Research

General introduction

In our laboratory, we are endeavoring to understand the mechanisms of germ cell development, particularly in oogenesis, in mammals. Among the more than 200 types of cells in the body, the germ cell lineage is the only lineage capable of transmitting genetic and epigenetic information to the next generation, thereby producing new individuals. Unlike somatic cells, which differentiate and lose plasticity over time and eventually die with the individual, germ cells can revert to their initial status, namely totipotent, with the fertilized egg, as time passes (Figure 1). Not only that, but they also create genetic diversity in individuals by mixing the genomes inherited from the parents. To accomplish these critical missions for perpetuating species, the germ cell lineage undergoes various specific processes. Therefore, understanding each of them is the goal in the field of reproductive biology.

We aim to understand the process by which the germ cell lineage, especially the oocyte lineage, is established during development and maintained in the body, utilizing cutting-edge techniques in cell culture, genome editing, imaging, and omics analysis. Building on the insights gained, we are also developing a culture system to reconstitute germ cell differentiation processes using pluripotent stem cells, thereby enabling the production of fertile eggs and sperm in culture, a process known as 'in vitro gametogenesis.' These research findings not only deepen our understanding of reproductive cell biology but also contribute to identifying and addressing the causes of infertility, improving breeding practices in livestock, and even facilitating the reproduction of endangered species. We invite you to explore each research topic further for additional details below. We hope you will take a closer look at each research topic for further details below.

Figure 1 Summary of germ cell development: A fertilized egg undergoes cleavage and then becomes a blastocyst. Subsequently, primordial germ cells (PGCs) differentiate from the pluripotent cell population in the embryo after implantation. Nascent primordial germ cells are located in an extraembryonic region, but as development progresses, they migrate to the gonads (future ovaries or testes). In the gonads, they receive sex-specific signals from surrounding somatic cells and differentiate into oocytes in the ovaries and sperm in the testes. This differentiation process is largely conserved in mammals, although the types of signals and the time required for development vary.

Research 01
Why are gametes far different between sexes?

The significant differences in gametes between sexes, called anisogamy, arise from the distinct reproductive strategies and roles each sex plays in sexual reproduction. In most species, including mammals, males typically produce smaller, motile gametes called sperm, while females produce larger, non-motile gametes called eggs or ova. These differences are primarily driven by evolutionary pressures related to fertilization and subsequent embryo development. In mammals, the morphological differences in gametes become prominent after meiotic transition: early oocytes and spermatocytes exhibit high similarity, but their morphology undergoes rapid changes in subsequent processes (Figure 2). Although it is well known that somatic cells of the gonads play critical roles in this morphological transformation, germ cell-intrinsic mechanisms make significant contributions. Based on algae models such as Chlamydomonas, Euglena, and Volvox, heteromorphic gametes appear early in the process of multicellularization, and genes involved in sex determination are expressed in germ cells per se, suggesting that endogenous mechanisms within germ cells may be a prototype for the formation of heteromorphic gametes. However, research focusing on the germ cell-intrinsic mechanisms for anisogamy in mammals is still limited. This limitation is in part due to the difficulty in reproducing the development process of germ cells in culture, making it challenging to establish robust experimental systems. In our laboratory, we have established a culture system to differentiate germ cells from pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem cells (iPS) cells. Using the experimental system, we identified oocyte-specific transcription factors (NOBOX, FIGLA, LHX8, TBPL2) that are essential and sufficient for oocyte formation: enforced expression of these transcription factors transformed ES/iPS cells into oocyte-like cells capable of fertilization. This raises a possibility that these four transcription factors orchestrate in forming the heteromorphic gametes in mice. In this research, we focus on identifying gene expression networks and epigenetic changes to elucidate how these transcription factors confer oocyte competency and to approach the mechanism constructing the sexual dimorphism of gametes.

Figure 2 Formation of heteromorphic gametes in mammals: (Top) In the gonads, gametes with different functions and morphologies between sexes differentiate in response to the environment constructed by surrounding somatic cells. During gamete differentiation, early oocytes and spermatocytes initially exhibit no apparent morphological differences. However, significant distinctions emerge during the subsequent growth of oocytes or after the completion of meiosis in spermatids. Transcription factors such as NOBOX, FIGLA, LHX8, and TBPL2 play crucial roles in the morphogenesis of mouse oocytes. (Bottom) Enforced expression of these transcription factors in ES cells leads to their transformation into oocyte-like cells.

Research 02
How do oocytes stay dormant?

