guide the formulation of a therapeutic plan. Uncarboxylated form of the osteocalcin (OCN) modulates fertility. In this study, we investigated the role of osteocalcin on SYCP3 expression in azoospermic mouse model.Methods Male mice (NMRI) ranging in age from 4 to 6 weeks (25 ± 5 g) were randomly divided into five groups (in all groups n = 5), Control, Sham I, Sham II, azoospermic model and azoospermic experimental OCN (3 ng/g/day) treated groups. At the end of the treatment period, (15th week age) the mice were sacrificed, left testes removed, weighted and put in fixative for morphology and IHC technique.Results Testis weight was reduced in azoospermic and azoospermic OCN treated group compared with Control and Sham groups, but in azoospermic OCN treated group is more than azoospermic mice (P ≤ 0.05). Daily injections of OCN improved spermatogenesis and SYCP3 expression in azoospermic OCN treated mice but not in azoospermic model.Conclusion Our results suggests that the osteocalcin overexpressed SYCP3 expression and improved spermatogenesis.

Induction of goat bone marrow mesenchymal stem cells into putative male germ cells using mRNA for STRA8, BOULE and DAZL.

Bone mesenchymal stem cells (BMSCs) have the capacity to differentiate into germ cells (GCs). This study was conducted to develop a non-integrated method of using RNA transfection to derive putative male GCs from goat BMSCs (gBMSCs) in vitro by overexpressing STRA8, BOULE and DAZL. The gBMSCs were induced by co-transfection these three mRNAs together (mi-SBD group) or sequential transfection according to their expression time order in vivo (mi-S + BD group). After transfection, a small population of gBMSCs transdifferentiated into early germ cell-like cells and had the potential to enter meiosis. These cells expressed primordial germ cell specific genes STELLA, C-KIT and MVH, as well as premeiotic genes DAZL, BOULE, STRA8, PIWIL2 and RNF17. Importantly, the expression level of meiotic marker synaptonemal complex protein 3 significantly increased in these transfected two groups compared with control cells by qRT-PCR, immunofluorescence and western blot analysis (P < 0.05). Moreover, the protein expression of MVH was significantly higher in mi-S + BD group than that in mi-SBD group (P < 0.05). In addition, compared with control group, the methylation rate of imprinted gene H19 decreased in these two transfected group (P < 0.05), and the rate was significantly lower in mi-S + BD group compared with mi-SBD group (P < 0.05). This study helps to understand the mechanisms of action of key genes in GCs differentiation and also provides a novel system for in vitro induction of male GCs from stem cells.

Pach-ing It in: The Peculiar Organization of Mammalian Pachytene Chromosomes

In this issue of Molecular Cell, Wang etal. (2019) use Hi-C to visualize at high resolution the complex reprogramming of chromatin architecture during spermatogenesis in rhesus monkeys and mice. They find that pachytene spermatocytes have a unique chromosome organization that may result from the presence of the synaptonemal complex and transcription-associated proteins.

Sohlh1 is required for synaptonemal complex formation by transcriptionally regulating meiotic genes during spermatogenesis in mice.

Gonad-specific transcription factor spermatogenesis- and oogenesis-specific helix-loop-helix transcription factor 1 (SOHLH1) plays a key role in the transcriptional regulation of the expression of differentiating spermatogonial genes. However, its role in spermatocytes (meiotic male germ cells) remains largely unknown. In this study, Sohlh1 knockout (KO) male mice displayed meiotic defects at the zygotene stage during spermatogenesis. Microarray analyses identified 66 upregulated genes and 139 downregulated genes in Sohlh1 KO testes compared with those in wild-type testes at postnatal Day 7.5. Among many of the downregulated genes, Sycp1 and Sycp3, which encode synaptonemal complex proteins 1 and 3 (SYCP1 and SYCP3), respectively, were significantly reduced in Sohlh1 knockout mice. Transmission electron microscopy revealed no formation of the synaptonemal complex in Sohlh1 KO spermatocytes. Luciferase reporter and chromatin-immunoprecipitation assays demonstrated that SOHLH1 enhanced the expression of the Sycp1 and Sycp3 genes by binding the -1276, -708, and -94 basepairs (bp) E-boxes upstream of the Sycp1 promoter and the -64 and -43 bp E-boxes upstream of the Sycp3 promoter. Our data suggest that SOHLH1 transcriptionally regulates the expression of many target genes critical for the meiotic phase of spermatogenesis.

