267 CLASSICAL AND NON-CLASSICAL MAJOR HISTOCOMPATABILITY COMPLEX CLASS I GENE EXPRESSION IN IN VITRO-DERIVED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 250
Author(s):  
J. Doyle ◽  
S. A. Ellis ◽  
P. Lonergan ◽  
T. Fair
Keyword(s):  
Gene Expression ◽  
Sequence Analysis ◽  
Mrna Expression ◽  
Complex Class ◽  
Mhc I ◽  
Expression Levels ◽  
Pcr Products ◽  
Bovine Oocytes ◽  
I Gene

The major histocompatibility complex class I (MHC-I) genes have been shown to play a role in the establishment of pregnancy in humans and mice. In particular, the secretion of the soluble isoform of the human non-classical HLA-G gene during in vitro embryo culture has been linked to higher pregnancy rates following embryo transfer. In contrast to the situation in humans, knowledge of the bovine MHC-I is quite limited. The objective of the current study was to characterize MHC-I gene expression during bovine pre-implantation development in vitro. Immature bovine oocytes were matured, fertilized, and cultured in vitro. Three replicate pools of 20 samples were collected at immature and mature oocyte, presumptive zygote, 2–4 cell, 8–16 cell, morula, blastocyst, and hatched blastocyst stages. Relative MHC-I mRNA expression levels were quantified using quantitative real-time PCR across pre-implantation development with a generic primer pair for global classical and non-classical MHC-I gene expression and primer pairs for two known non-classical MHC-I genes, N*50001 and N*50101 (NCBI accession numbers: X80936 and AY188807), cDNA sequencing, and wholemount immunocytochemistry with a Pan MHC-I-protein antibody (ILA88). MHC-I mRNA transcripts amplified by the generic primers were detected in greatest abundance in the oocyte and cumulus cells of immature oocytes (P < 0.05); following maturation, the expression levels decreased by 5-fold but remained stable until the 2- to 4-cell stage. Levels remained low until the morula and blastocyst stages when a small increase in relative expression was detected. Sequence analysis of oocyte and blastocyst PCR products amplified by the generic primers was carried out. The generic primers amplified a total of 6 discrete sequences, which are listed on the bovine MHC database (http://www.ebi.ac.uk/ipd/mhc/bola): (i) N*02601, (ii) N*02301, (iii) N*01301, (iv) N*04001, (v) N*01601, and (vi) non-classical N*5003. The mRNA expression pattern of Gene X differed from the global MHC-I mRNA profile in that mRNA abundance was highest in the matured oocytes (P < 0.05), after which abundance decreased 5-fold in the zygote and was not detected in the subsequent embryo stages until the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were Gene X. Relative HD15 mRNA expression was highest in immature oocytes (P < 0.05); however, there was a steep decrease in abundance following oocyte maturation (P < 0.05), followed by a transient increase at the presumptive zygote stage (P < 0.05). Thereafter, expression levels remained low up to the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were HD15. MHC-I protein expression was detected in immature and mature oocytes and their surrounding granulosa cells, in 2- and 4-cell embryos, and in blastocysts. In conclusion, these data demonstrate that classical and non-classical MHC-I genes are expressed in bovine oocytes and embryos at both the mRNA and the protein levels, and the expression pattern is gene- and stage-specific.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

Classical and non-classical Major Histocompatibility Complex class I gene expression in in vitro derived bovine embryos

2009 ◽  
Vol 82 (1) ◽  
pp. 48-56 ◽  
Author(s):  
Julie Doyle ◽  
Shirley A. Ellis ◽  
Grace M. O’Gorman ◽  
Ines Maria Aparicio Donoso ◽  
Patrick Lonergan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Class I ◽  
Complex Class ◽  
Bovine Embryos ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
I Gene

scholarly journals Expression of mRNAs for pro-and anti-apoptotic factors in granulosa cells and follicular fluid of women undergoing in vitro fertilization. A pilot study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
József Bódis ◽  
Endre Sulyok ◽  
Ákos Várnagy ◽  
Viktória Prémusz ◽  
Krisztina Gödöny ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract Background This observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF). Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3 ± 5.8 years, body mass index: 24.02 ± 3.12 kg/m2, duration of infertility: 4.2 ± 2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r = 0.393, p = 0.029), but the day of embryo transfer was negatively associated with GC LHR (r = − 0.414, p = 0.020) and GC FSHR transcripts (r = − 0.535, p = 0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs. Conclusion Our study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

scholarly journals Function of JARID2 in bovines during early embryonic development

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4189 ◽  
Author(s):  
Yao Fu ◽  
Jia-Jun Xu ◽  
Xu-Lei Sun ◽  
Hao Jiang ◽  
Dong-Xu Han ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Embryonic Stem ◽  
Early Embryonic Development ◽  
Mouse Development ◽  
Expression Levels ◽  
Relative Expression ◽  
Bovine Oocytes

Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC) differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV), MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05), and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc) after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the efficiency ofin vitroembryo culture as well as a theoretical basis for further studying the regulatory mechanisms involved in early embryonic development.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

scholarly journals In Vitro and in Vivo Calcitonin I Gene Expression in Parenchymal Cells: A Novel Product of Human Adipose Tissue

