Germline-soma Supply Mitochondria for mtDNA Inheritance in Mouse Oogenesis

Maternal transmission paradigm of mtDNA remains controversial in mammalian oogenesis. Germline-soma-to-oocyte communication by numerous transzonal nanotubes (TZTs) reminds whether intercellular mitochondrial transfer is associated with maternal inheritance. Here, we found that mouse oocytes egocentrically receive mitochondria via TZTs, which projected from germline-soma, to achieve 105 copies, instead of de novo synthesis of mtDNA subpopulation in growing oocytes. De novo assembled TZTs amongst germline-soma and oocytes accumulated mtDNA amounts of the oocytes in vitro. However, mitochondrial supplement from germline-soma gradually diminished along with oocyte growth and was terminated by meiosis resumption, in line with a decrease in the proportion of germline-soma with thriving mtDNA replication and FSH capture capability. Thus, germline-soma-to-oocyte mitochondrial transfer is responsible for mammalian mtDNA inheritance as well as oogenesis and aging.

Conserved meiotic mechanisms in the cnidarian Clytia hemisphaerica revealed by Spo11 knockout

During meiosis, each duplicated chromosome pairs and recombines with its unique homolog to ensure the shuffling of genetic information across generations. Functional studies in classical model organisms have revealed a surprising diversity in the chronology and interdependency of the earliest meiotic steps such as chromosome movements, pairing, association via Synaptonemal Complex formation (synapsis), recombination and the formation of chiasmata. A key player is Spo11, an evolutionarily conserved topoisomerase-related transesterase that initiates meiotic recombination via the catalysis of programmed DNA double stranded breaks (DSBs). While DSBs are required for pairing and synapsis in budding yeast and mouse, alternative pathways are employed during female meiosis of the fruit fly and nematode Caenorhabditis elegans. Here, to provide a comparative perspective on meiotic regulation from a distinct animal clade, we chart gametogenesis in Clytia hemisphaerica jellyfish and examine the role of Spo11 using CRISPR-Cas9 mutants, generated clonally from F0 polyp colonies. Spo11 mutant females fail to assemble synaptonemal complexes and chiasmata, such that homologous chromosome pairs disperse during oocyte growth. Subsequent meiotic divisions are abnormal but produce viable progeny. Clytia thus shares an ancient eukaryotic dependence of synapsis and chromosome segregation on Spo11-generated DSBs. It provides a valuable additional experimental model for dissecting meiotic mechanisms during animal gametogenesis, and for building a comparative framework for distinguishing evolutionarily conserved versus flexible features of meiosis.

Effects of the Sex Chromosome Complement, XX, XO, or XY, on the Transcriptome and Development of Mouse Oocytes During Follicular Growth

The sex chromosome complement, XX or XY, determines sexual differentiation of the gonadal primordium into a testis or an ovary, which in turn directs differentiation of the germ cells into sperm and oocytes, respectively, in eutherian mammals. When the X monosomy or XY sex reversal occurs, XO and XY females exhibit subfertility and infertility in the mouse on the C57BL/6J genetic background, suggesting that functional germ cell differentiation requires the proper sex chromosome complement. Using these mouse models, we asked how the sex chromosome complement affects gene transcription in the oocytes during follicular growth. An oocyte accumulates cytoplasmic components such as mRNAs and proteins during follicular growth to support subsequent meiotic progression, fertilization, and early embryonic development without de novo transcription. However, how gene transcription is regulated during oocyte growth is not well understood. Our results revealed that XY oocytes became abnormal in chromatin configuration, mitochondria distribution, and de novo transcription compared to XX or XO oocytes near the end of growth phase. Therefore, we compared transcriptomes by RNA-sequencing among the XX, XO, and XY oocytes of 50–60 µm in diameter, which were still morphologically comparable. The results showed that the X chromosome dosage limited the X-linked and autosomal gene transcript levels in XO oocytes whereas many genes were transcribed from the Y chromosome and made the transcriptome in XY oocytes closer to that in XX oocytes. We then compared the transcript levels of 3 X-linked, 3 Y-linked and 2 autosomal genes in the XX, XO, and XY oocytes during the entire growth phase as well as at the end of growth phase using quantitative RT-PCR. The results indicated that the transcript levels of most genes increased with oocyte growth while largely maintaining the X chromosome dosage dependence. Near the end of growth phase, however, transcript levels of some X-linked genes did not increase in XY oocytes as much as XX or XO oocytes, rendering their levels much lower than those in XX oocytes. Thus, XY oocytes established a distinct transcriptome at the end of growth phase, which may be associated with abnormal chromatin configuration and mitochondria distribution.

A novel mechanism of bulk cytoplasmic transport by cortical dynein in Drosophila ovary

Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. We have previously shown that cytoplasmic dynein is required for Drosophila oocyte growth, and assumed that it transports cargoes along microtubule tracks from nurse cells to the oocyte. Here we report that instead transporting cargoes along microtubules into the oocyte, cortical dynein actively moves microtubules in nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. We demonstrate this microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein can perform bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. We propose that the dynein-driven microtubule flow could serve as a novel mode of cargo transport for fast cytoplasmic transfer to support rapid oocyte growth.

