Mouse Male Germ Cells Research Articles

Mutagenicity of Acrylamide and Glycidamide in the Testes of Big Blue Mice

Acrylamide (AA) is an industrial chemical, a by-product of fried starchy foods, and a mutagen and rodent carcinogen. It can also cause damage during spermatogenesis. In this study, we investigated whether AA and its metabolite glycidamide (GA) induce mutagenic effects in the germ cells of male mice. Male Big Blue transgenic mice were administered 1.4 or 7.0mM of AA or GA in the drinking water for up to 4 weeks. Testicular cII mutant frequency (MF) was determined 3 weeks after the last treatment, and the types of the mutations in the cII gene were analyzed by DNA sequencing. The testes cII MFs in mice treated with either the low or high exposure concentrations of AA and GA were increased significantly. There was no significant difference in the cII MFs between AA and GA at the low exposure concentration. The mutation spectra in mice treated with AA (1.4mM) or GA (both 1.4 and 7.0mM) differed significantly from those of controls, but there were no significant differences in mutation patterns between AA and GA treatments. Comparison of the mutation spectra between testes and livers showed that the spectra differed significantly between the two tissues following treatment with AA or GA, whereas the mutation spectra in the two tissues from control mice were similar. These results suggest that AA possesses mutagenic effects on testes by virtue of its metabolism to GA, possibly targeting spermatogonial stem cells, but possibly via different pathways when compared mutations in liver.

Gynostemma pentaphyllum protects mouse male germ cells against apoptosis caused by zearalenone via Bax and Bcl-2 regulation

The objective of this study was to explore the effects of Gynostemma pentaphyllum on Zearalenone-induced apoptosis in mouse male germ cells. Fifty Kunming male mice at 25-days-old were classified into five groups: group A was the control (10% ethanol, 0.5 ml/day); group B with 10 µg Zearalenone/day; group C with 10 µg Zearalenone and 50 mg/kg/day Gynostemma pentaphyllum; group D with 10 µg Zearalenone and 100 mg/kg/day Gynostemma pentaphyllum; and group E with 10 µg Zearalenone and 200 mg/kg/day Gynostemma pentaphyllum. It was found that Gynostemma pentaphyllum has a marked effect on protecting male germ cells against Zearalenone-induced apoptosis, as evidenced by a reduced apoptosis rate of male germ cells and Bax expression as well as an enhancement of Bcl-2 expression in Gynostemma pentaphyllum-treated groups compared to the control. In addition, Gynostemma pentaphyllum remarkably improved pathologic changes of testicular tissue, reduced the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD) caused by Zearalenone. Taken together, these results suggest that Gynostemma pentaphyllum protects against toxicity caused by Zearalenone through anti-oxidation and anti-apoptosis via the regulation of Bax and Bcl-2 expression.

Microgravity Promotes Differentiation and Meiotic Entry of Postnatal Mouse Male Germ Cells

A critical step of spermatogenesis is the entry of mitotic spermatogonia into meiosis. Progresses on these topics are hampered by the lack of an in vitro culture system allowing mouse spermatogonia differentiation and entry into meiosis. Previous studies have shown that mouse pachytene spermatocytes cultured in simulated microgravity (SM) undergo a spontaneous meiotic progression. Here we report that mouse mitotic spermatogonia cultured under SM with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-positive spermatogonia under the RCCS condition enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-regulation of several pro-meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A PI3K inhibitor abolished Scp3 induction and meiotic entry stimulated by RCCS conditions. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation and might provide a tool to study the molecular mechanisms underlying the switch from mitosis to meiosis in mammals.

