90 ENDOGENOUS MODIFICATIONS OF AURORA B AND AURORA C KINASE EXPRESSION IN MOUSE OOCYTES AND EARLY EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 146
Author(s):  
C. A. Lima ◽  
V. Huntress ◽  
E. W. Overström
Keyword(s):  
Protein Localization ◽  
Somatic Cells ◽  
Early Embryo ◽  
Vital Role ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Mouse Oocytes ◽  
Reverse Transcription Pcr ◽  
Early Embryos

The Aurora (Aur) proteins are a family of serine/threonine kinases that play fundamental roles in controlling M-phase progression. Previous reports have shown that AurB is vital for proper completion of karyokinesis and cytokinesis in somatic cells. The role of AurC in somatic cells has been found to be much less significant, whereas it appears to play an important role in spermatogenesis. The role of these Aur proteins is not well characterized in mouse oocytes and early embryos. The objective of this study was to assess changes of AurB and AurC mRNA and protein expression in mouse oocytes and early embryos as development progresses through the activation of the zygotic genome. Oocytes and embryos were collected from the oviducts of hormone-stimulated CF-1 mice. After culturing for varying amounts of time, cumulus-denuded samples were either fixed for immunofluorescence microscopy studies or lysed for analysis of mRNA levels through the use of reverse transcription-PCR (RT-PCR). Samples were processed for immunofluorescence using markers of spindle morphology (tubulin) and AurB. Analysis of relative levels of AurB and AurC mRNA were assessed by RT-PCR methods. Marked differences were observed in the localization of AurB when unfertilized oocytes or prezygotic genome activation (ZGA) embryos were compared with post-ZGA samples. There was no evidence of AurB protein localized to the mitotic spindle or resultant midbody in oocyte and early embryo samples. Embryos fixed post-ZGA demonstrated AurB localization, as is conventionally found in somatic cells. The AurB protein was found co-localized with DNA in metaphase stage blastomeres and associated with the midbody in blastomeres near completion of cytokinesis. Relative levels of AurB mRNA were not found to be significantly different when pre- and post-ZGA samples were compared. A significant decrease in relative levels of AurC mRNA was observed in fertilized pre-ZGA samples when compared with unfertilized oocyte counterparts. These observations demonstrate significant differences in the status of AurB and AurC mRNA levels and protein localization in mouse oocytes and early embryos when compared with somatic cells. Given earlier reports showing the vital role of AurC in spermatogenesis, the elevated levels of AurC mRNA observed in prefertilization oocytes may be indicative of a similar role of AurC during oogenesis. Elucidating temporal and localization details of Aur expression is vital to gaining further understanding of cell-cycle regulation in oogenesis and early embryogenesis.

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

DIAGNOSTIC ROLE OF CHEST CT USING CO-RADS CATEGORIZATION IN SARS-COV-2 INFECTION WITH EMPHASIS ON IMPACT OF CT IN PATIENTS WITH DELAYED OR NEGATIVE INITIAL RTPCR TEST

2021 ◽  
pp. 51-52
Author(s):  
Tharani Putta ◽  
Kaushik Deconda
Keyword(s):  
Diagnostic Performance ◽  
Virus Disease ◽  
Vital Role ◽  
Chest Ct ◽  
Lung Involvement ◽  
Test Results ◽  
Rt Pcr ◽  
Diagnostic Role ◽  
Pcr Test

BACKGROUND AND OBJECTIVE: Role of chest CT in diagnosis of corona virus disease 2019 (COVID-19) has been controversial. The purpose of this study is to evaluate the diagnostic performance of chest CT when utilizing COVID-19 Reporting and Data System (CO-RADS). METHODOLOGY: Retrospective study including consecutive patients with positive SARS-CoV-2 RT-PCR test (initial or repeat test) and chest CT done in our institute between June and September 2020. Spectrum of CT ndings, CO-RADS score and 25 point CT severity score (CTSS) were recorded. RESULTS: A total of 300 consecutive patients with SARS-CoV-2 infection were included in the analysis. Out of the 168 patients who underwent CT prior to positive RT-PCR result, 125 (74.4%) had CO-RADS 3, 4 or 5 score on chest CT. 32 study patients (10.6%) had initial negative RT-PCR of which 24 (75%) had CO-RADS 4 or 5 score. Of the total patients with CO-RADS 3 to 5 score (227), 20 (8.8%) had severe lung involvement (CTSS 18-25), 83 (36.6%) had moderate lung involvement (CTSS 8-17) and 124 (54.6%) had mild lung involvement (CTSS 1-7). The mean CTSS was 7.9 with mean lobar score being higher in lower lobes (RLL=1.82, LLL=1.78) compared to the upper and middle lobes (RUL=1.61, RML=1.19, LUL=1.53). CONCLUSION:CT using CO-RADS scoring system has good diagnostic performance. In addition to assessing disease severity, it plays a vital role in triage of patients with suspected COVID-19 especially when there is limited availability of SARS-CoV-2 RT-PCR tests, delay in RT-PCR test results or in negative RT-PCR cases when there is high index of clinical suspicion.