A Novel Role for α-Importins and Akirin in Establishment of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans

During meiotic prophase I, sister chromatid cohesion is established in a way that supports the assembly of the synaptonemal complex (SC). The SC connects homologous chromosomes, directing meiotic recombination to create crossovers. In this paper, we identify two proteins that cooperate to import and load meiotic cohesins, thus indirectly promoting SC assembly. AKIR-1 is a protein with a previously identified meiotic role in SC disassembly. akir-1 mutants have no obvious defects in sister chromatid cohesion. We identified ima-2, a gene encoding for an α-importin nuclear transport protein, as a gene interacting with akir-1 Analysis of akir-1;ima-2 double mutants reveals a decrease in the number of germline nuclei and the formation of polycomplexes (PCs) (an SC protein aggregate). These PCs contain proteins that are part of the two main substructures of the SC: the central region and the lateral element. Unlike typical PCs, they also contain sister chromatid cohesion proteins. In akir-1;ima-2 double mutants, PCs are located in both the nucleus and the cytoplasm. This suggests that the defects observed in the double mutants are both in nuclear import and in the assembly of sister chromatid cohesion. PC formation is also associated with recombination defects leading to reduced numbers of crossovers. Similarly to cohesion mutants, the pairing center protein HIM-8 is mislocalized in akir-1;ima-2 double mutants, forming multiple foci. We propose that AKIR-1 and IMA-2 operate in parallel pathways to import and load chromosomally associated cohesin complex proteins in meiotic nuclei, a novel finding for both of these conserved proteins.

Effect of antiepileptic drug (Topiramate) and cold pressed ginger oil on testicular genes expression, sexual hormones and histopathological alterations in mice

Sexual dysfunction in the epileptic patient is difficult to confirm whether it is ailment or therapy related. Antiepileptic drugs often use in reproductive age, through reproductive progress and maturation. On the other side, cold-pressed oils are rich in bioactive phytochemicals with health-promoting traits. The target of this work was to appraise the sexual dysfunction of antiepileptic Topiramate (TPM) and cold pressed ginger oil (CPGO) as antiepileptic alternative medicine in male mice. Fifty-four adult male albino mice were divided into nine groups (n = 6 mice). One group given saline and used as negative control; another one was given corn oil as vehicle. Six groups administered orally with TPM or CPGO at 100, 200 and 400 mg/kg. Moreover, group of animals co-administrated orally CPGO with TPM (400 mg/kg) to study their interaction. Fatty acid profile and tocols composition of CPGO were determined. in vitro assays were undertaken to evaluate radical scavenging traits of CPGO utilizing sable 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and galvinoxyl radicals. The study investigated antioxidant and oxidative stress markers, sexual hormones levels, mRNA levels of vascular endothelial growth factor (Vegfa), synaptonemal complex protein (Sycp3), Wilms tumor gene (Wt1) as well as histopathological and immunohistochemical examination. Strong radical scavenging potential of CPGO against stable DPPH· and galvinoxyl radicals was recorded. The results revealed that TPM caused a dose-dependent reduction in the antioxidant activities and testosterone content, while, malonaldehyde (MDA) and nitric oxide (NO) as oxidative stress markers were elevated. Vegfa and Sycp3 mRNA expression down-regulated at all Topiramate tested doses, but Wt1 up-regulated at 400 mg/kg. TPM (400 mg/kg) revealed histological alterations associated with strong positive Bax immune reactive spermatogoneal and Leydig cells. Ginger oil elevated the CAT and SOD (antioxidant enzymes), serum testosterone and diminished the oxidative stress, up regulated the expression of Vegfa and Sycp3 and down-regulated the Wt1 expression. Meanwhile, CPGO revealed no histopathological alterations and no Bax immune-reactive cells. CPGO co-administration with TPM (400 mg/kg) attenuated the TPM toxicity. High doses of TPM may exhibit sexual dysfunction but CPGO is safe and has androgenic property. CPGO co-administration could protect the antiepileptic patient from the TPM sexual dysfunction.