180. THE CONSEQUENCES OF ACRYLAMIDE EXPOSURE ON THE MALE GERMLINE

Acrylamide is a common industrial compound that has recently been identified in cooked, carbohydrate-rich foods such as potato chips, breads and cereals. Acrylamide has been found to be a reproductive toxin in rodents, eliciting male infertility and transgenerational toxicity through the male germline; thus dietary exposure to the compound may have consequences for male fertility and reproduction in humans. The aim of this project was to elucidate the mechanisms of acrylamide toxicity in male germ cells of mice. Freshly isolated early male germ cells were exposed to acrylamide and assessed for cell viability and aberrant morphology. DNA damage in these cells was also investigated using a modified version of the Comet Assay, which allows for adduct specificity. Significant increases in cell death or aberrant morphology were not observed following acrylamide exposure (1 µM, 18 hours). However, a significant increase in DNA damage (125% increase in mean tail DNA assessed by Comet) was identified; which may originate from either the metabolic conversion of acrylamide to glycidamide, leading to glycidamide adducts, or from oxidative stress. Additionally, the regulation of cytochrome P450 gene expression was measured using real time PCR and early male germ cells were found to upregulate gene expression of cytochrome P450 enzymes in response to acrylamide exposure. Collectively, these results support a genotoxic mode of action of acrylamide toxicity, in addition to potential oxidative damage in male germ cells. However, the mechanism by which acrylamide elicits toxicity in germ cells requires further investigation. Future outcomes of this research may provide insight into factors necessary for the healthy development of offspring and aid in the risk assessment of dietary acrylamide exposure in humans.

In vitro investigations of glycidamide-induced DNA lesions in mouse male germ cells and in mouse and human lymphocytes

The industrial compound and food contaminant acrylamide (AA) is a probable human carcinogen, also known to induce male-mediated reproductive effects in animals. Most data suggest that its metabolite glycidamide (GA) is involved in the observed toxicity. We have investigated in vitro effects of AA/GA in mouse male germ cells (prior to spermatid elongation) and human and mouse peripheral blood lymphocytes, to assess inter-species and cell-type differences in susceptibility, and to explore the nature of the DNA lesion(s) as well as their potential repair. The comet assay was used in combination with the DNA-repair enzymes Fpg and hOGG1 to measure specific DNA lesions. In contrast to AA, GA induced significant levels of DNA lesions (strand breaks and alkali-labile sites) at millimolar concentrations in mouse testicular cells and human peripheral blood lymphocytes (hPBL). Using Fpg, the GA-induced DNA damage was measured at 20–50-fold higher sensitivity, in all cell types investigated. GA-induced DNA damage could not be recognised by hOGG1, suggesting that, based on the known affinities of these repair enzymes, alkylation of guanine is involved, rather than oxidation. Human lymphocytes appeared to be more susceptible to GA-induced lesions than both types of mouse cells. Mouse testicular cells and lymphocytes seemed to respond similarly to GA-induced Fpg-sensitive DNA lesions. The persistence of lesions was explored with cells from mice either proficient or deficient in Ogg1 (mouse 8-oxoguanine DNA glycosylase). Low in vitro repair of GA-induced Fpg-sensitive lesions was observed in primary male germ cells and lymphocytes from both Ogg1 +/+ and Ogg1 −/− mice. We conclude that there may be differences between mice and humans in AA/GA-induced genotoxicity, and DNA from mouse male germ cells does not appear to be more sensitive to GA than DNA from peripheral blood lymphocytes in vitro. The usefulness of the comet assay in combination with DNA-repair enzymes is demonstrated.

Study of The Transgenic Efficiency in Different Spermiogenesis Stages in Mice*

In the past 30 years, the research and application of transgenic technology in gene expression of animal had become a noticeable advancement in experimental biology and applied biology. Microinjection,retrovirus mediated method and embryonic stem cells method were all the traditional methods of producing transgenic animals. But these methods had defects and limited application in the research of transgenic animals in the future. Exogenous gene was transferred into germ cells of male mice, in order to study efficiency of transgenic mice in different spermiogenesis stages. The green fluorescence protein expression plasmid(pIRES2-EGFP) and liposomes were mixed and injected into male mouse testis and epididymis. Then the intratesticular mice were mated with female at day 7, 16, 30 and 42 after infection. Polymerase chain reaction(PCR) and Southern blot were applied to identify transgenic mice. The positive ratio was 6.82%, 0, 56.86%, 42.86% by PCR analysis and 6.82%, 0, 47.06%, 34.69% by Southern blot analysis . The transgenic mice showed green fluorescence in fluorescence imager and fluorescent microscope under EGFP excitation light (488 nm). Through comparing efficiency of transgenic mice in different spermiogenesis stages, it could provided an important theoretic foundation for efficient production of transgenic animals by testis mediated gene transfer.