scholarly journals Knockout and Overexpression of Pyrroloquinoline Quinone Biosynthetic Genes in Gluconobacter oxydans 621H

2006 ◽  
Vol 188 (21) ◽  
pp. 7668-7676 ◽  
Author(s):  
Tina Hölscher ◽  
Helmut Görisch
Keyword(s):  
Energy Source ◽  
Wild Type Strain ◽  
Gluconobacter Oxydans ◽  
Analysis Data ◽  
Pyrroloquinoline Quinone ◽  
Vital Role ◽  
Reverse Transcription Pcr ◽  
Membrane Bound ◽  
Pqq Biosynthesis

ABSTRACT In Gluconobacter oxydans, pyrroloquinoline quinone (PQQ) serves as the cofactor for various membrane-bound dehydrogenases that oxidize sugars and alcohols in the periplasm. Proteins for the biosynthesis of PQQ are encoded by the pqqABCDE gene cluster. Our reverse transcription-PCR and promoter analysis data indicated that the pqqA promoter represents the only promoter within the pqqABCDE cluster of G. oxydans 621H. PQQ overproduction in G. oxydans was achieved by transformation with the plasmid-carried pqqA gene or the complete pqqABCDE cluster. A G. oxydans mutant unable to produce PQQ was obtained by site-directed disruption of the pqqA gene. In contrast to the wild-type strain, the pqqA mutant did not grow with d-mannitol, d-glucose, or glycerol as the sole energy source, showing that in G. oxydans 621H, PQQ is essential for growth with these substrates. Growth of the pqqA mutant, however, was found with d-gluconate as the energy source. The growth behavior of the pqqA mutant correlated with the presence or absence of the respective PQQ-dependent membrane-bound dehydrogenase activities, demonstrating the vital role of these enzymes in G. oxydans metabolism. A different PQQ-deficient mutant was generated by Tn5 transposon mutagenesis. This mutant showed a defect in a gene with high homology to the Escherichia coli tldD gene, which encodes a peptidase. Our results indicate that the tldD gene in G. oxydans 621H is involved in PQQ biosynthesis, possibly with a similar function to that of the pqqF genes found in other PQQ-synthesizing bacteria.

Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1239-1249 ◽  
Author(s):  
C.A. Whittaker ◽  
D.W. DeSimone
Keyword(s):  
In Situ Hybridization ◽  
Rnase Protection Assay ◽  
Early Embryo ◽  
Mrna Levels ◽  
Rnase Protection ◽  
Dorsal Midline ◽  
Transmembrane Receptors ◽  
Early Embryos ◽  
Whole Mount

Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.

scholarly journals Developmental regulation of yolk sac hematopoiesis by Krüppel-like factor 6

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1357-1365 ◽  
Author(s):  
Nobuyuki Matsumoto ◽  
Atsushi Kubo ◽  
Huixian Liu ◽  
Kuniharu Akita ◽  
Friedrich Laub ◽  
...  
Keyword(s):  
Developmental Regulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Yolk Sac ◽  
Homozygous Mutation ◽  
Mesoderm Induction ◽  
Mrna Levels ◽  
Mouse Development

