Mad2 is dispensable for accurate chromosome segregation but becomes essential when oocytes are subjected to environmental stress.

Accurate chromosome segregation, monitored by the spindle assembly checkpoint (SAC), is crucial for the production of euploid cells. Previous in vitro studies by us and others showed that Mad2, a core member of the SAC, performs a checkpoint function in oocyte meiosis. Here, through an oocyte-specific knockout approach in mouse, we reconfirmed that Mad2-deficient oocytes exhibit an accelerated metaphase-to-anaphase transition caused by premature degradation of securin and cyclin B1 and subsequent activation of separase in meiosis I. However, it was surprising that the knockout mice were completely fertile and the resulting oocytes were euploid. In the absence of Mad2, other SAC proteins, including BubR1, Bub3 and Mad1, were normally recruited to the kinetochores, which likely explains the balanced chromosome separation. Further studies showed that the chromosome separation in Mad2-null oocytes was particularly sensitive to environmental changes and, when matured in vitro, showed chromosome misalignment, lagging chromosomes, and aneuploidy with premature separation of sister chromatids, which was exacerbated at a lower temperature. We reveal for the first time that Mad2 is dispensable for proper chromosome segregation but acts to mitigate environmental stress in meiotic oocytes.

PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development.

Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here, we show that protein phosphatase 4 catalytic subunit (PPP4C) deletion causes severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibited DNA lesions that inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of the essential HR factor RAD51. On the other hand, when inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods, which couple the checkpoint to HR.

The Impact of Centrosome Pathologies on Ovarian Cancer Development and Progression with a Focus on Centrosomes as Therapeutic Target.

The impact of centrosome abnormalities on cancer cell proliferation has been recognized as early as 1914 (Boveri, Zur Frage der Entstehung maligner Tumoren. Jena: G. Fisher, 1914), but vigorous research on molecular levels has only recently started when it became fully apparent that centrosomes can be targeted for new cancer therapies. While best known for their microtubule-organizing capabilities as MTOC (microtubule organizing center) in interphase and mitosis, centrosomes are now further well known for a variety of different functions, some of which are related to microtubule organization and consequential activities such as cell division, migration, maintenance of cell shape, and vesicle transport powered by motor proteins, while other functions include essential roles in cell cycle regulation, metabolic activities, signal transduction, proteolytic activity, and several others that are now heavily being investigated for their role in diseases and disorders (reviewed in Schatten and Sun, Histochem Cell Biol 150:303-325, 2018; Schatten, Adv Anat Embryol Cell Biol 235:43-50, 2022a; Schatten, Adv Anat Embryol Cell Biol 235:17-35, 2022b).Cancer cell centrosomes differ from centrosomes in noncancer cells in displaying specific abnormalities that include phosphorylation abnormalities, overexpression of specific centrosomal proteins, abnormalities in centriole and centrosome duplication, formation of multipolar spindles that play a role in aneuploidy and genomic instability, and several others that are highlighted in the present review on ovarian cancer. Ovarian cancer cell centrosomes, like those in other cancers, display complex abnormalities that in part are based on the heterogeneity of cells in the cancer tissues resulting from different etiologies of individual cancer cells that will be discussed in more detail in this chapter.Because of the critical role of centrosomes in cancer cell proliferation, several lines of research are being pursued to target centrosomes for therapeutic intervention to inhibit abnormal cancer cell proliferation and control tumor progression. Specific centrosome abnormalities observed in ovarian cancer will be addressed in this chapter with a focus on targeting such aberrations for ovarian cancer-specific therapies.

The Impact of Mitochondria in Ovarian Cancer Cell Metabolism, Proliferation, and Metastasis.