In Silico Analysis of Synaptonemal Complex Protein 1 (SYCP1) and Acrosin Binding Protein (ACRBP) Antigens to Design Novel Multiepitope Peptide Cancer Vaccine Against Breast Cancer

Multiepitope cancer vaccines are manifesting as the future of breast cancer immunotherapy. In the present study, high immunogenic fragments of synaptonemal complex protein 1 (SYCP1) and acrosin binding protein (ACRBP) antigens were selected according to MHCs binding affinity and CD8+ cytotoxic T lymphocytes’ (CTL) epitopes by various immunoinformatic servers. (RCQHKIAEMVALMEKHKHQYDKI) residue from p702–724 SYCP1 and the (YIQYPNYCSFKSQQCL) residue from p481–496 ACRBP were selected as the immunodominant fragments. Then, the selected fragments were fused together with a furin-sensitive linker to form the final peptide vaccine construct. The cleavage sites, TAP transport efficiency, CTL epitopes, post-translational modifications, and B cells epitopes were predicted for the final construct. The final construct contained appropriate CTLs epitopes and also, several linear and conformational B cell epitopes. Also, it exhibited 90.37% HLA population coverage in the world. Therefore, these preliminary results require validation by in vitro and in vivo experiments.

Expression and mutations of BRCA in breast cancer and ovarian cancer: Evidence from bioinformatics analyses.

Breast cancer1 (BRCA1) and breast cancer2 (BRCA2) are the most well‑known genes linked to breast cancer and ovarian cancer, which are crucial in DNA repair and transcriptional regulation. The present study aimed to elucidate the expression profiles, mutations and interaction networks of BRCA1 and BRCA2, which may provide insights to reveal the mechanisms of BRCA genes ultimately leading to breast or ovarian tumorigenesis. Bioinformatics analyses were performed in the present study. The mRNA levels of BRCA1 and BRCA2 were evaluated using FIREHOSE analysis, SAGE Genie tools and Oncomine analysis. cBioPortal analysis, and Catalogue Of Somatic Mutations In Cancer analysis were used to examine the BRCA1 and BRCA2 mutations. Kaplan‑Meier Plotter analysis was performed to identify the prognostic roles of BRCA1 and BRCA2 in breast cancer and ovarian cancer. The results of the present study showed that the mRNA expression levels of BRCA1 and BRCA2 were elevated in breast cancer and ovarian cancer tissues, compared with their matched normal tissues. Second, several common mutations of BRCA1 and BRCA2 genes were identified in breast cancer and ovarian cancer. Finally, neurofibromin1, synaptonemal complex protein 2 and tumor protein53 were predicted to be involved in the interaction network of BRCA1 and BRCA2 in breast cancer and ovarian cancer. Taken together, these results provide a significant insight into certain mutations and proteins involved in the interaction network of BRCA1 and BRCA2, which may have common roles in breast cancer and ovarian cancer. However, the complex mechanism underlying these observations remains to be fully elucidated, and further investigations are required in the future.

A ZIP1 separation-of-function allele reveals that centromere pairing drives meiotic segregation of achiasmate chromosomes in budding yeast.

In meiosis I, homologous chromosomes segregate away from each other–the first of two rounds of chromosome segregation that allow the formation of haploid gametes. In prophase I, homologous partners become joined along their length by the synaptonemal complex (SC) and crossovers form between the homologs to generate links called chiasmata. The chiasmata allow the homologs to act as a single unit, called a bivalent, as the chromosomes attach to the microtubules that will ultimately pull them away from each other at anaphase I. Recent studies, in several organisms, have shown that when the SC disassembles at the end of prophase, residual SC proteins remain at the homologous centromeres providing an additional link between the homologs. In budding yeast, this centromere pairing is correlated with improved segregation of the paired partners in anaphase. However, the causal relationship of prophase centromere pairing and subsequent disjunction in anaphase has been difficult to demonstrate as has been the relationship between SC assembly and the assembly of the centromere pairing apparatus. Here, a series of in-frame deletion mutants of the SC component Zip1 were used to address these questions. The identification of a separation-of-function allele that disrupts centromere pairing, but not SC assembly, has made it possible to demonstrate that centromere pairing and SC assembly have mechanistically distinct features and that the centromere pairing function of Zip1 drives disjunction of the paired partners in anaphase I.

