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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

SIRT1 reduces epigenetic and non-epigenetic changes to maintain the quality of postovulatory aged oocytes in mice

Highlights: • The intensity of H3K9ac and H3K4me2/3 increased in postovulatory aged oocytes. • SIRT1 overexpression inhibited the increase of H3K9ac and H3K4me2/3. • SIRT1 overexpression ameliorated aging-induced epigenetic and non-epigenetic changes in oocytes. Postovulatory oocyte aging has a major influence on the development potential of embryos. Many antioxidants can delay oocyte aging by regulating the expression of SIRT1. However, there is a lack of knowledge on SIRT1 function in postovulatory oocyte aging. In vitro transcribed RNA of Sirt1 was injected into fresh oocytes to investigate the function of SIRT1 during postovulatory oocyte aging. In the present study, SIRT1 was found to be down-regulated in aged oocytes compared with fresh oocytes. Meanwhile the intensity of acetylation of H3K9 (H3K9ac) and H3K4 methylation increased in postovulatory aged oocytes. After the oocytes were injected with SIRT1 and aged for 12 h, the intensity of H3K9ac and H3K4 methylation markedly decreased compared with controls. Furthermore, SIRT1 overexpression also reduced the aging-induced oocyte morphological changes and reactive oxygen species accumulation, maintained the spindle normal morphology and attenuated the aging-associated abnormalities of mitochondrial function. The role of SIRT1 in protecting oocyte aging was diminished when oocytes with overexpressed SIRT1 were cultured with SIRT1 inhibitor EX-527. Briefly, these present results show that SIRT1 not only reduced the non-epigenetic changes such as abnormal oocyte morphology, ROS accumulation, spindle defects and mitochondrial dysfunctions but also regulated the epigenetic changes in order to maintain the quality of postovulatory aged oocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes

Highlights: • Cortical granule exocytosis is sensitive to tetanus toxin in mouse oocytes. • VAMP1 and VAMP3 proteins participate in cortical granule exocytosis. • This work confirms that cortical granule exocytosis is mediated by SNARE complex. Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation

Highlights: • Inhibition of Chk1/2 has no significant effects on germinal vesicle breakdown. • Chk1/2 inhibition results in the first polar body extrusion defects. • Chk1/2 is critically involved in meiotic spindle organization. • Inhibition of Chk1/2 leads to abnormal chromosome condensation. • Chk1/2 is important for the development of MII stage oocytes. Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2000

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

NASA Discipline Plant Biology↗
NASA NTRS1985

Effects of Simulated Weightlessness on Mammalian Development. Part 2: Meiotic Maturation of Mouse Oocytes During Clinostat Rotation

In order to understand the role of gravity in basic cellular processes that are important during development, the effects of a simulated microgravity environment on mammalian gametes and early embryos cultured in vitro are examined. A microgravity environment is simulated by use of a clinostat, which essentially reorients cells relative to the gravity vector. Initial studies have focused on assessing the effects of clinostat rotation on the meiotic progression of mouse oocytes. Modifications centered on providing the unique in vitro culture of the clinostat requirements of mammalian oocytes and embryos: 37 C temperature, constant humidity, and a 5% CO2 in air environment. The oocytes are observed under the dissecting microscope for polar body formation and gross morphological appearance. They are then processed for cytogenetic analysis.

Wolgemuth, D. J.↗
NASA NTRS1984

Effects of simulated weightlessness on mammalian development. Part 1: Development of clinostat for mammalian tissue culture and use in studies on meiotic maturation of mouse oocytes

The effects of weightlessness on three aspects of mammalian reproduction: oocyte development, fertilization, and early embryogenesis was studied. Zero-gravity conditions within the laboratory by construction of a clinostat designed to support in vitro tissue culture were simulated and the effects of simulated weightlessness on meiotic maturation in mammalian oocytes using mouse as the model system were studied. The timing and frequency of germinal vesicule breakdown and polar body extrusion, and the structural and numerical properties of meiotic chromosomes at Metaphase and Metaphase of meiosis are assessed.

