Browsing by Author "Parada-Bustamante, A"

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    Effect of intrauterine insemination with spermatozoa or foreign protein on the mechanism of action of oestradiol in the rat oviduct
    (2003) Parada-Bustamante, A; Orihuela, PA; Croxatto, HB
    Previously, we showed that oestradiol accelerates oviductal egg transport through a non-genomic action involving oviductal protein phosphorylation in non-mated rats, and through a genomic action in mated rats. Thus, sensory stimulation, seminal fluid or sperm cells may be the source of signals that switch the mechanism of action of oestradiol in the oviduct to a genomic pathway. The present study examined the ability of spermatozoa to switch the mode of action of oestradiol in the absence of the sensory stimulation and seminal fluid provided by mating. Prooestrous rats were inseminated in each uterine horn with epididymal spermatozoa and 12 h later were injected subcutaneously with oestradiol and intrabursally with the mRNA synthesis inhibitor a-amanitin. The number of eggs in the oviduct, assessed 24 h later, showed that a-amanitin blocked the oestradiol-induced egg transport acceleration, indicating that the interaction of spermatozoa with the genital tract shifts the action of oestradiol from nongenomic to genomic. Other rats were inseminated with live or dead spermatozoa and then treated with the protein kinase inhibitor H-89, and oestradiol. Treatment with H-89 did not block the oestradiol-induced acceleration of egg transport in these rats, although dead spermatozoa did not enter the oviduct, indicating that the mere presence of spermatozoa in the uterus abrogated the non-genomic action of oestradiol in the oviduct. Treatment with H-89 also failed to prevent the acceleration of oviductal egg transport induced by oestradiol in rats inseminated with hamster spermatozoa or with BSA, whereas in rats inseminated with their own serum (autologous proteins), H-89 was able to prevent the effect of oestradiol. This finding reveals that the effect of insemination on the mode of action of oestradiol is neither species- nor sperm-specific and it is produced by foreign organic material. It can be concluded that the presence of spermatozoa or foreign protein in the uterus is one of the components of mating that is capable of switching the action of oestradiol in the oviduct from a non-genomic to a genomic mode.
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    Estrogen receptor, cyclic adenosine monophosphate, and protein kinase A are involved in the nongenomic pathway by which estradiol accelerates oviductal oocyte transport in cyclic rats
    (2003) Orihuela, PA; Parada-Bustamante, A; Cortés, PP; Gatica, C; Croxatto, HB
    This investigation examined the role of estrogen receptor (ER) on the stimulatory effect of estradiol (E) on protein phosphorylation in the oviduct as well as on E-2-induced acceleration of oviductal oocyte transport in cyclic rats. Estrous rats were injected with E-2 s.c. and with the ER antagonist 10 182 780 intrabursally (i.b.), and 6 h later, oviducts were excised and protein phosphorylation was determined by Western blot analysis. ICI 182780 inhibited the E-2-induced phosphorylation of some oviductal proteins. Other estrous rats were treated with E-2 s.c. and ICI 182 780 i.b. The number of eggs in the oviduct, assessed 24 h later, showed that ICI 182 780 blocked the E-2-induced egg transport acceleration. The possible involvement of adenylyl cyclase, protein kinase A (PK-A), protein kinase C (PK-C), or tyrosine kinases on egg transport acceleration induced by E-2 was then examined. Selective inhibitors of adenylyl cyclase or PK-A inhibited the E-2-induced egg transport acceleration, whereas PK-C or tyrosine kinase inhibitors had no effect. Furthermore, forskolin, an adenylyl cyclase activator, mimicked the effect of E-2 on ovum transport and E, increased the level of cAMP in the oviduct of cycling rats. Finally, we measured PK-A activity in vitro in the presence of E-2 or E-2-ER complex. Activity of PK-A in the presence of E-2 or E-2-ER was similar to PK-A alone, showing that E-2 or E-2-ER did not directly activate PK-A. We conclude that the nongenomic pathway by which E-2 accelerates oviductal egg transport in the rat requires absolute participation of ER and cAMP and partial participation of PK-A signaling pathways in the oviduct.
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    Inositol triphosphate participates in an oestradiol nongenomic signalling pathway involved in accelerated oviductal transport in cycling rats
    (2006) Orihuela, PA; Parada-Bustamante, A; Zuñiga, LM; Croxatto, HB
    Oestradiol (E,) accelerates oviductal transport of oocytes in cycling rats through a nongenomic pathway that involves the cAMP-PKA signalling cascade. Here we examined the role of the inositol triphosphate (IP3) and mitogen-activated protein kinase (MAPK) signalling cascades in this nongenomic pathway. Oestrous rats were injected with E-2 s.c. and intrabursally (i.b) with the selective inhibitors of phospholipase C (PLC) ET-18OCH(3) or MAPK PD98059. The number of eggs in the oviduct assessed 24 h later showed that ET-18-OCH3 blocked E-2-induced egg transport acceleration, whereas PD980559 had no effect. Other oestrous rats were treated with E-2 s.c. and 1, 3 or 6 h later oviducts were excised and the levels of IP3 and phosphorylated MAPK p44/42 (activated) were determined by radioreceptor assay and Western blot, respectively. Oestradiol administration increased IP3 level at 1 and 6 h after treatment, whereas activated MAPK p44/42 level was unchanged. Finally, e 0 1 we explored whether cAMP-PKA and PLC-IP3 signalling cascades are coupled. Inhibition of adenylyl cyclase by i.b. injection of SQ 22536 blocked the increase of IP3 levels induced by E, while inhibition of PLC by ET-18OCH(3) had no effect on E(2-)induced PKA activity. Furthermore, activation of adenylyl cylase by Forskolin increased oviducral IP3 levels. Thus, activation of PLC-IP3 by E, requires previous stimulation of cAMP-PKA. We conclude that the nongenomic pathway utilised by E, to accelerate oviductal transport of oocytes in cycling rats involves Successive activation of the cAMP-PKA and PLC-IP3 signalling cascades and does not require activation of MAPK. These findings clearly illustrate a non-genomic pathway triggered by E-2 that regulates a complex physiologic process accomplished by in entire organ.