The Role of Steroids, Growth Factors and CAMP Stimulators on the Gap Junction Activity in Cumulus Oocyte Complexes in the Rat

<p>Bidirectional communication between mammalian oocytes and their surrounding somatic cells is essential for oocyte maturation. Gap junctions promote the transfer of essential metabolites, nucleotides, amino acids and ions from cumulus cells to the oocyte that are crucial for oocyte growth and development. However, the range of factors present in the microenvironment of the developing antral follicle, which modulate gap junction activity of the cumulus-oocyte complexes (COCs), is largely unknown. The primary objective of this study was to determine the effects of various steroids, growth factors and cAMP stimulators on the gap junction activity in rat COCs. The gap junction activity was measured in presence or absence of different treatments using a fluorescence dye and in the presence of milrinone, a phosphodiesterase type 3 inhibitor. The major findings of this study were that cAMP stimulators increased the rate of dye transfer from cumulus cells to the oocyte. Under in vitro conditions it was established that neither steroids nor IGF1 by themselves were able to modulate gap junction activity in rat COCs. Furthermore, forskolin, a potent cAMP stimulator; caused a relative increase in Cx37 gene expression levels following a four hours incubation period. The outcomes from the present study may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes.</p>

The Role of Steroids, Growth Factors and CAMP Stimulators on the Gap Junction Activity in Cumulus Oocyte Complexes in the Rat

<p>Bidirectional communication between mammalian oocytes and their surrounding somatic cells is essential for oocyte maturation. Gap junctions promote the transfer of essential metabolites, nucleotides, amino acids and ions from cumulus cells to the oocyte that are crucial for oocyte growth and development. However, the range of factors present in the microenvironment of the developing antral follicle, which modulate gap junction activity of the cumulus-oocyte complexes (COCs), is largely unknown. The primary objective of this study was to determine the effects of various steroids, growth factors and cAMP stimulators on the gap junction activity in rat COCs. The gap junction activity was measured in presence or absence of different treatments using a fluorescence dye and in the presence of milrinone, a phosphodiesterase type 3 inhibitor. The major findings of this study were that cAMP stimulators increased the rate of dye transfer from cumulus cells to the oocyte. Under in vitro conditions it was established that neither steroids nor IGF1 by themselves were able to modulate gap junction activity in rat COCs. Furthermore, forskolin, a potent cAMP stimulator; caused a relative increase in Cx37 gene expression levels following a four hours incubation period. The outcomes from the present study may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes.</p>

Regulation of mouse primordial follicle formation by signaling through the PI3K pathway

Cell signaling mediated by the KIT receptor is critical for many aspects of oogenesis including the proliferation and migration of primordial germ cells, as well as the survival, growth, and maturation of ovarian follicles. We previously showed that KIT regulates cyst breakdown and primordial follicle formation, and in this study, have investigated the mechanisms downstream of the receptor by modulating the activity of two downstream signaling cascades: the phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways. E17.5 ovaries were cultured for five days with a daily dose of media supplemented with either the PI3K inhibitor LY294002, the MEK inhibitor U0126, or a DMSO vehicle control. Our histological observations aligned with the established role of PI3K in oocyte growth and primordial follicle activation but also revealed that LY294002 treatment delayed the processes of cyst breakdown and primordial follicle formation. U0126 treatment also led to a reduction in oocyte growth and follicle development but did not appear to affect cyst breakdown. The delay in cyst breakdown was mitigated when ovaries were dually dosed with LY294002 and KITL, suggesting that while KIT may signal through PI3K to promote cyst breakdown, other signaling networks downstream of the receptor could compensate. These observations unearth a role for PI3K signaling in the establishment of the ovarian reserve and suggest that PI3K might be the primary mediator of KIT-induced cyst breakdown and primordial follicle formation in the mouse ovary.

Mechanical mapping of mammalian follicle development using Brillouin microscopy

In early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation. Our work provides an alternative approach to study the role of mechanics in follicle morphogenesis and might pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

Promyelocytic leukemia nuclear body (PML-NB) -free intranuclear milieu facilitates development of oocytes in mice

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs), a class of membrane-less organelles in cells, are involved in multiple biological activities and are present throughout cells of adult organisms. Although the oocyte nucleus is an active region for the flux of multiple non-membranous organelles, PML-NBs have been predicted to be absent from oocytes. Here, we show that the deliberate assembly of PML-NBs during oocyte growth preferentially sequestered Small Ubiquitin-related Modifier (SUMO) protein from the nucleoplasm. SUMO not only was involved in the regulation of oocyte nuclear maturation but also was committed to the response, mediated by liquid droplet formation, to multiple stressors including nucleolar stress and proteotoxic stresses. Exogenous assembly of PML-NBs in the nucleus of oocytes affected the efficiency of the response of SUMO. These observations suggest that the PML-NB-free intranuclear milieu ensures that a reserve of SUMO remains available for emergent responses in oocyte development. This work demonstrated a benefit of the PML-NB-free intranuclear milieu, namely the ability to redirect the flux of SUMO otherwise needed to control PML-NB dynamics.