Endocrinology ◽  
2003 ◽  
Vol 144 (12) ◽  
pp. 5578-5584 ◽  
Author(s):  
Philippe Linscheid ◽  
Dalma Seboek ◽  
Eric S. Nylen ◽  
Igor Langer ◽  
Mirjam Schlatter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Human Adipose Tissue ◽  
Parenchymal Cells ◽  
I Gene

The Low Expression of DUSP1 and HSPA5 Inhibits Chicken Immune Response and Causes Decreased Immunity under Heat Stress

2019 ◽  
Author(s):  
Wenwu Zhang ◽  
Jiguo Xu ◽  
Hongmei Li ◽  
Lianghui Zhou ◽  
Qinghua Nie ◽  
...  
Keyword(s):  
Immune Response ◽  
Heat Stress ◽  
Mrna Expression ◽  
Production Performance ◽  
Bursa Of Fabricius ◽  
Mhc I ◽  
Expression Levels ◽  
Mhc Ii ◽  
Stress Related Genes ◽  
Immune Related Genes

Abstract Background As a major stressor, high temperatures negatively affect the poultry industry, through impairments to chicken immunity and production performance. The purpose of this study is to clarify how chicken immune systems responded to heat stress with and without immunization. In the present study, spleen and bursa of Fabricius of experimental chickens were subjected to RNA-seq. Key genes influencing immune response in heat-stressed chickens were identified and their functions validated. Results Immunized and heat-stressed chickens experienced a significant reduction in immune function. The expression of immune-related genes and heat stress-related genes in the spleen increased after immunization and decreased after heat stress, but in the bursa of Fabricius, few of these genes were differentially expressed after immunization and heat stress, indicating insensitivity to high temperature and the lack of vaccine processing. In the non-heat-stressed groups, spleen expression of DUSP1 and HSPA5 decreased significantly, suggesting their relationship to immunity. Upon DUSP1 or HSPA5 overexpression, the mRNA expression of MHC-I, MHC-II, CD80, CD86, CD1C, IL1B, IL6, and TLR4 was earlier than that under LPS stimulation only, indicating that DUSP1 or HSPA5 overexpression enhances HD11 recognition LPS. Inhibiting DUSP1 or HSPA5 expression, the mRNA expression levels of MHC-I, MHC-II, CD80, CD86, IL6 and TLR4 did not change significantly from LPS-stimulation-only levels but CD1C significantly decreased, suggesting that HD11 recognition of LPS is affected by DUSP1 or HSPA5 expression levels. Conclusions The inhibition of immune response due to lowly expressed DUSP1 and HSPA5 may be the cause of decreased immunity in chickens.

scholarly journals Therapeutic Potential of Thrombomodulin in Renal Fibrosis of Nephrotoxic Serum Nephritis in Wistar-Kyoto Rats

2020 ◽  
Vol 45 (3) ◽  
pp. 391-406
Author(s):  
Nobuhiro Kanazawa ◽  
Masayuki Iyoda ◽  
Shohei Tachibana ◽  
Kei Matsumoto ◽  
Yukihiro Wada ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Renal Fibrosis ◽  
Therapeutic Potential ◽  
The Body ◽  
Expression Levels ◽  
Nephrotoxic Serum Nephritis ◽  
Collagen Iii ◽  
Wistar Kyoto ◽  
Mrna Expression Levels

Background: Recombinant human soluble thrombomodulin (rhTM) was approved in 2008 and has been used for treatment of disseminated intravascular coagulation in Japan. The antifibrotic effects of rhTM in acute exacerbation of idiopathic pulmonary fibrosis are well established, but the therapeutic potential of rhTM in renal fibrosis remains poorly understood. Methods: Nephrotoxic serum nephritis (NTS-N) was induced in 22 female Wistar-Kyoto (WKY) rats on day 0. Rats were administered either rhTM or vehicle intraperitoneally, every day from day 4 to day 55. Rats were sacrificed on day 56 when renal fibrosis was established and renal morphological investigations were performed. In vitro, rat renal fibroblasts (NRK-49F) were pretreated with rhTM or saline, and expression levels of profibrogenic gene induced by thrombin were analyzed by real-time reverse transcription polymerase chain reaction. Results: Compared to WKY-GN-vehicle rats, the body weights of WKY-GN-rhTM rats were significantly greater on day 55. By day 56, rhTM had significantly reduced serum creatinine levels in NTS-N. On the other hand, urinary protein excretion was comparable between the two treatment groups throughout the study. The percentage of Masson trichrome-positive areas in WKY-GN-rhTM rats was significantly lower compared to that in WKY-GN-vehicle rats. Glomerular fibrin deposition was significantly reduced in WKY-GN-rhTM rats. In addition, rhTM significantly reduced the renal cortical mRNA expression levels of TNF-α, Toll-like receptor 4, MYD88, TGF-β, αSMA, collagen I, collagen III, fibronectin, and protease-activated receptor 1 (PAR1), a thrombin receptor. In vitro, thrombin stimulation of NRK-49F cells significantly enhanced the mRNA expression levels of αSMA and PAR1, and these upregulations were significantly reduced by pretreatment with rhTM. Conclusions: Administration of rhTM after establishment of crescentic glomerulonephritis (GN) attenuated the subsequent development of renal fibrosis in NTS-N, possibly in part by inhibiting thrombin-mediated fibrogenesis. Our results suggest that rhTM may offer a therapeutic option for limiting the progression of chronic kidney disease in crescentic GN.

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