Dataset Information

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

ABSTRACT:

Background

PGF2? is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2?. EGR1 is involved in the transactivation of many genes, including TGF?1, which plays an important role during luteal regression.

Methods

The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGF?1 during PGF2? induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2? for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3?HSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2? induced luteal regression via induction of TGF?1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9.

Result

The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2? treatment. Quantification of EGR1 and TGF?1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2? induction. In order to validate the role of PGF2? on stimulating the expression of TGF?1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2? and it was observed that EGR1 KO did not modulate the PGF2? induced expression of TGF?1. In PGF2? treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2? treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2? treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2?-treated wild type luteal cells.

Conclusion

These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2? induced TGF?1 signaling for luteolysis.

SUBMITTER: Punetha M

PROVIDER: S-EPMC7953609 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

Punetha Meeti M Kumar Sai S Paul Avishek A Jose Bosco B Bharati Jaya J Sonwane Arvind A Green Jonathan A JA Whitworth Kristin K Sarkar Mihir M

Biological research 20210312 1

<h4>Background</h4>PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFβ1, which plays an important role during luteal regression.<h4> ...[more]

PMID: 33712084

Similar Datasets

Project description:BackgroundThe corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.MethodsPorcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha.ResultsWe obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures.ConclusionOur data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.

Project description:BackgroundDuring female reproductive cycles, a rapid fall in circulating progesterone (P4) levels is one of the earliest events that occur during induced luteolysis in mammals. In rodents, it is well recognized that during luteolysis, P4 is catabolized to its inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP) by the action of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) enzyme and involves transcription factor, Nur77. Studies have been carried out to examine expression of 20alpha-HSD and its activity in the corpus luteum (CL) of buffalo cow.MethodsThe expression of 20alpha-HSD across different bovine tissues along with CL was examined by qPCR analysis. Circulating P4 levels were monitored before and during PGF2alpha treatment. Expression of 20alpha-HSD and Nur77 mRNA was determined in CL at different time points post PGF2alpha treatment in buffalo cows. The chromatographic separation of P4 and its metabolite, 20alpha-OHP, in rat and buffalo cow serum samples were performed on reverse phase HPLC system. To further support the findings, 20alpha-HSD enzyme activity was quantitated in cytosolic fraction of CL of both rat and buffalo cow.ResultsCirculating P4 concentration declined rapidly in response to PGF2alpha treatment. HPLC analysis of serum samples did not reveal changes in circulating 20alpha-OHP levels in buffalo cows but serum from pseudo pregnant rats receiving PGF2alpha treatment showed an increased 20alpha-OHP level at 24 h post treatment with accompanying decrease in P4 concentration. qPCR expression of 20alpha-HSD in CL from control and PGF2alpha-treated buffalo cows showed higher expression at 3 and 18 h post treatment, but its specific activity was not altered at different time points post PGF2alpha treatment. The Nur77 expression increased several fold 3 h post PGF2alpha treatment similar to the increased expression observed in the PGF2alpha-treated pseudo pregnant rats which perhaps suggest initiation of activation of apoptotic pathways in response to PGF2alpha treatment.ConclusionsThe results taken together suggest that synthesis of P4 appears to be primarily affected by PGF2alpha treatment in buffalo cows in contrast to increased metabolism of P4 in rodents.

Project description:In monoovulatory species like bovines, a striking diversity in regulation of the corpus luteum (CL) function exists within species during different stages of the luteal phase. The function and life span of CL appears to be regulated by the interplay of both luteotrophic and luteolytic factors, whose roles and actions are varied from one species to another. Although, studies continue to expand our current understanding of the cellular and molecular actions of prostaglandin F 2alpha (PGF2M-NM-1 - a physiological luteolysin)-induced luteolysis, knowledge of the mechanism whereby, PGF2M-NM-1 mediates its luteolytic actions remains poorly understood. The mechanism of PGF2M-NM-1-induced luteolysis is a complex process involving changes in expression of multiple genes encoding gene products that regulate steroidogenesis, stress related or apoptotic genes, factors involved in immune response and enzymes that stimulate PGF2M-NM-1 production. Thus, in the present study, efforts were made to identify the temporal changes in the global gene expression profile in the CL of buffalo cows in response to PGF2M-NM-1 treatment. The molecular signature of genome-wide transcriptional changes in the CL tissue subjected to PGF2M-NM-1-induced luteolysis will expand our knowledge on basic mechanism/s that regulates the luteolytic process. The results obtained in this study provide insight into the mechanisms underlying the PGF2M-NM-1- induced regression of CL.Keywords: CL, PGF2M-NM-1, gene expression The objective of this study was to identify the PGF2M-NM-1-induced temporal changes in transcriptome of bovine CL on day 11 of estrous cycle. In the experiments with buffalo cows, animals with history of normal estrous cyclicity were treated with Cloprostenol (an PGF2M-NM-1 analogue; 500 M-BM-5g i.m.) on day 11 of the estrous cycle to induce luteolysis and CL tissues were collected at (control; n=4 amimals/time point), 3 (n=2 animals/time point), 6 and 18 (n=3 animals/time point) h post PGF2M-NM-1 administration to examine gene expression changes associated with immediate early and delayed changes in the CL, respectively. Following retrieval of CL from the ovary, flash frozen in liquid nitrogen and stored at -70 M-BM-0C for the purpose of RNA isolation. The isolated RNA was labeled and hybridized to Affymetrix GeneChip Bovine Genome Arrays as per the manufacturerM-bM-^@M-^Ys instructions.

Dataset Information

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

Background

Methods

Result

Conclusion

Publications

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure