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Biomedical subjects

Mary Ann Handel

Publications and source records attributed to Mary Ann Handel.

17 recordsLinked to original sources
PubMed2006

Mutagenesis as an unbiased approach to identify novel contraceptive targets.

To accommodate diverse personal needs in family planning, diverse contraceptive approaches are desirable. This goal requires identification of new contraceptive targets. Phenotype-driven mutagenesis is an unbiased approach to identify novel genes and functions in reproductive processes. The ReproGenomics Program at The Jackson Laboratory is a United States National Institutes of Health resource for production, identification and distribution of mutant mouse models of infertility that can be used for identification of potential targets for contraception. The strategy of this program is whole genome, random ENU mutagenesis, coupled with a phenotype screen for breeding failure as the only phenotype. A three-generation breeding scheme selects recessive mutations affecting reproductive functions. G3 males and females that fail to reproduce by natural mating to wild-type animals undergo secondary phenotype screens to assess gonad and accessory organ histology, hormone production, gamete production and gamete function in fertilization. The genetic transmission of the infertility trait in each family is confirmed and each mutation is genetically mapped to a defined chromosome region, facilitating identification of candidate genes from sequence and expression databases. Genes essential for fertility in both males and females and acting both meiotically and post-meiotically have been identified by this strategy. Phenotypes include male infertility with normal sperm count, but failure in fertilization of oocytes. Phenotype descriptions of each mutation are posted on the program website, . These unique reproductive mutant mouse resources will lead to new discoveries in andrology (and gynecology) research, as well as reproductive medicine. Dissection of gene function in known and newly discovered reproductive pathways will expand our focus to reveal novel targets for contraception.

Alkylating Agents↗
PubMed2006

Spermatocyte responses in vitro to induced DNA damage.

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.

Animals↗
PubMed2005

Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis.

In postmeiotic male germ cells, TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. In contrast, in pachytene spermatocytes, TSN is found predominantly in nuclei. Tsn-null males show a reduced sperm count and high levels of apoptosis in meiotic cells, suggesting a critical function for TSN during meiosis. To identify meiotic target RNAs that associate in vivo with TSN, we reversibly cross-linked TSN to RNA in testis extracts from 17-day-old and adult mice and immunoprecipitated the complexes with an affinity-purified TSN antibody. Extracts from Tsn-null mice were used as controls. Cloning and sequencing the immunoprecipitated RNAs, we identified four new TSN target mRNAs, encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and ring finger protein 139. In contrast to the population of postmeiotic translationally delayed mRNAs that bind TSN, these four mRNAs are initially expressed in pachytene spermatocytes. In addition, anti-TSN also precipitated a nonprotein-coding RNA (ncRNA), which is abundant in nuclei of pachytene spermatocytes and has a putative polyadenylation signal, but no open reading frame. A second similar ncRNA is adjacent to a GGA repeat, a motif frequently associated with recombination hot spots. RNA gel-shift assays confirm that the four new target mRNAs and the ncRNA specifically bind to TSN in testis extracts. These studies have, for the first time, identified both mRNAs and a ncRNA as TSN targets expressed during meiosis.

Animals↗
PubMed2005

Regulation of meiotic cell divisions and determination of gamete quality: impact of reproductive toxins.

The events and tempo of mammalian meiosis show sexual dimorphism with gametogenic context having a significant influence on both chromosome dynamics and cell cycle transitions. However, although some regulators of the meiotic cell cycle may differ between males and females, there appears to be extraordinary conservation of key components in common with the mitotic cell cycle and between sexes in meiosis. Evidence is presented for the existence of meiotic checkpoints that might modify or ameliorate cellular error or damage by reproductive toxins. Although these checkpoints seem relatively inefficient, they may be more so in female meiosis than in male meiosis. Insight into mechanisms of meiotically acting reproductive toxins coupled with genetic models will ultimately bring about understanding of the basic mechanisms of meiotic cell divisions and chemically induced meiotic error.

Animals↗
PubMed2004

SUMO modified proteins localize to the XY body of pachytene spermatocytes.

The XY body is a specialized chromatin territory that forms during meiotic prophase of spermatogenesis and comprises the transcriptionally repressed sex chromosomes. Remodeling of the XY chromatin is brought about by recruitment of specific proteins to the X and Y chromosomes during meiosis, and also by post-translational modifications of histones and other chromatin-associated proteins. Here, we demonstrate that SUMO, a small ubiquitin-related modifier protein that regulates a wide variety of nuclear functions in somatic cells, dramatically localizes to the XY body. SUMO was first detected in the XY body of early pachytene spermatocytes and gradually accumulated, reaching maximal levels there during the mid to late pachytene stages. Several known SUMO substrates, including PML and DAXX, were also found to accumulate in the XY body of mid to late stage pachytene spermatocytes. These same proteins localize to PML nuclear bodies of somatic interphase nuclei. Together, these findings indicate a role for SUMO modification in regulating the structure and function of the XY body and reveal molecular similarities between the XY body and PML nuclear bodies.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The XY body: a specialized meiotic chromatin domain.

