Browsing by Author "Croxatto, HB"
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- ItemA segment and epithelium specific messenger ribonucleic acid fragment up-regulated by estradiol in the rat oviduct(2001) Rios, M; Ojeda, S; Velasquez, LA; Maisey, K; Croxatto, HBEstradiol accelerates oviductal embryo transport in the rat through changes of genomic expression in oviductal cells. However, the genes involved are unknown. We used a differential display by reverse transcription-polymerase chain reaction to detect estradiol (E-2)-dependent genes in the rat oviduct. Rats on day 2 of pregnancy were untreated or treated with 10 mug of E-2 and the oviducts were extracted at 30, 180 and 360 min later and used to isolate RNA. products of reverse transcriptase-PCR, made with pairs of arbitrary and oligo-deoxythymidine primers, were separated on denaturing polyacrylamide gels and candidate bands were excised and reamplified. Truly positive cDNA fragments determined by a single strand conformation polymorphism assay were cloned and sequenced. A ribonuclease protection assay confirmed that clone 25 is up-regulated by E-2 in the oviduct at 30, 180 and 360 min. This clone exhibited no homology with known genes and in situ hybridization showed it is only expressed in the epithelial cells of the isthmic segment. Clone 25 is likely to represent a new gene. which is upregulated by E-2 in the epithelium of the isthmic segment of the rat oviduct. Its rime frame of response is compatible with a mediator or the effect of E-2 on oviductal embryo transport.
- ItemAcceleration of oviductal transport of oocytes induced by estradiol in cycling rats is mediated by nongenomic stimulation of protein phosphorylation in the oviduct(2001) Orihuela, PA; Croxatto, HBIn order to explore nongenomic actions of estradiol (E,) and progesterone (P-4) in the oviduct, we determined the effect of E-2 and P-4 on oviductal protein phosphorylation. Rats on Day 1 of the cycle (C1) or pregnancy (P1) were treated with E-2, P-4, or E-2 + P-4, and 0.5 h or 2.5 h later their oviducts were incubated in medium with P-32-orthophosphate for 2 h. Oviducts were homogenized and proteins were separated by SDS-PAGE. Following autoradiography, protein bands were quantitated by densitometry. The phosphorylation of some proteins was increased by hormonal treatments, exhibiting steroid specificity and different individual time courses. Possible mediation of the E-2 effect by mRNA synthesis or protein kinases A (PK-A) or C (PK-C) was then examined. Rats on C1 treated with E-2 also received an intrabursal (i.b.) injection of alpha -amanitin (Am), or the PK inhibitors H-89 or GF 109203X, and 0.5 h later their oviducts were incubated as above plus the corresponding inhibitors in the medium. increased incorporation of P-32 into total oviductal protein induced by E-2 was unchanged by Am, whereas it was completely suppressed by PK inhibitors. Local administration of H-89 was utilized to determine whether or not E-2-induced egg transport acceleration requires protein phosphorylation. Rats on C1 or P1 were treated with E-2 s.c. and H-89 Lb. The number and distribution of eggs in the genital tract assessed 24 h later showed that H-89 blocked the E-2-induced oviductal egg loss in cyclic rats and had no effect in mated rats. It is concluded that E-2 and P-4 change the pattern of oviductal protein phosphorylation. Estradiol increases oviductal protein phosphorylation in cyclic rats due to a nongenomic action mediated by PK-A and PK-C. In the abscence of mating, this action is essential for its oviductal transport accelerating effect. Mating changes the mechanism of action of E-2 in the oviduct by waiving this nongenomic action as a requirement for E-2-induced embryo transport acceleration.