The impact of low‐intensity extracorporeal shock wave therapy on testicular function in adult rats

Low-intensity extracorporeal shock wave therapy (Li-ESWT) has been introduced as a treatment for penile diseases. Its impact on testicular function during treatment remains unknown. To clarify whether Li-ESWT impairs testicular function during the treatment of penile diseases by investigating the impact of Li-ESWT on testosterone synthesis and spermatogenesis in adult rats. Twenty-four male Sprague-Dawley rats were equally divided into the following three groups: control group, 1.6 BAR group, and 3.2 BAR group. Rats in the experimental groups were treated with Li-ESWT at different energy levels (300 shocks at 1.6 BAR or 3.2 BAR, 2Hz frequency) three times a week for 3weeks. The control group did not receive any treatment during the same period of time. One day after the last shock wave treatment, serum and testicular tissue testosterone concentrations were measured, and sperm quality was assayed. Histologic examination of the testes and quantitative real-time PCR were performed. Testosterone levels in both the serum and testicular tissue did not change after Li-ESWT exposure. The expression levels of steroidogenic enzymes (steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD)) were not impacted by Li-ESWT. The 3.2 BAR group showed a significantly lower sperm count and lower expression of synaptonemal complex protein 3 (SYCP3) in testicular tissue than the control group. No significant differences in sperm quality or SYCP3 expression were observed between the control group and the 1.6 BAR group. Li-ESWT exposure at 3.2 BAR inhibited spermatogenesis and decreased sperm quality, which indicated that male patients with a desire to preserve fertility should undergo low-energy Li-ESWT or other treatment modalities.

Abstract 4725: Blockade of cyclin D-CDK4/6 sensitizes immune-refractory tumor cells to tumor antigen-specific T cell-mediated cytotoxicity by repressing SCP3-NANOG axis

Abstract Antitumor immune drives tumor cell to acquire immune-refractory phenotypes. Previously, we demonstrated tumor antigen-specific T cells edited them resistant to CTL killing and enriched NANOGhigh cancer stem cell-like (CSC-like) cells. In this study, in an effort to elucidate targetable pathways of immune-edited tumor cells with the multiaggressiveness, we show synaptonemal complex protein 3 (SCP3), a member of Cor1 family, which is overexpressed in the immune-edited cells, upregulates NANOG by hyperactivating cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and, notably, negatively correlated with the progression-free survival of cervical cancer patients. Moreover, targeting CDK4/6 with its inhibitor, palbociclib, succeeded in reversing the multiaggressive phenotypes of immune-edited tumor cells, and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6, and identify CDK4/6 inhibitors as an actionable drug for controlling SCP3high immune-refractory cancer. Citation Format: Se Jin Oh, Tae Woo Kim. Blockade of cyclin D-CDK4/6 sensitizes immune-refractory tumor cells to tumor antigen-specific T cell-mediated cytotoxicity by repressing SCP3-NANOG axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4725.

Targeting Cyclin D-CDK4/6 Sensitizes Immune-Refractory Cancer by Blocking the SCP3-NANOG Axis.

Immunoediting caused by antitumor immunity drives tumor cells to acquire refractory phenotypes. We demonstrated previously that tumor antigen-specific T cells edit these cells such that they become resistant to CTL killing and enrich NANOGhigh cancer stem cell-like cells. In this study, we show that synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, is overexpressed in immunoedited cells and upregulates NANOG by hyperactivating the cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and correlated negatively with progression-free survival of cervical cancer patients. Targeting CDK4/6 with the inhibitor palbociclib reversed multiaggressive phenotypes of SCP3high immunoedited tumor cells and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6 and identify CDK4/6 inhibitors as actionable drugs for controlling SCP3high immune-refractory cancer.Significance: These findings reveal cyclin D1-CDK4/6 inhibition as an effective strategy for controlling SCP3high immune-refractroy cancer. Cancer Res; 78(10); 2638-53. ©2018 AACR.