Krüppel-like factor 6 (KLF6) is a member of a growing family of transcription factors that share a common 3 C2H2 zinc finger DNA binding domain and have broad activity in regulating proliferation and development. We have previously established that Klf6 is expressed in neuronal tissue, hindgut, heart, lung, kidney, and limb buds during midgestation. To explore the potential role of Klf6 in mouse development, we analyzed Klf6-/- mice and found that the homozygous mutation is embryonic lethal by embryonic day (E) 12.5 and associated with markedly reduced hematopoiesis and poorly organized yolk sac vascularization. Additionally, mRNA levels of Scl and Gata1 were reduced by approximately 80% in Klf6-/- yolk sacs. To further analyze this phenotype, we generated Klf6-/- embryonic stem (ES) cells by homologous recombination, and compared their capacity to differentiate into the hematopoietic lineage with that of either Klf6+/- or Klf6+/+ ES cells. Consistent with the phenotype in the early embryo, Klf6-/- ES cells displayed significant hematopoietic defects following differentiation into EBs. Prolongation of epiblast-like cells and delays in mesoderm induction were also observed in the Klf6-/- EBs, associated with delayed expression of Brachyury, Klf1, and Gata1. Forced expression of KLF6 using a tet-inducible system enhanced the hematopoietic potential of wild-type EBs. Collectively, these findings implicate Klf6 in ES-cell differentiation and hematopoiesis.

Correlation between mRNA levels and functional role of α1-adrenoceptor subtypes in arteries: evidence of α1L as a functional isoform of the α1A-adrenoceptor

2005 ◽  
Vol 289 (5) ◽  
pp. H1923-H1932 ◽  
Author(s):  
Daniel Martí ◽  
Raquel Miquel ◽  
Khalid Ziani ◽  
Regina Gisbert ◽  
M. Dolores Ivorra ◽  
...  
Keyword(s):  
Mesenteric Artery ◽  
Mesenteric Arteries ◽  
Main Role ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Response Curves ◽  
Inhibitory Potency ◽  
Adrenoceptor Antagonist ◽  
Relevant Role

The mRNA levels for the three α1-adrenoceptor subtypes, α1A, α1B, and α1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The α1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), α1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both α1A- and α1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the α1B-subtype were a minority in all vessels (1.7–11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to α1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 μmol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro ( 4 , 5 ) decane-7-dionedihydrochloride} (BMY-7378, an α1D-adrenoceptor antagonist) confirm the relevant role of α1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an α1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of α1A- and α1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of α1A/α1L-adrenoceptors with minor participation of the α1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the α1L-adrenoceptor could be a functional isoform of the α1A-subtype.

scholarly journals Interleukin-1 Receptors Are Differentially Expressed in Normal and Psoriatic T Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Attila Bebes ◽  
Ferenc Kovács-Sólyom ◽  
Judit Prihoda ◽  
Róbert Kui ◽  
Lajos Kemény ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Interleukin 1 ◽  
Effector T Cells ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Healthy Control

This study was carried out to examine the possible role of interleukin-1 (IL-1) in the functional insufficiency of regulatory T cells in psoriasis, by comparing the expression of IL-1 receptors on healthy control and psoriatic T cells. Patients with moderate-to-severe chronic plaque psoriasis and healthy volunteers, matched in age and sex, were selected for all experiments. CD4+CD25−effector and CD4+CD25+CD127lowregulatory T cells were separated and used for the experiments. Expression of the mRNA of IL-1 receptors (IL-1R1, IL-1R2, and sIL-1R2) was determined by quantitative real-time RT-PCR. Cell surface IL-1 receptor expression was assessed by flow cytometry. Relative expression of the signal transmitting IL-1 receptor type 1 (IL-1R1) mRNA is higher in resting psoriatic effector and regulatory T cells, and activation induces higher IL-1R1 protein expression in psoriatic T cells than in healthy cells. Psoriatic regulatory and effector T cells express increased mRNA levels of the decoy IL-1 receptors (IL-1R2 and sIL-1R2) upon activation compared to healthy counterparts. Psoriatic T cells release slightly more sIL-1R2 into their surrounding than healthy T cells. In conclusion, changes in the expression of IL-1 receptors in psoriatic regulatory and effector T cells could contribute to the pathogenesis of psoriasis.

245 EXPRESSION AND FUNCTIONAL ROLE OF CELL DIVISION CYCLE 42 IN MOUSE OOCYTES MATURATION AND PRE-IMPLANTATION DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 230
Author(s):  
X.-S. Cui ◽  
X.-Y. Li ◽  
N.-H. Kim
Keyword(s):  
Protein Synthesis ◽  
Cell Division ◽  
Mrna Expression ◽  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Cell Division Cycle ◽  
Mrna Levels ◽  
Mouse Oocytes