- ItemAn estradiol metabolite accelerates ovum transport in cyclic rats through non-genomic pathways(W B SAUNDERS CO LTD, 2006) Ambriz, GD; Parada Bustamante, A; Orihuela, PA; Ortiz, ME; Villalon, MJ; Croxatto, HB
- ItemBasic aspects of oviduct function(PARTHENON PUBLISHING GROUP LTD, 1997) Croxatto, HB; Ortiz, ME; Villalon, M; Cardenas, H; Imarai, M; Hermoso, M; Velasquez, L; Orihuela, P; Coutifaris, C; Mastroianni, L
- ItemDisparate effects of estradiol on egg transport and oviductal protein synthesis in mated and cyclic rats(2001) Orihuela, PA; Ríos, M; Croxatto, HBPreviously, we found that the dose of estradiol (E-2) require to accelerate egg transport increases 5- to 10-fold, in mated compared to cyclic rats. Here we examined protein synthesis in the oviduct of mated and cyclic rats following a single injection of E-2 known to accelerate oviductal egg transport or after concomitant treatment with progesterone (P-4) known to block this acceleration. On Day 1 of the cycle or pregnancy, E-2, P-4, or E-2 + P-4 were injected s.c., and 4 h later oviducts were removed and incubated for 8 h in medium with S-35-methionine. Tissue proteins were separated by SDS-PAGE, and protein bands were quantitated by fluorography and densitometry. In mated rats, E-2 and P-4 increased different protein bands and P-4 did not affect the fluorographic pattern induced by E-2. In contrast with mated rats, none of these treatments changed the fluorographic pattern of the oviductal proteins in cyclic rats. Estradiol-induced egg transport acceleration was then compared under conditions in which oviductal protein synthesis was suppressed. Mated and cyclic rats treated with equipotent doses of E-2 for accelerating egg transport also received actinomycin D (Act D) locally. Estradiol-induced oviductal egg loss was partially blocked by Act D in mated but had no effect in cyclic rats. We conclude that the oviduct of mated and cyclic rats differs in that only the former responds with increased protein synthesis to a pulse of exogenous E-2 and P-4 and requires an intact protein synthesis machinery in order to accelerate egg transport in response to E-2.
- ItemEffect 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, HBPreviously, 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.
- ItemEstrogen 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, HBThis 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.
- ItemExpression of platelet-activating factor receptor in the hamster oviduct: Localization to the endosalpinx(1997) Velasquez, LA; Ojeda, SR; Croxatto, HBPlatelet-activating factor (PAF) is a lipid mediator that has a range of biological effects on various cells and tissues. PAF-like activity has been detected in the spent media of two-cell to morula stage hamster embryos, leading to the suggestion that PAF may be the embryonic signal that hastens embryo transport to the uterus in this species. The present study was undertaken to examine whether the PAF receptor (PAFr) gene is expressed in hamster oviduct, and to identify the cell types in which the gene is expressed. DNA fragments complementary to the coding region of mRNA encoding hamster PAFr were cloned by reverse transcription-polymerase chain reaction (RT-PCR), identified by sequencing and used to prepare hamster specific cRNA probes. The presence of mRNA transcripts encoding the PAFr receptor in the oviduct was investigated by subjecting oviduct mRNA to RT-PCR. Southern blot analysis of the RT-PCR products verified the identity of the presumptive PAFr cDNAs. The cloned cDNA fragment of hamster PAFr was found to be highly conserved with respect to the receptor of other species, having 94.3% sequence similarity to the rat PAFr receptor. Hybridization histochemistry demonstrated that PAFr is expressed in the subepithelial cells and occasionally in the epithelium. In conclusion, expression of PAFr in the hamster oviduct is compatible with the proposed paracrine role of early embryo-derived PAF.
- ItemIn vivo expression of β-galactosidase by rat oviduct exposed to naked DNA or messenger RNA(2002) Rios, M; Venegas, A; Croxatto, HBIntra-oviductal administration of RNA obtained from oviducts of estradiol-treated rats resulted in accelerated egg transport (Rios et al., 1997). It is probable that estradiol-induced messenger RNA (mRNA) entered oviductal cells and was translated into the proteins involved in accelerated egg transport. In order to test this interpretation we deposited in vivo 50 mug of pure beta-galactosidase (beta-gal) mRNA, 50 mug of pure DNA from the reporter gene beta-gal under SV40 promoter or the vehicle (control oviducts) into the oviductal lumen of rats. Twenty four hours later the beta-gal activity was assayed in oviductal tissue homogenates using o-nitrophenyl-beta-D-galactopyranoside as a substrate. The administration of beta-gal mRNA and pSVBgal plasmid increased beta-gal activity by 71% and 142%, respectively, over the control oviducts. These results indicate that naked DNA and mRNA coding for beta-gal can enter oviductal cells and be translated into an active enzyme. They are consistent with the interpretation that embryo transport acceleration caused by the injection of estradiol-induced RNA in the oviduct involves translation of the injected mRNA.
- ItemInositol 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, HBOestradiol (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.