Mitochondrial dysfunctions are significantly implicated in cancer initiation, progression, and metastasis, which have been shown for several cancers including ovarian cancer.An increase in mitochondrial dysfunction is also associated with drug resistance along with cancer progression, which in part is related to its specific microenvironment that is characterized by ascites, low glucose levels, and hypoxia that causes ovarian cancer cells to switch to mitochondrial respiration to enable their survival. Peritoneal ascitic fluid accumulation is a specific feature of ovarian cancer, and it is a major cause of its metastatic spread that also presents challenges for effective treatment. Among the treatment difficulties for ovarian cancer is the mutation rate and frequency of mtDNA in ovarian cancer tissue that can affect the efficiency of chemotherapeutic drugs. The varied and multiple mutations of different types enable metabolic reprogramming, cancer cell proliferation, and drug resistance.New specific information on mechanisms underlying several of the mitochondrial dysfunctions has led to proposing various mitochondrial determinants as targets for ovarian cancer therapy, which include targeting specific mitochondrial proteins and phosphoproteins as well as reactive oxygen species (ROS) that accumulate abnormally in cancer cells. Because of the genetically and histologically heterogeneous nature of the disease, combination therapy approaches will be necessary to combat the disease and achieve progress in effective treatment of ovarian cancer. This chapter will address (1) mitochondrial vulnerabilities underlying dysfunction and disease; (2) mitochondrial dysfunction in ovarian cancer; (3) present treatment difficulties for ovarian cancer and new potential treatment strategies to target ovarian cancer mitochondrial metabolism; and (4) biobehavioral factors influencing ovarian cancer development.

SRSF2 is required for mRNA splicing during spermatogenesis.

BACKGROUND: RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. RESULTS: Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. CONCLUSIONS: Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

Maternal SMC2 is essential for embryonic development via participating chromosome condensation in mice.

During the oocyte growth, maturation and zygote development, chromatin structure keeps changing to regulate different nuclear activities. Here, we reported the role of SMC2, a core component of condensin complex, in oocyte and embryo development. Oocyte-specific conditional knockout of SMC2 caused female infertility. In the absence of SMC2, oocyte meiotic maturation and ovulation occurred normally, but chromosome condensation showed defects and DNA damages were accumulated in oocytes. The pronuclei were abnormally organized and micronuclei were frequently observed in fertilized eggs, their activity was impaired, and embryo development was arrested at the one-cell stage, suggesting that maternal SMC2 is essential for embryonic development.

Single-cell RNA sequencing reveals species-specific time spans of cell cycle transitions in early oogenesis.

Oogenesis is a highly regulated process and its basic cellular events are evolutionarily conserved. However, the time spans of oogenesis differ substantially among species. To explore these interspecies differences in oogenesis, we performed single-cell RNA-sequencing on mouse and monkey female germ cells and downloaded the single-cell RNA-sequencing data of human female germ cells. The cell cycle analyses indicate that the period and extent of cell cycle transitions are significantly different between the species. Moreover, hierarchical clustering of critical cell cycle genes and the interacting network of cell cycle regulators also exhibit distinguished patterns across species. We propose that differences in the regulation of cell cycle transitions may underlie female germ cell developmental allochrony between species. A better understanding of the cell cycle transition machinery will provide new insights into the interspecies differences in female germ cell developmental time spans.

Mitochondrial E3 ubiquitin ligase MARCH5 is required for mouse oocyte meiotic maturation†.

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore-microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.

Kinetochore scaffold 1 regulates SAC function during mouse oocyte meiotic maturation.

Precise regulation of chromosome separation through spindle assembly checkpoint (SAC) during oocyte meiosis is critical for mammalian reproduction. The kinetochore plays an important role in the regulation of SAC through sensing microtubule tension imbalance or missing microtubule connections. Here, we report that kinetochore scaffold 1 (KNL1, also known as CASC5), an outer kinetochore protein, plays a critical role in the SAC function of mouse oocytes. KNL1 localized at kinetochores from GVBD to the MII stage, and microinjection of KNL1-siRNA caused accelerated metaphase-anaphase transition and premature first meiosis completion, producing aneuploid eggs. The SAC was prematurely silenced in the presence of unstable kinetochore-microtubule attachments and misaligned chromosomes in KNL1-depleted oocytes. Additionally, KNL1 and MPS1 had a synergistic effect on the activation and maintenance of SAC. Taken together, our results suggest that KNL1, as a kinetochore platform protein, stabilizes SAC to ensure timely anaphase entry and accurate chromosome segregation during oocyte meiotic maturation.

