The Inactivation of Arabidopsis UBC22 Results in Abnormal Chromosome Segregation in Female Meiosis, but Not in Male Meiosis

Protein ubiquitination is important for the regulation of meiosis in eukaryotes, including plants. However, little is known about the involvement of E2 ubiquitin-conjugating enzymes in plant meiosis. Arabidopsis UBC22 is a unique E2 enzyme, able to catalyze the formation of ubiquitin dimers through lysine 11 (K11). Previous work has shown that ubc22 mutants are defective in megasporogenesis, with most ovules having no or abnormally functioning megaspores; furthermore, some mutant plants show distinct phenotypes in vegetative growth. In this study, we showed that chromosome segregation and callose deposition were abnormal in mutant female meiosis while male meiosis was not affected. The meiotic recombinase DMC1, required for homologous chromosome recombination, showed a dispersed distribution in mutant female meiocytes compared to the presence of strong foci in WT female meiocytes. Based on an analysis of F1 plants produced from crosses using a mutant as the female parent, about 24% of female mutant gametes had an abnormal content of DNA, resulting in frequent aneuploids among the mutant plants. These results show that UBC22 is critical for normal chromosome segregation in female meiosis but not for male meiosis, and they provide important leads for studying the role of UBC22 and K11-linked ubiquitination.

iPLA2-VIA is required for healthy aging of neurons, muscle, and the female germline in Drosophila melanogaster

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is critical for mitochondrial integrity in the Drosophila female germline, which may provide a novel context to investigate its functions with parallels to PLAN.

Altered germline cyst formation and oogenesis in Tex14 mutant mice

ABSTRACT During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.

iPLA2-VIA is required for healthy aging in neurons, muscle, and female germline in Drosophila melanogaster

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. We have generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). Our mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in our mutants is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, we find that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is important for germline mitochondrial integrity in Drosophila, which may be relevant for understanding how PLAN develops.

A Phosphotyrosine Switch in Estrogen Receptor β Is Required for Mouse Ovarian Function

The two homologous estrogen receptors ERα and ERβ exert distinct effects on their cognate tissues. Previous work from our laboratory identified an ERβ-specific phosphotyrosine residue that regulates ERβ transcriptional activity and antitumor function in breast cancer cells. To determine the physiological role of the ERβ phosphotyrosine residue in normal tissue development and function, we investigated a mutant mouse model (Y55F) whereby this particular tyrosine residue in endogenous mouse ERβ is mutated to phenylalanine. While grossly indistinguishable from their wild-type littermates, mutant female mice displayed reduced fertility, decreased ovarian follicular cell proliferation, and lower progesterone levels. Moreover, mutant ERβ from female mice during superovulation is defective in activating promoters of its target genes in ovarian tissues. Thus, our findings provide compelling genetic and molecular evidence for a role of isotype-specific ERβ phosphorylation in mouse ovarian development and function.

Cerebellar Morphology and Behavioral Profiles in Mice Lacking Heparan Sulfate Ndst Gene Function

Disruption of the Heparan sulfate (HS)-biosynthetic gene N-acetylglucosamine N-Deacetylase/N-sulfotransferase 1 (Ndst1) during nervous system development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. However, the role of Ndst function in adult brain physiology is less explored. Therefore, we generated mice bearing a Purkinje-cell-specific deletion in Ndst1 gene function by using Cre/loxP technology under the control of the Purkinje cell protein 2 (Pcp2/L7) promotor, which results in HS undersulfation. We observed that mutant mice did not show overt changes in the density or organization of Purkinje cells in the adult cerebellum, and behavioral tests also demonstrated normal cerebellar function. This suggested that postnatal Purkinje cell development and homeostasis are independent of Ndst1 function, or that impaired HS sulfation upon deletion of Ndst1 function may be compensated for by other Purkinje cell-expressed Ndst isoforms. To test the latter possibility, we additionally deleted the second Purkinje-cell expressed Ndst family member, Ndst2. This selectively abolished reproductive capacity of compound mutant female, but not male, mice, suggesting that ovulation, gestation, or female reproductive behavior specifically depends on Ndst-dependent HS sulfation in cells types that express Cre under Pcp2/L7 promotor control.

Loss of Ikbkap/Elp1 in mouse oocytes causes spindle disorganization, developmental defects in preimplantation embryos and impaired female fertility

Elongator complexes are well known to be involved in a wide variety of cellular processes; however, their functions in mammalian oocytes have not been characterized. Here, we demonstrated in mice that specific deletion of one of the core subunits, Ikbkap/Elp1, in oocytes resulted in spindle defects and chromosome disorganization without affecting folliculogenesis. In accordance with these findings, we observed that Ikbkap mutant female mice are subfertile. Further analyses uncovered that kinetochore–microtubule attachments are severely compromised in Ikbkap-deficient oocytes. Moreover, we revealed that Ikbkap modulates the acetylation status of α-tubulin in oocytes, which may at least in part mediate the meiotic phenotypes described above by affecting microtubule dynamics and kinetochore function. Finally, we showed that embryos derived from Ikbkap-deficient oocytes exhibit an increased frequency of aneuploidy, digyny, progressive delays in preimplantation development, and severe degeneration before reaching the blastocyst stage. In summary, we identify Ikbkap as an important player in regulating oocyte meiosis by modulating tubulin acetylation for chromosome/spindle organization.