- ItemMuc1 and glycan expression in the oviduct and endometrium of a new world monkey, Cebus apella(OXFORD UNIV PRESS INC, 2001) Jones, CJP; Ortiz, ME; Croxatto, HB; Manzur, A; Slevin, G; Aplin, JDCebus apella is a New World monkey that has a menstrual cycle of 18-23 days with implantation at approximately luteal Day 5, The aim of this study was to characterize by lectin- and antibody-labeling the distribution of Mud and associated glycans on the endometrial and oviductal epithelium during the luteal phase of the cycle, Endometrial histology showed a thin endometrium, with glands extending deeply into the myometrium, No obvious evidence of secretory differentiation in cells of either the superficial or the basal segments of glands could be obtained using a panel of antibodies and lectins that marked epithelial glycoprotein, and glycosylation changes observed in some other primate endometrial cycles were not observed in this study, Antibodies to human MUC1 were shown to cross-react with C, apella, and Mud was localized to the apical epithelial surfaces of both the endometrial and the tubal epithelium, with stronger expression in the latter. Again, no cyclic changes were noted, Antibodies specific to the isoform Muc1/Sec showed strong staining at the apical tubal epithelium, but no reactivity was detectable in the luminal epithelium of the uterus. This observation suggests differences between the two glycocalyces and could help to explain why C, apella embryos do not implant in this location.
- ItemNorplant® implants and progesterone vaginal rings do not affect maternal bone turnover and density during lactation and after weaning(1999) Díaz, S; Reyes, MV; Zepeda, A; González, GB; López, JM; Campino, C; Croxatto, HBBone density and turnover was assessed in a longitudinal study of healthy lactating women who initiated use of Norplant(R) implants (NOR, n = 29), progesterone vaginal rings (PVR, n = 28) or Copper T 380A intrauterine devices (T-Cu, n = 51, control group) around day 60 postpartum. Bone density, serum calcium, phosphorus, alkaline phosphatases, parathyroid hormone (PTH), follicle stimulating hormone (FSH), oestradiol and prolactin, and urinary hydroxyproline and creatinine were measured at postpartum months 1 (PM1), and 12 (PM12) and 6 or 12 months after weaning; at month 6 postpartum (PM6) serum and urine tests alone were performed. Baseline characteristics and lactation performance were similar between groups. Biochemical markers of bone turnover were higher at PM1, PM6 and PM12 than after weaning, with no differences between groups. Bone density in the lumbar spine (L2-L4) and femoral neck at PM1 and PM12 (similar to 1.11 g/cm(2)) was similar in three groups. Lumbar spine values were found to be lower in lactating women than those present in nonlactating women, but increased after weaning to similar values, The two progestin-only contraceptives studied appear to have no deleterious effect upon bone density and metabolism in healthy lactating women.
- ItemPost-coital administration of levonorgestrel does not interfere with post-fertilization events in the new-world monkey Cebus apella(2004) Ortiz, ME; Ortiz, RE; Fuentes, MA; Parraguez, VH; Croxatto, HBBACKGROUND: Experimental evidence to disprove the belief that emergency contraception with levonorgestrel (LNG) prevents pregnancy by interfering with post-fertilization events is lacking. Here we determined the effect of post-coital and pre-ovulatory administration of LNG on fertility and ovulation, respectively, in the Cebus monkey. METHODS: To determine the effect on fertility, LNG 0.75 mg or vehicle were administered orally or s.c. once or twice within the first 24 h after mating occurring very close to the time of ovulation. Females that became pregnant were aborted with mifepristone and re-entered the study after a resting cycle until each of 12 females had contributed, in a randomized order, two LNG and two vehicle-treated cycles. To determine the effect on ovulation, LNG 0.75 mg or vehicle were injected twice coinciding with follicles smaller or larger than 5 mm in diameter. Six females contributed five treated cycles each. RESULTS: The pregnancy rate was identical in vehicle- and LNG-treated cycles. LNG inhibited or delayed ovulation only when treatment coincided with a follicle <5 mm diameter. CONCLUSION: In Cebus monkeys, LNG can inhibit or delay ovulation but, once fertilization has taken place, it cannot prevent the establishment of pregnancy. These findings do not support the hypothesis that emergency contraception with LNG prevents pregnancy by interfering with post-fertilization events.