Scraping the tip of Zip1's role in meiotic chromosome dynamics: Using lacO/LacI Corecruitment to identify crossover promoting factors that interface with the N‐terminus of a synaptonemal complex protein

During the first division of meiosis, diploid nuclei divide to produce nuclei with only one set of chromosomes. Accurate chromosome segregation during this so‐called “reductional” division relies on the prior establishment of recombination‐based associations between homologous chromosomes (homologs). Typically, homolog pairing and interhomolog recombination take place in conjunction with the assembly of a conserved tripartite chromosomal structure, the Synaptonemal Complex (SC), and interhomolog crossover recombination intermediates are processed within the physical context of SC. However, the molecular underpinnings of the relationship between SC and meiotic recombination remain obscure. The SC is assembled by rod‐like transverse filament proteins that bridge aligned homolog axes; transverse filaments interface with a central element substructure at the midline of the SC. In the yeast Saccharomyces cerevisiae, a major component of the SC transverse filament is the coiled‐coil protein, Zip1. Our prior work found that small, adjacent regions within Zip1's N terminus act to independently promote crossover recombination and SC assembly, and that versions of Zip1 missing residues 2–9 or 10–14 support SC assembly but not SC‐associated crossover formation. Mutants expressing zip1‐[Δ2–9] and zip1‐[Δ10–14] alleles assemble SC, but form remarkably large Zip1 polycomplex structures and display hyper‐SUMOylation of another SC protein, Ecm11, two relatively unusual meiotic phenotypes that are also observed in meiotic cells missing the crossover‐promoting factor, Zip3. These observations suggest that residues 2–14 of Zip1 somehow affect Zip3 behavior. Zip1 has previously been found to be essential for the recruitment of Zip3 protein to sites of recombination initiation on meiotic chromosomes, and chromatin immunoprecipitation experiments revealed that Zip3 fails to localize to all surveyed meiotic recombination initiation sites in zip1‐D1[Δ2–9] and zip1‐D2[Δ10–14] mutants, suggesting that the crossover defect exhibited by these mutants is based at least in part on the inability of the Zip1[Δ2–9] or Zip1[Δ10–14] proteins to recruit Zip3 to recombination initiation sites. Furthermore, cytological analysis indicated that Zip3 is excluded from the polycomplex structures assembled by Zip1‐[Δ2–9] or Zip1‐[Δ10–14] protein, suggesting that Zip1's residues 2–14 somehow interface with Zip3. I am currently creating a lacO/LacI co‐recruitment assay to investigate whether Zip1's N‐terminus is sufficient for recruiting Zip3 to an ectopic site in meiotic nucleus. I have created diploid strains in which the Zip1's N terminal residues 1–17, 1–33, 1–171, or 1–348 are fused to the N‐terminus of GFP‐LacI and that carry lacO on chromosome IV. Using immunofluorescence on surface spread meiotic chromosomes, I will ask whether Zip3‐MYC is recruited to a chimeric Zip1‐GFP‐LacI protein bound to a lacO array positioned on chromosome IV within the meiotic nucleus. The data from this experiment will not only provide strong evidence for a direct interaction between Zip1 and Zip3, but also establish a starting point for future structure‐function studies aimed at identifying residues that are critical for, and the meiotic genes that might regulate, a putative Zip1‐Zip3 interaction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro differentiation of human oocyte-like cells from oogonial stem cells: single-cell isolation and molecular characterization.