Cell division cycle 42 (Cdc42), a member of the Rho family of small guanosine triphosphatase (GTPase) proteins, regulates multiple cell functions, including motility, proliferation, apoptosis, and cell morphology. In order to gain insight into the role of Cdc42 in embryo development, we first characterized mRNA and protein levels of Cdc42 in mouse oocytes and early embryogenesis. We then examined the possible role of the gene in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundance of Cdc42 transcripts were measured by real time RT-PCR. After normalization with histone H2a mRNA levels, the mRNA expression of Cdc42 was abundant in immature oocytes and reduced slightly in zygotes and 2- to 8-cell stage embryos. The expression levels were significantly increased during the morula and blastocyst stages. Indirect immunocytochemistry showed protein synthesis of Cdc42 in oocytes and embryos of all stages. Introducing small interference RNA (siRNA) of Cdc42 into germinal vesicle stage oocytes or zygotes specifically reduce both mRNA expression and protein synthesis of Cdc42 in metaphase II stage oocytes and early embryos developing in vitro. Meiotic maturation was significantly reduced following siRNA injection into germinal vesicle stage oocytes. It is evident that actin distribution in siRNA treated blastocysts is morphologically abnormal following injection of siRNA for Cdc42. Injection of siRNA into zygotes did not influence cleavage, but significantly decreased in vitro development to morulae and blastocysts. While housekeeping genes such as tissue plasminogen activator were not altered by siRNA, wiskott-aldrich syndrome protein family 1 (WASP1) mRNA was down-regulated in the morula. Interestingly, mRNA of WASP1, tubulin alpha 1 (Tuba1), and actin-related protein 2/3 complex subunit V (Arpc5) increased at the blastocyst stage following siRNA injection. These results suggest that Cdc42 plays an important role during oocyte maturation and early pre-implantation development, likely through linkage with several other genes. This work was funded by a grant from National Research Laboratory Program in Korea.

Role of E2F3 and shugoshin I in epithelial-to-mesenchymal transition and cell invasion in breast cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13100-e13100
Author(s):  
Shirley Jusino ◽  
Srikumar P. Chellappan ◽  
Harold I. Saavedra
Keyword(s):  
Breast Cancer ◽  
Cell Invasion ◽  
Cancer Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Cellular Proliferation ◽  
Protein Localization ◽  
Breast Cancer Metastasis ◽  
Mrna Levels ◽  
Mesenchymal Transition

e13100 Background: Triple-negative breast cancer (TNBC) is the most aggressive and poorly prognostic breast cancer subtype, yet there are currently no biological therapies against this subtype. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. While the deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT), the underlining mechanism of how E2Fs drive EMT in TNBC remains unknown. We recently published that the E2F transcriptional activators (E2Fs) are overexpressed in the vast majority of TNBC and that their overexpression upregulates mitotic kinases such as TTK, which we have shown to induce EMT and invasion in TNBC cells. We also demonstrated that the E2Fs maintain genomic integrity in part through Shugoshin I (SGO1), which normally controls chromosome cohesion; however, the role of SGO1 in EMT in breast cancer is unknown. Our hypothesis is that E2F3 and SGO1 are highly expressed in TNBC and that their overexpression modulates EMT genes, thus promoting cell invasion. Methods: To test our hypothesis, we conducted siRNA transfection to knockdown E2F3 and SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after E2F3 or SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Results: Our results demonstrate that E2F3 and SGO1 depletion decrease MMP3 mRNA levels. Moreover, E2F3 and SGO1 depletion restore E-cadherin expression and localization. Furthermore, E2F3 and SGO1 depletion significantly reduce cell invasion in MDA-MB-231 and Hs578t cells. Conclusions: Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis and E2F3 is presently undruggable.

scholarly journals Autophagic activation in vitrified–warmed mouse oocytes

Reproduction ◽  
2014 ◽  
Vol 148 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Soyoung Bang ◽  
Hyejin Shin ◽  
Haengseok Song ◽  
Chang Suk Suh ◽  
Hyunjung Jade Lim
Keyword(s):  
Slight Reduction ◽  
Mrna Levels ◽  
Imaging Analysis ◽  
Response Mechanism ◽  
Rt Pcr ◽  
Mouse Oocytes ◽  
Lc3 Puncta ◽  
First Time ◽  
Cellular Stressors ◽  
Warming Process

Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN2), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified–warmed oocytes has not been examined. In this work, we investigated whether the vitrification–warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN2for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that severalAtggenes such asAtg5,Atg7,Atg12,LC3a(Map1lc3a),LC3b(Map1lc3b), andBeclin1were expressed in MII mouse oocytes. Slight reduction in mRNA levels ofAtg7andAtg12in vitrified–warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified–warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified–warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified–warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified–warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein.

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