Shen Yin

DAZ Family Proteins, Key Players for Germ Cell Development.

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.

Regulation of primordial follicle recruitment by cross-talk between the Notch and phosphatase and tensin homologue (PTEN)/AKT pathways

The growth of oocytes and the development of follicles require certain pathways involved in cell proliferation and survival, such as the phosphatidylinositol 3-kinase (PI3K) pathway and the Notch signalling pathway. The aim of the present study was to investigate the interaction between Notch and the PI3K/AKT signalling pathways and their effects on primordial follicle recruitment. When the Notch pathway was inhibited by L-685,458 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT) in vitro, the expression of genes in the pathway and the percentage of oocytes in growing follicles decreased significantly in mouse ovaries. By 2 days postpartum, ovaries exposed to DAPT, short interference (si) RNA against Notch1 or siRNA against Hairy and enhancer of split-1 (Hes1) had significantly decreased expression of HES1, the target protein of the Notch signalling pathway. In contrast, expression of phosphatase and tensin homologue (Pten), a negative regulator of the AKT signalling pathway, was increased significantly. Co immunoprecipitation (Co-IP) revealed an interaction between HES1 and PTEN. In addition, inhibition of the Notch signalling pathway suppressed AKT phosphorylation and the proliferation of granulosa cells. In conclusion, the recruitment of primordial follicles was affected by the proliferation of granulosa cells and regulation of the interaction between the Notch and PI3K/AKT signalling pathways.

DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.

Post-ovulatory aging of mouse oocytes in vivo and in vitro: Effects of caffeine on exocytosis and translocation of cortical granules

The developmental potential of post-ovulatory oocytes decreases with aging in vivo and in vitro. In this study, we aimed to investigate the effects of a potent antioxidant caffeine on cortical granules (CGs) distribution in mouse oocytes aging in vivo and in vitro. We found that in vivo administration of 150u2009mg/kg caffeine caused ovulation of some morphologically abnormal oocytes showing premature exocytosis or congregation of CGs, but significantly decreased abnormal distribution of CGs in oocytes aging for 6u2009h, 12u2009h and 18u2009h in vivo compared to those without caffeine treatment. Unexpectedly, supplementation of oocyte culture medium with 10u2009mmol/L caffeine accelerated CGs release of oocytes and the normal CG distribution rate dramatically decreased from 6u2009h in oocytes aging in vitro. It appeared that oocytes showed a high degree of abnormal CG distribution by aging for 18u2009h, and caffeine might delay oocyte CG exocytosis in vivo, but accelerates CG exocytosis in vitro. Our findings may have implications for improving assisted reproduction technologies.

The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells in vitro

Primordial germ cells (PGCs) are founder cells of the germ cell lineage, and can be differentiated from stem cells in an induced system in vitro. However, the induction conditions need to be optimized in order to improve the differentiation efficiency. Activin A (ActA) is a member of the TGF-β super family and plays an important role in oogenesis and folliculogenesis. In the present study, we found that ActA promoted PGC-like cells (PGCLCs) formation from mouse skin-derived stem cells (SDSCs) in both embryoid body-like structure (EBLS) differentiation and the co-culture stage in a dose dependent manner. ActA treatment (100 ng/ml) during EBLS differentiation stage and further co-cultured for 6 days without ActA significantly increased PGCLCs from 53.2% to 82.8%, and as well as EBLS differentiation without ActA followed by co-cultured with 100 ng/ml ActA for 4 to 12 days with the percentage of PGCLCs increasing markedly in vitro. Moreover, mice treated with ActA at 100 ng/kg body weight from embryonic day (E) 5.5–12.5 led to more PGCs formation. However, the stimulating effects of ActA were interrupted by Smad3 RNAi, and in an in vitro cultured Smad3−/− mouse skin cells scenario. SMAD3 is thus likely a key effecter molecule in the ActA signaling pathway. In addition, we found that the expression of some epiblast cell markers, Fgf5, Dnmt3a, Dnmt3b and Wnt3, was increased in EBLSs cultured for 4 days or PGCLCs co-cultured for 12 days with ActA treatment. Interestingly, at 16 days of differentiation, the percentage of PGCLCs was decreased in the presence of ActA, but the expression of meiosis-relative genes, such as Stra8, Dmc1, Sycp3 and Sycp1, was increased. In conclusion, our data here demonstrated that ActA can promote PGCLC formation from SDSCs in vitro, at early stages of differentiation, and affect meiotic initiation of PGCLCs in later stages

Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro

Previous studies suggest that hydrogen sulfide (H2S) and ammonia (NH3) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na2S and/or NH4Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na2S treatment but not after NH4Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3u202fB expression level was higher in oocytes treated with Na2S and/or NH4Cl, which might be caused by ROS elevation. Additionally, exposure to Na2S and/or NH4Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H2S and/or NH3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy.

Repeated superovulation may affect mitochondrial functions of cumulus cells in mice

Controlled ovarian stimulation by exogenous gonadotrophins is a key procedure during the in vitro fertilization cycle to obtain a sufficient number of oocytes in humans. Previous studies demonstrated that repeated superovulation had deleterious effects on the ovaries. However, whether repeated superovulation adversely affects the mitochondrial functions of cumulus cells remains unclear. In this study, mice were divided into three groups: superovulation once (R1); superovulation three times (R3), and superovulation five times (R5). We evaluated the effects of repeated superovulation on mitochondrial DNA copies (mtDNA) and observed decreased mtDNA copies per cell with increasing number of superovulation cycles. Further, we investigated the DNA methylation status in exon 2 and the mRNA expression level of nuclear-encoded DNA polymerase gamma A (PolgA). The results showed that the DNA methylation levels of PolgA in R1 and R5 were slightly lower than in R3. Additionally, the altered DNA methylation in PolgA coincided with the changes in PolgA expression in cumulus cells. We also found that the mRNA expression of COX1, CYTB, ND2, and ND4 was altered by repeated superovulation in cumulus cells. Thus, repeated superovulation had adverse effects on mitochondrial function.

Interaction of the transforming growth factor-? and Notch signaling pathways in the regulation of granulosa cell proliferation.

The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

The role of L-type calcium channels in mouse oocyte maturation, activation and early embryonic development

Calcium ion fluctuation is closely related to the transformation of cell cycle. However, little is known about the function of L-type calcium channel in mammalian oocyte and embryo development. We thus studied the roles of L-type calcium channel in mouse oocyte meiotic maturation, parthenogenetic activation and early embryonic development. We used the antagonist Amlodipine to block L-type calcium channel. Oocytes or zygotes were cultured to different time points with 0xa0μM, 10xa0μM, 30xa0μM and 50xa0μM Amlodipine. Then we checked the rate of first polar body extrusion, spindle formation, asymmetric division parthenogenetic activation and early embryo cleavage. The results showed that Amlodipine treatment did not affect germinal vesicle breakdown, but caused disruption of cytoskeleton organization, symmetric division, formation of mature oocytes with a large polar body, or reduced the first polar body extrusion, depending on its concentrations. Amlodipine treatment also resulted in decreased parthenogenetic activation and arrested early embryonic development. Overall, these data suggest that proper function of L-type calcium channel is critical for oocyte maturation, activation, and early embryonic development.