The sex chromosomes of mammalian spermatocytes form a specialized nuclear territory known as the XY body, where both transcription and homologous recombination are restricted. The array of proteins assembled into the XY body is typical of heterochromatin. This special subnuclear domain is in distinct contrast to the autosomal domain of the spermatocyte nucleus, where both homologous recombination and transcription occur. The special features of the XY body might reflect absence of homology between the sex chromosomes, rather than any form of dosage compensation, and may also serve to mark parental origin of the paternal X chromosome.

Animals↗
PubMed2004

The relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells.

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP.

Animals↗
PubMed2004

Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes.

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.

4-Butyrolactone↗
PubMed2004

Reductions in linker histone levels are tolerated in developing spermatocytes but cause changes in specific gene expression.

H1 linker histones are involved in packaging chromatin into 30-nm fibers and higher order structures. Most eukaryotic cells contain nearly one H1 molecule for each nucleosome core particle. Male germ cells in mammals contain large amounts of a germ cell-specific linker histone, HIST1HT, herein denoted H1t, which is particularly abundant in pachytene spermatocytes. Despite its abundance in male germ cells and significant divergence in primary sequence from other H1 subtypes, inactivation of the H1t gene in mice showed that it is not required for spermatogenesis. Analysis of germ cell chromatin from H1t null mice showed that other H1 subtypes, especially the testis-enriched HIST1H1A, herein denoted as the H1a subtype, were able to compensate for the absence of H1t to maintain a normal total H1 to nucleosome core ratio. To disrupt the compensation, we generated H1t and H1a double null mice by two sequential gene-targeting steps in embryonic stem cells. Elimination of both H1t and H1a led to a 25% decrease in the ratio of H1 to nucleosome cores in double null germ cells. Surprisingly, the reduction in H1 did not perturb spermatogenesis or produce detectable defects in meiotic processes. Microarray analysis of gene expression showed that the reduced linker histone levels did not affect global gene expression, but it did cause changes in expression of specific genes. Our results indicate that a partial reduction in linker histone-nucleosome core particle stoichiometry is tolerated in developing male germ cells.

Animals↗
PubMed2004

A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse.

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.

Anaphase↗
PubMed2004

Mouse strains with an active H2-Ea meiotic recombination hot spot exhibit increased levels of H2-Ea-specific DNA breaks in testicular germ cells.

We devised a sensitive method for the site-specific detection of rare meiotic DNA strand breaks in germ cell-enriched testicular cell populations from mice that possess or lack an active recombination hot spot at the H2-Ea gene. Using germ cells from adult animals, we found an excellent correlation between the frequency of DNA breaks in the 418-bp H2-Ea hot spot and crossover activity. The temporal appearance of DNA breaks was also studied in 7- to 18-day-old mice with an active hot spot during the first waves of spermatogenesis. The number of DNA breaks detected rose as leptotene and zygotene spermatocytes populate the testis with a peak at day 14 postpartum, when leptotene, zygotene, and early pachytene spermatocytes are the most common meiotic prophase I cell types. The number of DNA breaks drops precipitously 1 day later, when middle to late pachytene spermatocytes become the dominant subtype. The recombination-related breaks in the hot spot likely reflect SPO11-induced double-strand breaks and/or recombination intermediates containing free 3' hydroxyl groups.

Animals↗
PubMed2003

BRCA2 deficiency in mice leads to meiotic impairment and infertility.

The role of Brca2 in gametogenesis has been obscure because of embryonic lethality of the knockout mice. We generated Brca2-null mice carrying a human BAC with the BRCA2 gene. This construct rescues embryonic lethality and the mice develop normally. However, there is poor expression of the transgene in the gonads and the mice are infertile, allowing examination of the function of BRCA2 in gametogenesis. BRCA2-deficient spermatocytes fail to progress beyond the early prophase I stage of meiosis. Observations on localization of recombination-related and spermatogenic-related proteins suggest that the spermatocytes undergo early steps of recombination (DNA double strand break formation), but fail to complete recombination or initiate spermiogenic development. In contrast to the early meiotic prophase arrest of spermatocytes, some mutant oocytes can progress through meiotic prophase I, albeit with a high frequency of nuclear abnormalities, and can be fertilized and produce embryos. Nonetheless, there is marked depletion of germ cells in adult females. These studies provide evidence for key roles of the BRCA2 protein in mammalian gametogenesis and meiotic success.