The endocannabinoid system and pivotal role of the CB 2 receptor in mouse spermatogenesis

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB(2) receptors. In fact, we found that the selective CB(2) receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.

Centrobin/Nip2 expression in vivo suggests its involvement in cell proliferation.

Centrobin/Nip2 was initially identified as a centrosome protein that is critical for centrosome duplication and spindle assembly. In the present study, we determined the expression and subcellular localization of centrobin in selected mouse tissues. Immunoblot analysis revealed that the centrobin-specific band of 100 kDa was detected in all tissues tested but most abundantly in the thymus, spleen and testis. In the testis, centrobin was localized at the centrosomes of spermatocytes and early round spermatids, but no specific signal was detected in late round spermatids and elongated spermatids. Our results also revealed that the centrosome duplication occurs at interphase of the second meiotic division of the mouse male germ cells. The centrobin protein was more abundant in the mitotically active ovarian follicular cells and thymic cortex cells than in non-proliferating corpus luteal cells and thymic medullary cells. The expression pattern of centrobin suggests that the biological functions of centrobin are related to cell proliferation. Consistent with the proposal, we observed reduction of the centrobin levels when NIH3T3 became quiescent in the serum-starved culture conditions. However, a residual amount of centrobin was also detected at the centrosomes of the resting cells, suggesting its role for maintaining integrity of the centrosome, especially of the daughter centriole in the cells.

Testicular cell suspensions of the mouse in vitro.

A method is presented which allows survival and continued differentiation of male mouse germ cells for up to 12 days in culture. The system uses continuous flow perfusion at 20 degrees C in a completely chemically defined medium. To show differentiation through meiosis, mice were treated with hydroxyurea (HU) which creates a gap in the spermatogenic line. Suspensions of testicular cells of these mice, completely lacking pachytene/diplotene stages, were put into culture. After 7 or 10 days culturing, differentiation was demonstrated by the appearance of pachytene and diplotene stages and round spermatids. The velocity of the differentiation in vitro was found the same as would be expected in vivo. The number of cells which show differentiation throughout meiosis is less than would be expected in cultures of testicular cells of mice which were not treated with hydroxyurea.

Transient expression of theArftumor suppressor during male germ cell and eye development inArf-Crereporter mice

The Arf tumor suppressor is expressed transiently during mouse male germ cell and eye development. Its inactivation compromises spermatogenesis as mice age and leads to aberrant postnatal proliferation of cells in the vitreous of the eye, resulting in blindness. In the testis, expression of p19(Arf) is limited to spermatogonia but is extinguished completely in spermatocytes, suggesting that Arf plays a physiologic role in setting the balance between mitotic and meiotic germ cell division. A knock-in allele encoding Cre recombinase regulated by the mouse cellular Arf promoter was used to trace Arf gene induction in vivo. Interbreeding to a reporter strain that expresses Cre-dependent YFP provided proof-of-principle that the Arf-Cre allele was appropriately expressed in the male germ cell lineage. However, Cre expression resulted in male sterility, limiting germ line transmission of the knock-in allele to females. Arf-null mice fail to resorb the hyaloid vasculature within the ocular vitreous where pericyte-like cells that express the PDGF-beta receptor (Pdgfrbeta) proliferate aberrantly and destroy the retina and lens. Interbreeding of Arf-Cre females to males containing "floxed" (FL) Arf alleles yielded Arf(Cre/FL) progeny that exhibited variably penetrant defects in visual acuity ranging to total blindness. Crossing the Arf(Cre/FL) alleles onto a Pdgfrbeta(FL/FL) background normalized all histopathology and restored vision fully.

Two-step oligoclonal development of male germ cells

During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the "deciduous" germ cells are lost, most likely by apoptosis, before the reproductive period. The second "actual" founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.

Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice

We have generated a transgenic mouse line,Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia and is detected through preleptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/GFP)21Lbe (Z/EG) reporter females and compared the expression of enhanced green fluorescent protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF-a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development.

