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1.
Article in English | MEDLINE | ID: mdl-39023827

ABSTRACT

PURPOSE: Ovarian aging is closely related to a decrease in follicular reserve and oocyte quality. The precise molecular mechanisms underlying these reductions have yet to be fully elucidated. Herein, we examine spatiotemporal distribution of key proteins responsible for DNA double-strand break (DSB) repair in ovaries from early to older ages. Functional studies have shown that the γH2AX, RAD51, BRCA1, and RPA70 proteins play indispensable roles in HR-based repair pathway, while the KU80 and XRCC4 proteins are essential for successfully operating cNHEJ pathway. METHODS: Female Balb/C mice were divided into five groups as follows: Prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). The expression of DSB repair proteins, cellular senescence (ß-GAL) and apoptosis (cCASP3) markers was evaluated in the ovaries using immunohistochemistry. RESULT: ß-GAL and cCASP3 levels progressively increased from prepuberty to aged groups (P < 0.05). Notably, γH2AX levels varied in preantral and antral follicles among the groups (P < 0.05). In aged groups, RAD51, BRCA1, KU80, and XRCC4 levels increased (P < 0.05), while RPA70 levels decreased (P < 0.05) compared to the other groups. CONCLUSIONS: The observed alterations were primarily attributed to altered expression in oocytes and granulosa cells of the follicles and other ovarian cells. As a result, the findings indicate that these DSB repair proteins may play a role in the repair processes and even other related cellular events in ovarian cells from early to older ages.

2.
J Assist Reprod Genet ; 40(4): 929-941, 2023 Apr.
Article in English | MEDLINE | ID: mdl-36823316

ABSTRACT

Expression of the embryonic poly(A)-binding protein (EPAB) in frog, mouse, and human oocytes and early-stage embryos is maintained at high levels until embryonic genome activation (EGA) after which a significant decrease occurs in EPAB levels. Studies on the vertebrate oocytes and early embryos revealed that EPAB plays key roles in the translational regulation, stabilization, and protection of maternal mRNAs during oocyte maturation and early embryogenesis. However, it remains elusive whether EPAB interacts with other cellular proteins and undergoes phosphorylation to perform these roles. For this purpose, we identified a group of Epab-interacting proteins and its phosphorylation status in mouse germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, and in 1-cell, 2-cell, and 4-cell preimplantation embryos. In the oocytes and early preimplantation embryos, Epab-interacting proteins were found to play roles in the translation and transcription processes, intracellular signaling and transport, maintenance of structural integrity, metabolism, posttranslational modifications, and chromatin remodeling. Moreover, we discovered that Epab undergoes phosphorylation on the serine, threonine, and tyrosine residues, which are localized in the RNA recognition motifs 2, 3, and 4 or C-terminal. Conclusively, these findings suggest that Epab not only functions in the translational control of maternal mRNAs through binding to their poly(A) tails but also participates in various cellular events through interacting with certain group proteins. Most likely, Epab undergoes a dynamic phosphorylation during the oocyte maturation and the early embryo development to carry out these functions.


Subject(s)
Serine , Tyrosine , Humans , Animals , Mice , Phosphorylation , Tyrosine/metabolism , Serine/metabolism , Threonine/metabolism , Oocytes , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism
3.
Histochem Cell Biol ; 159(2): 127-147, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36241856

ABSTRACT

DNA double-strand breaks (DSBs) are commonly appearing deleterious DNA damages, which progressively increase in male germ cells during biological aging. There are two main pathways for repairing DSBs: homologous recombination (HR) and classical nonhomologous end joining (cNHEJ). Knockout and functional studies revealed that, while RAD51 and RPA70 proteins are indispensable for HR-based repair, KU80 and XRCC4 are the key proteins in cNHEJ repair. As is known, γH2AX contributes to these pathways through recruiting repair-related proteins to damaged site. The underlying reasons of increased DSBs in male germ cells during aging are not fully addressed yet. In this study, we aimed to analyze the spatiotemporal expression of the Rad51, Rpa70, Ku80, and Xrcc4 genes in the postnatal mouse testes, classified into young, prepubertal, pubertal, postpubertal, and aged groups according to their reproductive features and histological structures. We found that expression of these genes significantly decreased in the aged group compared with the other groups (P < 0.05). γH2AX staining showed that DSB levels in the germ cells from spermatogonia to elongated spermatids as well as in the Sertoli cells remarkably increased in the aged group (P < 0.05). The RAD51, RPA70, KU80, and XRCC4 protein levels exhibited predominant changes in the germ and Sertoli cells among groups (P < 0.05). These findings suggest that altered expression of the Rad51, Rpa70, Ku80, and Xrcc4 genes in the germ and Sertoli cells may be associated with increasing DSBs during biological aging, which might result in fertility loss.