Animals↗
PubMed2003

Meiosis in male PL/J mice: a genetic model for gametic aneuploidy.

Sperm from mice of the PL/J strain have a high frequency of sperm-head morphology abnormalities. Fluorescence in situ hybridization (FISH) methods revealed that PL/J sperm are also characterized by a high frequency of aneuploidy. The traits of abnormal sperm head morphology and aneuploidy are associated with numerous meiotic abnormalities. Spermatocytes of PL/J mice exhibit chromosome asynapsis during meiotic prophase as well as reduced crossing over, revealed by analysis of both MLH1 foci in pachytene spermatocytes and chiasmata seen at the first meiotic metaphase. During the first meiotic division, roughly one-third of the PL/J spermatocytes exhibit aberrant spindle morphology, with abnormalities including monopolar spindles, split spindle poles, and incomplete spindle formation and centrosomal abnormalities. F1 progeny of a cross between PL/J and C57BL/6J did not exhibit a high frequency of either sperm aneuploidy or sperm head morphology aberrations, as would be expected if the PL/J traits were dominant. Among progeny of a backcross of F1 mice to PL/J, none of 16 males assessed exhibited elevated frequencies of sperm head morphology abnormalities. Four of the individuals exhibited elevated sperm aneuploidy, but not at the levels of the PL/J parents. Thus, it is likely that the aberrant PL/J traits are due to several genes and/or modifiers affecting the generation of both sperm aneuploidy and abnormal sperm head morphology.

Aneuploidy↗
PubMed2002

Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: insights into regulation of spermatogenic progress.

The MLH1 protein is required for normal meiosis in mice and its absence leads to failure in maintenance of pairing between bivalent chromosomes, abnormal meiotic division, and ensuing sterility in both sexes. In this study, we investigated whether failure to develop foci of MLH1 protein on chromosomes in prophase would lead to elimination of prophase spermatocytes, and, if not, whether univalent chromosomes could align normally on the meiotic spindle and whether metaphase spermatocytes would be delayed and/or eliminated. In spite of the absence of MLH1 foci, no apoptosis of spermatocytes in prophase was detected. In fact, chromosomes of pachytene spermatocytes from Mlh1(-/-) mice were competent to condense metaphase chromosomes, both in vivo and in vitro. Most condensed chromosomes were univalents with spatially distinct FISH signals. Typical metaphase events, such as synaptonemal complex breakdown and the phosphorylation of Ser10 on histone H3, occurred in Mlh1(-/-) spermatocytes, suggesting that there is no inhibition of onset of meiotic metaphase in the face of massive chromosomal abnormalities. However, the condensed univalent chromosomes did not align correctly onto the spindle apparatus in the majority of Mlh1(-/-) spermatocytes. Most meiotic metaphase spermatocytes were characterized with bipolar spindles, but chromosomes radiated away from the microtubule-organizing centers in a prometaphase-like pattern rather than achieving a bipolar orientation. Apoptosis was not observed until after the onset of meiotic metaphase. Thus, spermatocytes are not eliminated in direct response to the initial meiotic defect, but are eliminated later. Taken together, these observations suggest that a spindle assembly checkpoint, rather than a recombination or chiasmata checkpoint, may be activated in response to meiotic errors, thereby ensuring elimination of chromosomally abnormal gamete precursors.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The mouse meiotic mutation mei1 disrupts chromosome synapsis with sexually dimorphic consequences for meiotic progression.

mei1 (meiosis defective 1) is the first meiotic mutation in mice derived by phenotype-driven mutagenesis. It was isolated by using a novel technology in which embryonic stem (ES) cells were chemically mutagenized and used to generate families of mice that were screened for infertility. We report here that mei1/mei1 spermatocytes arrest at the zygotene stage of meiosis I, exhibiting failure of homologous chromosomes to properly synapse. Notably, RAD51 failed to associate with meiotic chromosomes in mutant spermatocytes, despite evidence for the presence of chromosomal breaks. Transcription of genes that are markers for the leptotene and zygotene stages, but not genes that are markers for the pachytene stage, was observed. mei1/mei1 females are sterile, and their oocytes also show severe synapsis defects. Nevertheless, unlike arrested spermatocytes, a small number of mutant oocytes proved capable of progressing to metaphase I and attempting the first meiotic division. However, their chromosomes were unpaired and were not organized properly at the metaphase plate or along the spindle fibers during segregation. mei1 was genetically mapped to chromosome (Chr) 15 in an interval that is syntenic to human Chr 22q13. This region, which has been completely sequenced, contains no known homologs of genes specifically required for meiosis in model organisms. Thus, mei1 may be a novel meiotic gene.

Animals↗