GermSAGE: a comprehensive SAGE database for transcript discovery on male germ cell development

GermSAGE is a comprehensive web-based database generated by Serial Analysis of Gene Expression (SAGE) representing major stages in mouse male germ cell development, with 150 000 sequence tags in each SAGE library. A total of 452 095 tags derived from type A spermatogonia (Spga), pachytene spermatocytes (Spcy) and round spermatids (Sptd) were included. GermSAGE provides web-based tools for browsing, comparing and searching male germ cell transcriptome data at different stages with customizable searching parameters. The data can be visualized in a tabulated format or further analyzed by aligning with various annotations available in the UCSC genome browser. This flexible platform will be useful for gaining better understanding of the genetic networks that regulate spermatogonial cell renewal and differentiation, and will allow novel gene discovery. GermSAGE is freely available at http://germsage.nichd.nih.gov/

Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1 x Milano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D(28k) (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.

ICSI and Nuclear Transfer Using Male Germ Cells.

In vitro fertilization (IVF) in mammals was achieved in the middle of the last century by the pioneers of fertilization studies, Drs. Austin, Chan, and Yanagimachi (Yana). Soon after the success of IVF, a sperm injection technique (later called ICSI, or microinsemination in a broad sense) was developed to simplify the fertilization process and determine what happens within the oocyte. In its first decade, ICSI provided invaluable information on the mechanisms of mammalian fertilization including sperm nucleus stability. Then, around 1990, normal ICSI offspring were reported in rabbits, cattle, and humans. The first reliable application of mouse ICSI was achieved by Yana's group in 1995. Since then, the mouse model has held a leading role in the field of microinsemination. We have also employed the technique in mouse genetic studies and found that elongated and round spermatids, and even primary spermatocytes, could be used to fertilize oocytes to produce normal pups. The consistent fecundity enables us to apply the mouse microinsemination technique to transgenesis, gene therapy, germline stem cell, and gene-targeting studies. In contrast, in other mammals, immaturity of male germ cells significantly decreases the efficiency of microinsemination. No, or very few, offspring have been born following ICSI using spermatids in these species. This is not due to immaturity of their genome, but to their physically unstable chromatin structure and low oocyte-activating capacity. Our recent ICSI study was also undertaken to simplify the cryopreservation technique for mouse male germ cells. Whole mouse testes and even whole bodies can be frozen and then used as a source of sperm. Spermatozoa retrieved from male bodies frozen for 15 years at −20°C support full-term development following ICSI, indicating unexpectedly high stability of sperm nucleus integrity. Younger male germ cells include spermatogonia and primordial germ cells (PGCs). Their nuclei are diploid and therefore cannot be used for microinsemination. Construction of diploid embryos with these early male germ cells requires a cloning technique using nuclear transfer (NT). Cloning spermatogonia or PGCs has two scientific significances: analysis of genomic imprinting status and genomic reprogrammability (not reprogramming ability). The former is based on the fact that the imprinting memory of the donor cell genome is maintained after transfer into MII oocytes under well-controlled experimental conditions. By analyzing the fetuses thus produced, we clearly demonstrated that the major erasing process for the imprinting memory occurred around day 11.5 in mouse PGCs and that the PGC genome before imprinting erasure could support full-term development after NT. As to genome reprogrammability, we found that the some proportion of male PGCs gradually acquired the ability to be reprogrammed normally following nuclear transfer, as revealed by the gene expression patterns of the reconstructed embryos. We hope that this experimental model may allow identification of some keys or epigenetic markers that permit differentiation between somatic cell and germ cell genomes. Thus, ICSI and nuclear transfer using male germ cells have great potential as tools for a broad range of genetic and epigenetic research in mammals.