The mechanisms for maintaining reproductive capacity differ between males and females. In males, self-renewing spermatogonial stem cells continuously support the production of numerous sperm. In contrast, in females, oocytes do not proliferate further after entering meiosis during fetal development; instead, the majority of oocytes remain dormant, while a portion periodically resumes growth (Figure 3). As the number of oocytes is limited, the balance between dormancy and growth determines the reproductive lifespan of females. Despite its crucial function, the mechanisms controlling this balance remain largely unknown. Based on genetic analysis in mice, FOXO3, a member of the FOXO transcription factor family, has been identified as a key factor for oocyte dormancy: in FOXO3-deficient mice, oocyte dormancy was abrogated, leading to the precocious disappearance of immature oocytes from the ovary (similar to the state of premature ovarian insufficiency). The FOXO family has been noted for its function in extending the lifespan of organisms in species such as nematodes and fruit flies, suggesting that placing immature oocytes in a dormant state may prevent oocyte aging and/or improve oocyte quality (Figure 3). In this research, we are analyzing the genetic network orchestrated by FOXO3, along with elucidating environmental factors that induce oocyte dormancy. Understanding this genetic network not only contributes to the understanding of the maintenance mechanism of immature oocytes in the ovary but also develops methodologies to extend reproductive lifespan of females.

Figure 3 Maintenance of dormant state of oocytes: (Top) Most oocytes in the ovary remain in a dormant state to prepare for upcoming periodic activation. The balance between dormancy and activation is poorly understood. In mice, the transcription factor FOXO3 plays an important role: when FOXO3 is in the nucleus, oocytes remain dormant, but activation occurs upon its translocation to the cytoplasm. (Bottom) The FOXO family primarily contributes to responses related to maintaining cellular quality in response to growth factors and cellular stress. However, the genetic cascade of FOXO3 in oocytes remains unclear.

Research 03
How is the quality of the germ cell lineage secured?

Eggs inherits not only the genome but also maternal proteins and organelles stored in the cytoplasm to the next generation. Among maternal inheritance, how the quality of mitochondria is secured attracts our attention. Mitochondria harbor their own circular DNA (mtDNA) encoding proteins essential for the electron transport chain, and mutations in mtDNA can cause mitochondrial diseases. Since mtDNA is located within mitochondria, which produce abundant reactive oxygen species, mutations occur at a higher rate compared to the nuclear genome. Furthermore, unlike the nuclear genome, there is no extensive DNA repair process during meiosis. Despite these unfavorable conditions, mtDNA is somehow maintained stably across generations (Figure 4). Why? Several models have been proposed to explain this phenomenon, but definitive insights are still lacking, leaving this mechanism a longstanding mystery in reproductive biology. In collaboration with the University of Tsukuba, our laboratory is conducting research to track mtDNA in the germ cell lineage in culture using iPS cells derived from mice with mtDNA mutations. This research is expected to contribute not only to addressing the longstanding mystery but also to the prevention of mitochondrial diseases.

Figure 4 Maintenance of mitochondrial DNA (mtDNA) integrity in the germ cell lineage: mtDNA is maternally inherited, but its mutation rate varies between generations. In mouse models, it is known that mutated mtDNA is eliminated in the germ cell lineage, but it is unclear when and how this occurs. (The figure is merely a schematic diagram and does not mean the disappearance of mutated mtDNA during oogenesis.)

Research 04
In vitro gametogenesis: Creating eggs and sperm in a dish.

ES/iPS cells have the ability to become any cell type in the body. Taking advantage of this property, research on cell, tissue, and organ regeneration has been actively conducted. Our laboratory, pioneering in the world, has successfully induced the differentiation of primordial germ cells, the precursors of gametes, from mouse ES/iPS cells, and further succeeded in inducing the differentiation of functional eggs and spermatogonial stem cells from mouse ES/iPS cells. Recently, we have also succeeded in inducing differentiation of gonadal somatic cells from mouse ES/iPS cells, enabling the production of functional eggs entirely in culture without using any embryonic or animal tissues (Figure 5). The reconstitution of the germ cell differentiation process using pluripotent stem cells, named "in vitro gametogenesis," is expected to provide extremely useful experimental tools for uncovering the complicated process of germ cell differentiation and serve as an alternative source of gametes for animal and human reproduction. However, in vitro gametogenesis still needs to be improved to fulfill these expectations. For example, there is still a room to improve the efficiency of germ cell differentiation and the quality of resultant gametes, especially in terms of developmental potency. Another issue is application of the technology developed in mice to other mammalian species. In our laboratory, we are making efforts to improve the quality of gametes produced by in vitro gametogenesis and conducting research to apply in vitro gametogenesis to animal species other than mice, such as humans, monkeys, cattle, and white rhinoceros. Studies on white rhinoceros are conducted through international collaborations involving Germany, the United States, Italy, the Czech Republic, Kenya, and others, and we are developing techniques to produce eggs for repopulating endangered species.

Figure 5 In vitro gametogenesis is a technology that produces functional gametes by replicating the differentiation process of germ cells in vitro, using pluripotent stem cells such as ES cells and iPS cells. This technique serves not only as a tool for elucidating the mechanisms of germ cell differentiation but also contributes significantly to the development of assisted reproductive technologies.