Wolegemuth, D. J.↗
NASA NTRS1992

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

Non-NASA Center↗
DOE OSTI2021

Cold shock induces novel nuclear bodies in Xenopus oocytes

Here we describe novel spherical structures that are induced by cold shock on the lampbrush chromosomes (LBCs) of Xenopus laevis oocytes. We call these structures cold bodies or C-bodies. C-bodies are distributed symmetrically on homologous LBCs, with a pattern similar to that of 5S rDNA. Neither active transcription nor translation is necessary for their formation. Similar protrusions occur on the edges of some nucleoli. Endogenous LBCs as well as those derived from injected sperm form C-bodies under cold shock conditions. The function of C-bodies is unknown.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1986

Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

Schatten, Heide↗
DOE OSTI2022

Zinc dynamics regulate early ovarian follicle development

Zinc fluctuations regulate key steps in late oocyte and pre-implantation embryo development; however, roles for zinc in preceding stages in early ovarian follicle development, when cooperative interactions exist between the oocyte and somatic cells, are unknown. To understand the roles of zinc during early follicle development, we applied single cell X-ray fluorescence microscopy, a radioactive zinc tracer, and a labile zinc probe to measure zinc in individual mouse oocytes and associated somatic cells within early follicles. Here, we report a significant stage-specific increase and compartmental redistribution in oocyte zinc content upon the initiation of early follicle growth. The increase in zinc correlates with the increased expression of specific zinc transporters, including two that are essential in oocyte maturation. While oocytes in follicles exhibit high tolerance to pronounced changes in zinc availability, somatic survival and proliferation are significantly more sensitive to zinc chelation or supplementation. Finally, transcriptomic, proteomic, and zinc loading analyses reveal enrichment of zinc targets in the ubiquitination pathway. Overall, these results demonstrate that distinct cell type- specific zinc regulations are required for follicle growth and indicate that physiological fluctuation in the localization and availability of this inorganic cofactor has fundamental functions in early gamete development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A cooperative network at the nuclear envelope counteracts LINC-mediated forces during oogenesis in C. elegans

Oogenesis involves transduction of mechanical forces from the cytoskeleton to the nuclear envelope (NE). In Caenorhabditis elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to collapse under forces mediated through LINC (linker of nucleoskeleton and cytoskeleton) complexes. Here, we use cytological analysis and in vivo imaging to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We also use a mechano-node-pore sensing device to directly measure the effect of genetic mutations on oocyte nuclear stiffness. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein, which induces polarization of a LINC complex composed of Sad1 and UNC-84 homology 1 (SUN-1) and ZYGote defective 12 (ZYG-12). Lamins contribute to oocyte nuclear stiffness and cooperate with other inner nuclear membrane proteins to distribute LINC complexes and protect nuclei from collapse. We speculate that a similar network may protect oocyte integrity during extended oocyte arrest in mammals.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2017

Perfluorinated Alkyl Acids and Fecundity Assessment in Striped Mullet (Mugil Cephalus) at Merritt Island National Wildlife Refuge

This study investigated wild caught Striped mullet (Mugil cephalus) at Merritt Island National Wildlife Refuge (MINWR) for levels of 15 perfluoroalkyl acids (PFAAs) in tandem with fecundity measurements (n 42) and oocyte developmental stages (n 128). PFAAs measurements were undertaken for liver (n 128), muscle (n 49), and gonad (n 10). No significant negative impacts of liver PFAA burden on wild-caught, mullet fecundity endpoints are observed in this study; however, changes in PFAAs are seen in the liver as mullet progress through different sub-stages of oocyte development. Of the PFAAs with significant changes by sub-stage of oocyte development, the carboxylic acids (perfluorooctanoic acid, perfluorononanoic acid, and perfluorotridecanoic acid) increase in the liver with increasing sub-stage while the sulfonic acid and its precursor (perfluorooctanesulfonic acid (PFOS) and perfluorooctanesulfonamide, respectively) decrease in the liver with increasing stage of oocyte development. This is a unique find and suggests PFAAs change location of compartmentalization as mullet progress towards spawning. Investigations also revealed higher than expected median muscle and gonad levels of PFOS in Striped mullet collected at MINWR (9.01 ngg and 80.2 ngg, respectively).

Jacqueline T Bangma↗
NASA NTRS1985

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

Schatten, G.↗