Septin 4 controls CCNB1 stabilization via APC/CCDC20 during meiotic G2/M transition in mouse oocytes.

In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/CCDC20 . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.

Miro1 regulates mitochondrial homeostasis and meiotic resumption of mouse oocyte.

Miro1, a mitochondrial Rho GTPase1, is a kind of mitochondrial outer membrane protein involved in the regulation of mitochondrial anterograde transport and its subcellular distribution. Mitochondria influence reproductive processes of mammals in some aspects. Mitochondria are important for oocyte maturation, fertilization and embryonic development. The purpose of this study was to evaluate whether Miro1 regulates mouse oocyte maturation by altering mitochondrial homeostasis. We showed that Miro1 was expressed in mouse oocyte at different maturation stages. Miro1 mainly distributed in the cytoplasm and around the spindle during oocyte maturation. Small interference RNA-mediated Miro1 depletion caused significantly abnormal distribution of mitochondria and endoplasmic reticulum as well as mitochondrial dysfunction, resulting in severely impaired germinal vesicle breakdown (GVBD) of mouse oocytes. For those oocytes which went through GVBD in the Miro1-depleted group, part of them were inhibited in meiotic prophase I stage with abnormal chromosome arrangement and scattered spindle length. Our results suggest that Miro1 is essential for maintaining the maturation potential of mouse oocyte.

CENP-T regulates both the G2/M transition and anaphase entry by acting through CDH1 in meiotic oocytes.

Oocyte meiotic maturation failure is one of the major causes for female infertility. Meiotic resumption (the G2/M transition) and progression through metaphase I (MI) are two critical stages of oocyte meiotic maturation. Here, we report that centromere protein T (CENP-T), an internal kinetochore protein, plays a critical role in meiotic resumption of mouse oocytes. Depletion of CENP-T by siRNA injection increased the CDH1 (also known as FZR1) level, resulting in increased activity of the anaphase-promoting complex (APC)-CDH1 complex, and further leading to decreased levels of the cyclin protein CCNB1, attenuated maturation-promoting factor (MPF) activity, and finally severely compromised meiotic resumption. The impaired meiotic resumption caused by CENP-T depletion could be rescued by overexpression of exogenous CCNB1 or knockdown of endogenous CDH1. Overexpression of exogenous CENP-T resulted in decreased CDH1 levels, which accelerated the progression of G2/M transition, and accelerated meiotic cell cycle progression after germinal vesicle breakdown (GVBD). Unexpectedly, spindle organization after GVBD was not affected by the overexpression, but the distribution of chromosomes was affected. Our findings reveal a novel role for CENP-T in regulating meiotic progression by acting through CDH1.

Cell and Molecular Biology of Centrosome Structure and Function.

The centrosome field has seen enormous progress during the past few decades which spans the large areas of cell biology with new information on cell cycle controls and cellular health; immunology with centrosomes being essential for the formation of the immunological synapse; neurobiology with new insights into centrosome dysfunctions leading to disorders and disease; stem cell biology with fate-determining distribution of centrosomal material during asymmetric cell division; cancer biology with huge insights into the role of centrosomes in disease initiation, progression, and manifestation; reproductive biology with essential centrosome functions in oocytes, during fertilization and embryo development in which centrosome dysfunctions can be related back to abnormal centrosomal material in the meiotic spindle of oocytes; and several others that will be highlighted in the specific chapters of this book.

The Centrosome Cycle within the Cell Cycle.

The synchronized distribution of centrosomal and genetic materials to the dividing daughter cells is critically important and depends on precisely orchestrated processes on structural and molecular levels. Structural and functional relationships between the nucleus and centrosomes facilitate cellular communication and coordination of cell cycle control and progression which becomes especially important during the transition from interphase to mitosis when synchrony between centrosomes and nuclear events is critical.

Centrosome Dysfunctions in Cancer.