Are the large cells derived from cultured DEAD box polypeptide 4 (DDX4)-positive oogonial stem cells (OSCs), isolated from the ovarian cortex of non-menopausal and menopausal women, oocyte-like cells? Under appropriate culture conditions, DDX4-positive OSCs from non-menopausal and menopausal women differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. The recent reports of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, several questions remain regarding the isolation of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes. DDX4-positive OSCs were obtained from 19 menopausal and 13 non-menopausal women (who underwent hysterectomy for uterine fibroma, ovarian cyst, or other benign pathologies) and cultured for up to 3 weeks. Large and small cells were individually isolated and typed for early and late differentiation markers. Ovarian cortex fragments were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and the positive populations were measured by assessing both FRAGILIS and stage-specific embryonic antigen 4 (SSEA-4) expression. After 3 weeks in culture, large oocyte-like cells were individually isolated by DEPArray based on PKH26 red staining and cell size determination. GDF-9 and SYCP3 as final, and developmental pluripotency-associated protein 3 (DPPA3) as primordial, germline markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content of chromosomes X and 5 was investigated using fluorescence in situ hybridization (FISH). SSEA-4+ and FRAGILIS+ subsets of DDX4-positive populations were present at lower mean levels in menopausal (SSEA-4+: 46.7%; FRAGILIS+: 47.5%) than in non-menopausal (SSEA-4+: 64.9%; FRAGILIS+: 64.8) women (P < 0.05). A comparison of the women's age with the ratio of DDX4-positive cells/cm3 of ovarian cortex revealed an inverse correlation with OSC number (P < 0.05). Once cultured, cells from both groups differentiated to form large (up to 80 μm) mature oocyte-like cells with typical oocyte morphology. Despite the higher numbers of these cells in cultures from non-menopausal women (37.4 versus 23.7/well; P < 0.001), the intra-culture percentages of large oocyte-like cells did not differ significantly between the two groups. Single large oocyte-like cells isolated from non-menopausal and menopausal women expressed equivalent levels of GDF-9 (e.g. 2.0 and 2.6 copies/μl RNA, respectively) and SYCP3 (e.g. 1.2 and 1.5 copies/μl RNA, respectively) mRNA. The remaining small cells isolated from the cultures expressed large amounts of DPPA3 mRNA (e.g. 5.0 and 5.1 copies/μl RNA, from menopausal and non-menopausal women, respectively), which was undetectable in the large oocyte-like cells. FISH analysis of the large and small cells using probes for chromosomes X and 5 revealed a single signal in the large cells, indicative of chromosome haploidy, whereas in the small cells two distinct signals for each chromosome indicated diploidy. Not applicable. Our study demonstrated the final differentiation of OSCs, collected from the ovarian cortex of adult women, to oocyte-like cells. However, because the rate of differentiation was low, a major role of the stem cell niche housing these OSCs cannot be ruled out. Since the ability of OSCs to generate mature oocytes in vitro is highly variable, the viability of these cells in the ovarian cortex of non-menopausal and menopausal women may well be determined by the stem cell niche and the woman's concurrent reproductive state. Our study showed that the oocyte-like cells obtained by OSC differentiation in vitro, including those from the OSCs of menopausal women, express markers of meiosis. This model of ovarian neo-oogenesis will contribute to the development of approaches to treat female infertility. The study was funded by Italian Association for Cancer Research (IG grant 17536), and from the Apulia Region ('Oncogenomic Project' and 'Jonico-Salentino Project'). All Authors declare no competing interests.

Superresolution imaging of the synaptonemal complex.

This chapter describes how two different superresolution microscopy techniques, namely, structured illumination microscopy and direct stochastic optical reconstruction microscopy, can be used to analyze the molecular architecture of the synaptonemal complex. The experimental protocols provided allow the construction of precise localization maps for different synaptonemal complex proteins.

Dwarfs and Giants of Parathyroid Adenomas - No Difference in Outcome after Parathyroidectomy

Although anti-Mullerian hormone (AMH) is present in seminiferous tubules, its physiological roles in testis remain unclear. We defined expression patterns of AMH and its type II receptor (AMHR2) in germ cells during spermatogenic cycles. To this end, we performed in situ hybridization of Amh and Amhr2, and immunohistochemical staining using antibodies against AMH and AMHR2 in testes from rats aged 10, 15, 21, 35, and 49 days (d). In addition, quantitative RT-PCR analyses were performed to determine relative expression levels of Amh, Amhr2, synaptonemal complex protein 1 (Scp1) and round spermatid basic protein 1 (Rsbn1), and Smads 1, 5, and 8 in germ cells isolated from 49d testes. In 49d testes, synthesis of AMH and AMHR2 corresponded with spermatocyte expression of Amh and Amhr2. Spermatocytes of stages VII-XIV synthesized AMH, and AMH staining was decreased in round spermatids and was not detectable in elongated spermatids. Spermatocytes of stages VII-XIV synthesized AMHR2, and round and elongated spermatids continued expressing AMHR2. The strongest staining of AMH was observed in spermatocytes at stage VIII, and that of AMHR2 was observed in round spermatids at the same stage. When isolated germ cells were cultured with anti-AMH antibody, expression levels of Amh and Amhr2 were significantly increased, those of Smads 1, 5 and Scp1 increased simultaneously, and that of Rsbn1 decreased. We speculate that the AMH-SMADs axis is associated with morphological change from round to elongated spermatids at stage VIII.