- ItemPostcoital treatment with levonorgestrel does not disrupt postfertilization events in the rat(2003) Müller, AL; Llados, CM; Croxatto, HBLevonorgestrel (LNG), a progestin widely used for regular hormonal contraception, is also used for emergency contraception (EC) to prevent pregnancy after unprotected intercourse. However, its mode of action in EC is only partially understood. One unresolved question is whether or not EC prevents pregnancy by interfering with postfertilization events. Here, we report the effects of acute treatment with LNG upon ovulation. fertilization and implantation in the rat. LNG inhibited ovulation totally or partially, depending on the timing of treatment and/or total dose administered, whereas it had no effect on fertilization or implantation when it was administered shortly before or after mating. or before implantation. It is concluded that acute postcoital administration of LNG at doses several-fold higher than those used for EC in women, which are able to inhibit ovulation, had no postfertilization effect that impairs fertility in the rat. (C) 2003 Elsevier Inc. All rights reserved.
- ItemThe expression of αv and β3 integrin subunits in the normal human Fallopian tube epithelium suggests the occurrence of a tubal implantation window(1998) Sülz, L; Valenzuela, JP; Salvatierra, AM; Ortiz, ME; Croxatto, HBThe co-expression of alpha(1)beta(1), alpha(4)beta(1) and alpha(nu)beta(3) integrins in the human endometrium coincides with the implantation window The alpha(nu)beta(3) integrin is expressed in the apical surface of the luminal epithelium and may serve to anchor trophoblast cells in the adhesion phase of implantation. Using immunohistochemistry, we compared the expression of alpha, alpha(1), alpha(4) and beta(3) integrin subunits in samples of normal human Fallopian tube and endometrium obtained from five women in the non-receptive period (luteal phase days 2-4) and from another five women in the receptive period (luteal phase days 6-8), The staining was quantified visually on a scale of 0 to ++, according to the intensity and density of stained cells. The alpha(nu) subunit is expressed in the Fallopian tube epithelium during both periods in a pericellular distribution. The beta(3) subunit is also expressed in the same location, but it is up-regulated during the period of endometrial receptivity, The other subunits are expressed in localizations which are not relevant to trophoblast adhesion and exhibit little or no difference in the level of expression between the non-receptive and receptive periods. Based on these results we postulate that the expression of the beta(3) subunit in the human tubal epithelium is under the same systemic controlling signals as in the endometrium and that the normal tubal epithelium may have an implantation window, at about the same time as the endometrium, that affords the opportunity for trophoblast attachment should a 5-7 day embryo be unduly retained in the tube.
- ItemVariability of breast sucking, associated milk transfer and the duration of lactational amenorrhoea(1999) Prieto, CR; Cardenas, H; Croxatto, HBQuantitative relationships between physical parameters of sucking, milk transfer and the duration of amenorrhoea were examined in normal mother-baby pairs under exclusive breastfeeding. Sucking pressures were recorded twice on the second and once on the fifth month after birth, during complete breastfeeding episodes, by means of a catheter attached to the nipple and connected to a pressure transducer, the signals of which were analysed by computer. Babies were weighed before and after each sucking episode to estimate milk transfer. In the first nursing episode after noon, 2-month-old babies sucked from 140 to > 800 times during 4-15 min from the first breast, obtaining from 20 to > 100 g milk. The physical parameters of sucking and milk transfer exhibited high inter-individual but low intra-individual variabilities. There were significant differences in the physical parameters of sucking and-milk transfer efficiency between first and second breast and between the second and fifth months after birth. Milk transfer efficiency was inversely correlated with time occupied by non-sucking pauses greater than or equal to 1.5 s, and was directly correlated with mean intersuck intervals in the first breast and with duration of the sucking episode, number of sucks, mean pressure and area under the pressure curve in the second bl:east. There was no correlation between the physical parameters of sucking and duration of lactational amenorrhoea (n = 62). However, significantly more mothers had amenorrhoea lasting > 180 days among those whose babies spent a longer proportion of the nursing episode in non-sucking pauses greater than or equal to 1.5 s. This finding indicates that sensory stimulation of the nipple produced during a nursing episode by stimuli other than sucking itself may have an important role in sustaining lactational amenorrhoea. It is concluded that nursing episodes have a complex structure that allows the development of a breastfeeding phenotype in each mother-baby pair, exhibiting important inter-individual variability The present analysis does not support the contention that this source of variability accounts for the variability in the duration of lactational amenorrhoea.