As major accomplishments and breakthroughs in centrosome research had been achieved by Theodor Boveri in reproductive cells with the invertebrate sea urchin being an ideal model system for such studies on fertilization, cell division, and embryo development, these studies also gave rise to Boveri's brilliant concept regarding cancer cells. He discovered that eggs fertilized with two sperm resulted in tripolar mitosis and abnormal cell division, similar to cells observed in cancer tissue.

Centrosome as Center for Proteolytic Activity and Dysfunctions Associated with Pathogenesis of Human Disease.

Among the multiple and intriguing roles of centrosomes in cellular functions is the ubiquitin-proteasome-mediated protein degradation. It has been shown that proteasomes are concentrated at the mammalian centrosome which led to further studies to view the centrosome as a proteolytic center (Wojcik et al. 1996; Wigley et al. 1999; reviewed in Badano et al. 2005). Proteasomal components that are concentrated around the centrosome include ubiquitin, the 20S and 19S subunits of the proteasome, as well as the E3 enzyme parkin. These proteasomal components colocalize with the centrosomal marker γ-tubulin and co-purify with γ-tubulin in the centrosomal fractions after sucrose-gradient ultracentrifugation (Wigley et al. 1999). The localization, accumulation, and concentration of proteasomal components around centrosomes appear to be microtubule independent which has been shown experimentally by inhibiting microtubule functions. When intracellular levels of misfolded proteins were experimentally increased by either proteasome inhibition with drugs such as lactacystin, or by overexpression of misfolded mutant proteins, the centrosome-associated proteasome network became expanded and proteolytic components were recruited from the cytosol without involvement of microtubules. These studies revealed a critical role of centrosomes in the organization and subcellular localization of proteasomes (Wigley et al. 1999; Fabunmi et al. 2000).

Virus Exploitation (Hijacking) of Centrosomes.

One of the most interesting aspects of host cell-viral interactions is how the pathogen exploits the host cell cytoskeleton and centrosomes for survival in the host cell.

Centrosomes in Reproduction.

Centrosome functions are vitally important for all aspects of reproduction with essential functions during meiosis, fertilization, cell division, centrosome remodeling during cellular polarization for tissue formation, and all stages of subsequent embryo development. Any defects in centrosome organization and dynamics can result in meiotic spindle formation errors, meiotic division errors, infertility, subfertility, arrested or failed development, and predisposition to various diseases including cancer. These aspects of reproduction will be addressed in more detail in the following sections.

Transitions from Centrosomal to Non-centrosomal Microtubule Organization During Cellular Polarization.

Cellular polarization involves significant remodeling and decentralization of the nucleus-associated centrosome to focal points at the apical and basolateral surfaces which is associated with major remodeling of the microtubule system in which individual microtubules become nucleated and organized from the polarizing cell surfaces, as studied in polarizing epithelial cells (reviewed in Müsch 2004; Muroyama and Lechler 2017). These changes are associated with cellular asymmetry in preparation for cellular differentiation of previously non-committed cells. During this process, the previously nucleus-associated centrosome becomes deconstructed into specific centrosomal components which are now referred to as "non-centrosomal." At the present time we still only have limited information about this process and to understanding the mechanisms underlying the centrosome decentralization process. Gaining detailed insights is further complicated by the fact that there is considerable diversity in the molecular mechanisms of centrosome and microtubule reorganization.

External and Environmental Effects on Centrosomes.

The effects of ionizing radiation on centrosomes have been well documented and reviewed by Saladino et al. (2012) and are only briefly addressed here. These results showed that exposure of tumor cells to ionizing radiation causes centrosome overduplication and the formation of multipolar mitotic spindles, resulting in nuclear fragmentation and subsequent cell death (Sato et al. 2000). By using a variety of cell lines derived from different types of human solid tumors, it was shown that exposure to 10 Gy γ-radiation resulted in a substantial increase in cells containing an abnormally high number of aberrant centrosomes that formed multipolar spindles, resulting in imbalanced chromosome separation followed by mitotic cell death and formation of multi- or micronucleated cells.