Subject(s)
DNA Repair , Rad51 Recombinase , Male , Mice , Animals , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Mice, Knockout , DNA Repair/genetics , Homologous Recombination/genetics , Germ Cells/metabolism
4.
DNA Repair (Amst) ; 118: 103386, 2022 10.
Article in English | MEDLINE | ID: mdl-35963140

ABSTRACT

Spermatogenesis is a complex developmental process. During this process, male germ cells from spermatogonia to sperm cells encounter a number of DNA damages. The most severe form of these damages is double-strand breaks (DSBs) deriving from exogenous and endogenous genotoxic insults. DSBs must be correctly repaired in a short time to maintain genomic integrity in the male germ cells. For this purpose, there are four pathways working in repair of DSBs: homologous recombination (HR), classical non-homologous end joining (cNHEJ), alternative end joining (aEJ), and single strand annealing (SSA). While the HR pathway repairs DSBs with a homology-based and error-free manner, the cNHEJ, aEJ, and SSA pathways join free ends in a sequence-independent mechanism. Possible impairments in these DSB repair mechanisms can lead to cell cycle arrest, abnormal meiotic recombination, and ultimately male infertility. In this review, we comprehensively introduce DSB repair pathways being used by male germ cells during spermatogenesis. Also, potential relationship between dysfunction in these pathways and male infertility development are discussed in the light of existing studies.


Subject(s)
DNA Breaks, Double-Stranded , Infertility, Male , DNA , DNA End-Joining Repair , DNA Repair , Germ Cells , Humans , Infertility, Male/genetics , Male , Semen , Spermatogenesis
5.
Histochem Cell Biol ; 158(1): 79-95, 2022 Jul.
Article in English | MEDLINE | ID: mdl-35445296

ABSTRACT

Histone methylation is one of the main epigenetic mechanisms by which methyl groups are dynamically added to the lysine and arginine residues of histone tails in nucleosomes. This process is catalyzed by specific histone methyltransferase enzymes. Methylation of these residues promotes gene expression regulation through chromatin remodeling. Functional analysis and knockout studies have revealed that the histone lysine methyltransferases SETD1B, SETDB1, SETD2, and CFP1 play key roles in establishing the methylation marks required for proper oocyte maturation and follicle development. As oocyte quality and follicle numbers progressively decrease with advancing maternal age, investigating their expression patterns in the ovaries at different reproductive periods may elucidate the fertility loss occurring during ovarian aging. The aim of our study was to determine the spatiotemporal distributions and relative expression levels of the Setd1b, Setdb1, Setd2, and Cxxc1 (encoding the CFP1 protein) genes in the postnatal mouse ovaries from prepuberty to late aged periods. For this purpose, five groups based on their reproductive periods and histological structures were created: prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). We found that Setd1b, Setdb1, Setd2, and Cxxc1 mRNA levels showed significant changes among postnatal ovary groups (P < 0.05). Furthermore, SETD1B, SETDB1, SETD2, and CFP1 proteins exhibited different subcellular localizations in the ovarian cells, including oocytes, granulosa cells, stromal and germinal epithelial cells. In general, their levels in the follicles, oocytes, and granulosa cells as well as in the germinal epithelial and stromal cells significantly decreased in the aged groups when compared the other groups (P < 0.05). These decreases were concordant with the reduced numbers of the follicles at different stages and the luteal structures in the aged groups (P < 0.05). In conclusion, these findings suggest that altered expression of the histone methyltransferase genes in the ovarian cells may be associated with female fertility loss in advancing maternal age.


Subject(s)
Histone-Lysine N-Methyltransferase , Histones , Ovary , Trans-Activators , Animals , Female , Granulosa Cells/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Lysine/metabolism , Mice , Oocytes/metabolism , Ovary/metabolism , Sexual Maturation , Trans-Activators/metabolism
6.
Histochem Cell Biol ; 157(1): 7-25, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34599660

ABSTRACT

Histone methylation is a key epigenetic mechanism and plays a major role in regulating gene expression during oocyte maturation and early embryogenesis. This mechanism can be briefly defined as the process by which methyl groups are transferred to lysine and arginine residues of histone tails extending from nucleosomes. While methylation of the lysine residues is catalyzed by histone lysine methyltransferases (KMTs), protein arginine methyltransferases (PRMTs) add methyl groups to the arginine residues. When necessary, the added methyl groups can be reversibly removed by histone demethylases (HDMs) by a process called histone demethylation. The spatiotemporal regulation of methylation and demethylation in histones contributes to modulating the expression of genes required for proper oocyte maturation and early embryonic development. In this review, we comprehensively evaluate and discuss the functional importance of dynamic histone methylation in mammalian oocytes and early embryos, regulated by KMTs, PRMTs, and HDMs.


Subject(s)
Histones , Protein Processing, Post-Translational , Animals , Histones/metabolism , Lysine/metabolism , Methylation , Oocytes/metabolism
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