FMNL1, a key regulator for asymmetric cell division

Asymmetric division plays a crucial role during oogenesis, characterized by extrusion of a small polar body in the first meiosis.1 Oocytes preserve most of their maternal stores by expelling a minimal cytoplasmic content with half chromosomes, which is essential for early embryonic development by maintaining nutrition and energy resources due to extremely asymmetric division.2 Spindle positioning is important for the asymmetric or symmetric division in cell cycle.3 Cytoplasmic actin is involved in chromosome gathering and spindle positioning in mitosis, because randomization of spindle positioning occurs after disassembling actin with disrupting drugs.4 Also, astral microtubules emanating from centrosomes interact with cortical anchor proteins decide the spindle position along the longest cell axis, which helps the symmetric division.5 However, this rule is not suitable for oocytes because they are round and lack of centrosome and astral microtubules. In contrast to symmetric mitotic divisions, oocytes undergo asymmetric divisions with half homologous chromosomes segregated to the small polar body during anaphase of the first meiotic division. To compensate for the lack of centrosome and astral microtubules, actin assembly is very important for the spindle positioning by transmitting forces over long distances during oocytes meiosis. A recent paper in Cell Cycle describes a key factor, Formin-like 1 (FMNL1), associated with RhoA signal pathway, regulates asymmetric division by controlling actin assembly and spindle positioning in mouse oocytes (Fig. 1).6 n nFMNL1 is a member of Formin family proteins which are the actin nucleators. It has been shown that FMNL1 is essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis. The authors discovered the functions of FMNL1 in meiotic mouse oocytes by using specific morpholino microinjection and live cell imaging. FMNL1 knockdown caused disrupted asymmetric division, characterized by the large polar bodies and low rate of polar body extrusion. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD), which implies that it may be involved in spindle organization and positioning. Next, the authors found various morphologically defective spindles in FMNL1 knockdown oocytes by co-staining FMNL1, α-tubulin and DNA. The wrong localization and lower expression level of phospho-p44/42 mitogen-activated protein kinase (p-MAPK) further confirmed the FMNL1s roles in spindle organization, because MAPK has been previously shown to be required for proper spindle formation. In addition, same results were observed in mammalian diaphanous1 (mDia1) knockdown oocytes. FMNL1 depletion resulted in reduced mDia1 expression, which implied that FMNL1 might be upstream to mDia1.7 n nSubsequently, time-lapse microscopic and immunofluorescence intensity analysis demonstrated that the wrong spindle positioning was due to the aberrant actin assembly and expression level in FMNL1 knockdown oocytes. Similar results have been observed in other members of the Formin family members. Meantime, cortical polarity was disrupted as shown by a loss of actin cap and cortical granule-free domain (CGFD) formation. Because spindle migration is actin-dependent, FMNL1 may regulate spindle positioning by its effect on actin nucleation, which can further affect oocyte cortical polarity formation. Also, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130, which caused disrupted Golgi apparatus.6 Because Golgi mediates the long-range transport of vesicles, FMNL1 may modulate vesicle-based mechanism of actin network for asymmetric division by regulating Golgi complex. In addition, mDia1 and Profilin1 had similar localization patterns in mouse oocytes and mDia1 knockdown resulted in reduced Profilin1 expression, which also partially explained the wrong actin assembly.7 n nInhibition of the small GTPase RhoA activity resulted in decreased FMNL1 expression, which indicates that RhoA may be the upstream molecule of FMNL1.6 However, RhoA inhibition did not alter mDia1 expression, which indicates that there is a FMNL1-mDia1-Profilin1 signaling pathway. Taken together, there are RhoA-FMNL1/GM130 and RhoA-FMNL1-dMia1- Profilin1 pathways for the spindle positioning and actin assembly during mouse oocyte meiosis.7 n n nIn summary, the identification of FMNL1 as a key regulator for spindle positioning and actin assembly in the current study strongly implies that the actin-based spindle positioning is vital for asymmetric division during oocyte maturation. These recent studies, on one hand clarify the mechanism for asymmetric division in mouse oocytes, and on the other hand show a RhoA-FMNL1 pathway. It will contribute to understanding how physical parameters including cell size and shape are controlled in the cell cycle. n n n nFigure 1. n nFMNL1 regulates asymmetric division by controlling actin assembly and spindle positioning in mouse oocytes. Actin network modulation is essential for the asymmetric positioning of meiotic spindle. FMNL1 is important for spindle organization with the help ...