Adrenocorticotropic hormone affects nonapoptotic cell death of undifferentiated germ cells in the fetal mouse testis: In vivo study by exo utero transplantation of corticotropic tumor cells into embryos

Adrenocorticotropic hormone (ACTH) has been suggested to have possible roles in the fetal testes, one of the organs that express its specific receptors, melanocortin type 2 and 5 receptors (MC2R and MC5R), during the fetal period. We investigated the effect of ACTH on the cells in the testis cord of the fetal mouse testis by inducing ACTH-secreting AtT20 tumor cells in mouse fetuses. We first identified that mouse testicular germ cells at embryonic day (E) 16.5 and E18.5 spermatogonia were entirely CDH1 (E-cadherin)-positive by immunohistochemistry. We next performed AtT20-cell transplantation into the mouse fetus at E12.5, and analyzed ACTH effects on the development of fetal male mouse germ cells that express MC2R and MC5R at E16.5 and E18.5. The spermatogonia in the testis of AtT20-implanted embryos exhibited morphological changes, including pyknotic nuclei and swollen cytoplasm. In the AtT20-implanted embryos, the number of spermatogonia per unit area of the testis cord was significantly lower, but there were more pyknotic spermatogonia than in the controls. Single-stranded DNA-positive (apoptotic) and histone H3-positive (mitotic) spermatogonia were rarely observed and their numbers did not significantly differ in the two groups. Anti-Müllerian hormone (AMH)-positive Sertoli cells, another cell type that constitutes the fetal testis cord but does not express MC2R or MC5R, showed no apparent morphological changes compared with controls, nor were their numbers in the two groups significantly different between the two groups. These results suggest that ACTH, via MC2R and/or MC5R, may be involved in the nonapoptotic cell death of fetal mouse spermatogonia that is observed during the normal perinatal period.

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells – spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.

Role of hPHF1 in H3K27 Methylation and Hox Gene Silencing

Polycomb group (PcG) proteins are required for maintaining the silent state of the homeotic genes and other important developmental regulators. The silencing function of the PcG proteins has been linked to their intrinsic histone modifying enzymatic activities. The EED-EZH2 complex, containing the core subunits EZH2, EED, SUZ12, and RbAp48, functions as a histone H3K27-specific methyltransferase. Here we describe the identification and characterization of a related EED-EZH2 protein complex which is distinguished from the previous complex by the presence of another PcG protein, hPHF1. Consistent with the ability of hPHF1 to stimulate the enzymatic activity of the core EED-EZH2 complex in vitro, manipulation of mPcl1, the mouse counterpart of hPHF1, in NIH 3T3 cells and cells of the mouse male germ cell line GC1spg results in global alteration of H3K27me2 and H3K27me3 levels and Hox gene expression. Small interfering RNA-mediated knockdown of mPcl1 affects association of the Eed-Ezh2 complex with certain Hox genes, such as HoxA10, as well as Hox gene expression concomitant with an alteration on the H3K27me2 levels of the corresponding promoters. Therefore, our results reveal hPHF1 as a component of a novel EED-EZH2 complex and demonstrate its important role in H3K27 methylation and Hox gene silencing.

Acetylcholinesterase‐R increases germ cell apoptosis but enhances sperm motility

Changes in protein subdomains through alternative splicing often modify protein-protein interactions, altering biological processes. A relevant example is that of the stress-induced up-regulation of the acetylcholinesterase (AChE-R) splice variant, a common response in various tissues. In germ cells of male transgenic TgR mice, AChE-R excess associates with reduced sperm differentiation and sperm counts. To explore the mechanism(s) by which AChE-R up-regulation affects spermatogenesis, we identified AChE-R's protein partners through a yeast two-hybrid screen. In meiotic spermatocytes from TgR mice, we detected AChE-R interaction with the scaffold protein RACK1 and elevated apoptosis. This correlated with reduced scavenging by RACK1 of the pro-apoptotic TAp73, an outcome compatible with the increased apoptosis. In contrast, at later stages in sperm development, AChE-R's interaction with the glycolytic enzyme enolase-α elevates enolase activity. In transfected cells, enforced AChE-R excess increased glucose uptake and adenosine tri-phosphate (ATP) levels. Correspondingly, TgR sperm cells display elevated ATP levels, mitochondrial hyperactivity and increased motility. In human donors' sperm, we found direct association of sperm motility with AChE-R expression. Interchanging interactions with RACK1 and enolase-α may hence enable AChE-R to affect both sperm differentiation and function by participating in independent cellular pathways.