Paper 3 by Velásquez Cumplido

About: Mating changes the Subcellular distribution and the functionality of estrogen receptors in the rat oviduct



Abstract

  • Background: Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS).
  • Methods: Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats.
  • Results: Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c- fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats.
  • Conclusion: Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct.

Conclusion

Estrogen receptors ESR1 and ESR2 present a subcellular distribution in oviductal epithelial cells that is compatible with genomic and nongenomic actions of E2 in the rat ovi- duct. Mating is associated with changes in the basal and E2-induced subcellular distribution of ESR1 and ESR2 in these cells although it did not clearly explain IPS. Further- more, mating affected signaling of both ER in the oviduct and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the intraoviductal ER sig- nalling pathways exerted by mating.

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Paper 2 by Velásquez Cumplido

About: Participation of the oviductal s100 calcium binding protein G in the genomic effect of estradiol that accelerates oviductal embryo transport in mated rats.



Abstract

  • Background: Mating changes the mechanism by which E2 regulates oviductal egg transport, from a non-genomic to a genomic mode. Previously, we found that E2 increased the expression of several genes in the oviduct of mated rats, but not in unmated rats. Among the transcripts that increased its level by E2 only in mated rats was the one coding for an s100 calcium binding protein G (s100 g) whose functional role in the oviduct is unknown.
  • Methods: Herein, we investigated the participation of s100 g on the E2 genomic effect that accelerates oviductal transport in mated rats. Thus, we determined the effect of E2 on the mRNA and protein level of s100 g in the oviduct of mated and unmated rats. Then, we explored the effect of E2 on egg transport in unmated and mated rats under conditions in which s100 g protein was knockdown in the oviduct by a morpholino oligonucleotide against s100 g (s100 g-MO). In addition, the localization of s100 g in the oviduct of mated and unmated rats following treatment with E2 was also examined.
  • Results: Expression of s100 g mRNA progressively increased at 3-24 h after E2 treatment in the oviduct of mated rats while in unmated rats s100 g increased only at 12 and 24 hours. Oviductal s100 g protein increased 6 h following E2 and continued elevated at 12 and 24 h in mated rats, whereas in unmated rats s100 g protein increased at the same time points as its transcript. Administration of a morpholino oligonucleotide against s100 g transcript blocked the effect of E2 on egg transport in mated, but not in unmated rats. Finally, immunoreactivity of s100 g was observed only in epithelial cells of the oviducts of mated and unmated rats and it was unchanged after E2 treatment.
  • Conclusions: Mating affects the kinetic of E2-induced expression of s100 g although it not changed the cellular localization of s100 g in the oviduct after E2 . On the other hand, s100 g is a functional component of E2 genomic effect that accelerates egg transport. These findings show a physiological involvement of s100 g in the rat oviduct.

Mariana Ríos1, Alexis Parada-Bustamante1, Luis A Velásquez Cumplido 2,3, Horacio B Croxatto2,3,4 and Pedro A Orihuela2,3*

Conclusions

IMating changes the kinetic of E2-induced expression of s100 g in the oviduct although cellular localization of s100 g was not affected by mating following E2 treat- ment. Furthermore, the E2 intraoviductal genomic path- way that accelerates embryo transport in mated rats Page 8 of 9 requires functional participation of s100 g. These find- ings provide evidence of a physiological involvement of s100 g in the rat oviduct.

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Paper 1 by Velasquez Cumplido

About: Differences in the endometrial transcript profile during the receptive period between women who were refractory to implantation and those who achieved pregnancy.

Gene expression profiling of normal receptive endometrium has been characterized, but intrinsic defects in endometrial gene expression associated with implantation failure have not been reported. METHODS: Women who had previously participated as recipients in oocyte donation cycles and repeatedly exhibited implantation failure (Group A, study group) or had at least one successful cycle (Group B, control group) and spontaneously fertile women (Group C, normal fertility group) were recruited. All were treated with exogenous estradiol and progesterone to induce an endometrial cycle, and an endometrial biopsy was taken on the seventh day of progesterone adminis- tration. RNA from each sample was analysed by cDNA microarrays to identify differentially expressed genes between groups. RESULTS: 63 transcripts were differentially expressed (􏰀2-fold) between Groups A and B, of which 16 were subjected to real time RT–PCR. Eleven of these were significantly decreased in Group A with regard to Groups B and C. Among the dysregulated genes were MMP-7, CXCR4, PAEP and C4BPA. CONCLUSIONS: Repeated implantation failure in some oocyte recipients is associated with an intrinsic defect in the expression of multiple genes in their endometrium. Significantly decreased levels of several transcripts in endome- tria without manifest abnormalities is demonstrated for the first time and shown to be associated with implantation failure.

Funding


Funding for this work was provided by CONRAD (CIG-02-83 to L.V.); CONICYT (Beca Apoyo para realizacio ́n de Tesis Doctoral 2002 to A.T.); Beca Fulbright-CONICYT (to A.T.); DICYT (to L.V.) and with Federal Funds from the National Cancer Institute, National Institutes of Health, under contract no. N01-CO-12400 (Article H.36 of the Prime Contract). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Ser- vices, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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