Acceleration Of Egg Transport Research Articles

Sperm utilize tumor necrosis factor alpha to shut down a 2-methoxyestradiol nongenomic pathway that accelerates oviductal egg transport in the rat.

Mating shuts down the 2-methoxyestradiol (2ME) nongenomic pathway that accelerates oviductal egg transport in the rat. This study shows that sperm cells, but not vaginocervical stimulation, utilize TNF-α to shut down this 2ME nongenomic pathway. The transport of oocytes or embryos throughout the oviduct to the implantation site in the uterus is defined as egg transport. In the rat, 2-methoxyestradiol (2ME) accelerates egg transport through the oviduct via a nongenomic pathway. Mating is known to shut down this 2ME pathway and then trigger an estradiol genomic pathway that accelerates egg transport. Here, we tested whether intrauterine insemination (IUI) or vaginocervical stimulation (VCS) shuts down the 2ME nongenomic pathway that accelerates egg transport, and if these mating components require tumor necrosis factor alpha (TNF-α). Levels of TNF-α and the mRNA for TNF-α receptors were measured in the oviduct of IUI or VCS rats. The tissue distribution of TNF-α receptor proteins and the concentration of the mRNA for catechol-O-methyl transferase (Comt) and 2ME were also analyzed in the oviduct. Finally, we assessed whether 2ME accelerates egg transport in IUI or VCS rats previously treated with the TNF-α antagonist W9P9QY. Results show that IUI, but not VCS, increased TNF-α and their receptors in the oviduct. IUI and VCS did not change the tissue distribution of TNF-α receptors; however, both decreased the oviductal concentration of Comt and 2ME. IUI and VCS each blocked the 2ME-induced egg transport acceleration; however, only the IUI was antagonized by the TNF-α antagonist. We concluded that IUI and VCS inhibit the 2ME nongenomic pathway that accelerates egg transport; however, the vias of action are distinct, with a TNF-α increase on spermatozoa presence being required for the shutdown of the 2ME pathway.

Tumour necrosis factor-α is the signal induced by mating to shutdown a 2-methoxyestradiol nongenomic action necessary to accelerate oviductal egg transport in the rat

Mating shut down a 2-methoxyestradiol (2ME) nongenomic action necessary to accelerate egg transport in the rat oviduct. Herein, we investigated whether tumour necrosis factor-α (TNF-α) participates in this mating effect. In unmated and mated rats, we determined the concentration of TNF-α in the oviductal fluid and the level of the mRNA for Tnf-a (Tnf) and their receptors Tnfrsf1a and Tnfrsf1b in the oviduct tissues. The distribution of the TNFRSF1A and TNFRSF1B proteins in the oviduct of unmated and mated was also assessed. Finally, we examined whether 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a rat recombinant TNF-α alone or concomitant with a selective inhibitor of the NF-κB activity. Mating increased TNF-α in the oviductal fluid, but Tnf transcript was not detected in the oviduct. The mRNA for TNF-α receptors as well as their distribution was not affected by mating, although they were mainly localized in the endosalpinx. Administration of TNF-α into the oviduct of unmated rats prevented the effect of 2ME on egg transport. However, the NF-κB activity inhibitor did not revert this effect of TNF-α. These results indicate that mating increased TNF-α in the oviductal fluid, although this not associated with changes in the expression and localization of TNF-α receptors in the oviductal cells. Furthermore, TNF-α mimicked the effect of mating on the 2ME-induced egg transport acceleration, independently of the activation of NF-κB in the oviduct. We concluded that TNF-α is the signal induced by mating to shut down a 2ME nongenomic action in the rat oviduct.

Differential participation of endothelin receptors in estradiol-induced oviductal egg transport acceleration in unmated and mated rats

Objective To determine the role of endothelin (EDN) signaling in the estradiol (E2) nongenomic and genomic mechanism that regulates egg transport in the oviduct.

Genital sensory stimulation shifts estradiol intraoviductal signaling from nongenomic to genomic pathways, independently from prolactin surges.

Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.

Inositol triphosphate participates in an oestradiol nongenomic signalling pathway involved in accelerated oviductal transport in cycling rats

Oestradiol (E(2)) 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-18-OCH(3) or MAPK PD98059. The number of eggs in the oviduct assessed 24 h later showed that ET-18-OCH(3) blocked E(2)-induced egg transport acceleration, whereas PD98059 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, 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(2), while inhibition of PLC by ET-18-OCH(3) had no effect on E(2)-induced PKA activity. Furthermore, activation of adenylyl cyclase by Forskolin increased oviductal IP3 levels. Thus, activation of PLC-IP3 by E(2) requires previous stimulation of cAMP-PKA. We conclude that the nongenomic pathway utilised by E(2) 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 an entire organ.

Effect of intrauterine insemination with spermatozoa or foreign protein on the mechanism of action of oestradiol in the rat oviduct.

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. Pro-oestrous 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 alpha-amanitin. The number of eggs in the oviduct, assessed 24 h later, showed that alpha-amanitin blocked the oestradiol-induced egg transport acceleration, indicating that the interaction of spermatozoa with the genital tract shifts the action of oestradiol from non-genomic 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.

Effect of intrauterine insemination with spermatozoa or foreign protein on the mechanism of action of oestradiol in the rat oviduct

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. Pro-oestrous 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 alpha-amanitin. The number of eggs in the oviduct, assessed 24 h later, showed that alpha-amanitin blocked the oestradiol-induced egg transport acceleration, indicating that the interaction of spermatozoa with the genital tract shifts the action of oestradiol from non-genomic 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.

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.

This investigation examined the role of estrogen receptor (ER) on the stimulatory effect of estradiol (E2) on protein phosphorylation in the oviduct as well as on E2-induced acceleration of oviductal oocyte transport in cyclic rats. Estrous rats were injected with E2 s.c. and with the ER antagonist ICI 182 780 intrabursally (i.b.), and 6 h later, oviducts were excised and protein phosphorylation was determined by Western blot analysis. ICI 182 780 inhibited the E2-induced phosphorylation of some oviductal proteins. Other estrous rats were treated with E2 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 E2-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 E2 was then examined. Selective inhibitors of adenylyl cyclase or PK-A inhibited the E2-induced egg transport acceleration, whereas PK-C or tyrosine kinase inhibitors had no effect. Furthermore, forskolin, an adenylyl cyclase activator, mimicked the effect of E2 on ovum transport and E2 increased the level of cAMP in the oviduct of cycling rats. Finally, we measured PK-A activity in vitro in the presence of E2 or E2-ER complex. Activity of PK-A in the presence of E2 or E2-ER was similar to PK-A alone, showing that E2 or E2-ER did not directly activate PK-A. We conclude that the nongenomic pathway by which E2 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.

Acceleration of oviductal transport of oocytes induced by estradiol in cycling rats is mediated by nongenomic stimulation of protein phosphorylation in the oviduct.

In order to explore nongenomic actions of estradiol (E2) and progesterone (P4) in the oviduct, we determined the effect of E2 and P4 on oviductal protein phosphorylation. Rats on Day 1 of the cycle (C1) or pregnancy (P1) were treated with E2, P4, or E2 + P4, and 0.5 h or 2.5 h later their oviducts were incubated in medium with 32P-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 E2 effect by mRNA synthesis or protein kinases A (PK-A) or C (PK-C) was then examined. Rats on C1 treated with E2 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 32P into total oviductal protein induced by E2 was unchanged by Am, whereas it was completely suppressed by PK inhibitors. Local administration of H-89 was utilized to determine whether or not E2-induced egg transport acceleration requires protein phosphorylation. Rats on C1 or P1 were treated with E2 s.c. and H-89 i.b. The number and distribution of eggs in the genital tract assessed 24 h later showed that H-89 blocked the E2-induced oviductal egg loss in cyclic rats and had no effect in mated rats. It is concluded that E2 and P4 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 absence of mating, this action is essential for its oviductal transport accelerating effect. Mating changes the mechanism of action of E2 in the oviduct by waiving this nongenomic action as a requirement for E2-induced embryo transport acceleration.

Disparate effects of estradiol on egg transport and oviductal protein synthesis in mated and cyclic rats.

Previously, we found that the dose of estradiol (E2) required 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 E2 known to accelerate oviductal egg transport or after concomitant treatment with progesterone (P4) known to block this acceleration. On Day 1 of the cycle or pregnancy, E2, P4, or E2 + P4 were injected s.c., and 4 h later oviducts were removed and incubated for 8 h in medium with 35S-methionine. Tissue proteins were separated by SDS-PAGE, and protein bands were quantitated by fluorography and densitometry. In mated rats, E2 and P4 increased different protein bands and P4 did not affect the fluorographic pattern induced by E2. 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 E2 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 E2 and P4 and requires an intact protein synthesis machinery in order to accelerate egg transport in response to E2.

Effect of clomiphene on nuclear estrogen receptor of the fallopian tube during ovum transport in rabbits.

The effect of clomiphene on nuclear estrogen receptors of the Fallopian tube during ovum transport in the rabbit has been studied. Nuclear binding capacity was observed in ampulla (A), ampullary-isthmic junction (AIJ), isthmus (I), uterine-isthmic junction (UIJ) and uterus (U). Receptor concentration decreased in all segments of the tube after administration of clomiphene in mated animals. The bindings are of high affinity and low capacity. Important alterations were observed during transport when compared to that of 14, 24, 34, 48, 72, 144 and 168 hr post-coitum (p.c). At 24 hr p.c binding increased only in I and decreased in A and AIJ. Retention of eggs at I at 24 hr p.c showed as increase in binding at I. Egg transport was accelerated and eggs reached prematurely in the uterus due to the influence of clomiphene. Binding in I remained constant from 48 hr p.c to 144 hr p.c but concurrently the binding level increased in U from 34 hr p.c. The elevation of nuclear estrogen receptor level was maximum at 24 hr p.c which coincided with increased plasma estrogen level. The result of such study showed that clomiphene depleted nuclear estrogen receptor complex in the fallopian tube before transfer to the uterus. Further, observation indicated that clomiphene acted directly on the rate of egg transport because of the variations in estrogen receptors during different time periods. Thus, clomiphene reduced the quantity of estrogen receptor i.e., insensitiveness to estrogen. The variations in estrogen binding to its receptor and plasma level at different post-coital periods are modulated by clomiphene resulting in the acceleration of egg transport and prevention of pregnancy.

Effects of 17 beta-hydroxy-7 alpha-methylandrost-5-en-3-one on early pregnancy in the rat.

A single sc injection of 1 mg 17-beta-hydroxy-7 alpha-methylandrost-5-en-3-one (RMI-12,936) given a few hours after mating interrupted pregnancy in all the treated rats. Circulating progesterone (delta 4P) levels were higher in RMI-12,936 treated females than in controls on the corresponding days during the course of termination of pregnancy. Higher levels of delta 4P were recorded on day-4 (P less than 0.01) and day-6 (P less than 0.05) of pregnancy. In addition to the changes in serum delta 4P, an acceleration of egg transport was encountered. The eggs were prematurely expelled from the uterus within 48 h of the treatment. Although the oestrous cycle of the RMI-12,935 treated females was disturbed, they were found sexually receptive. Successful matings resulting in normal gestation and morphologically normal foetuses were recorded 20-26 days after RMI-12,936 induced pregnancy termination. These results suggest that in addition to its mid-pregnancy terminating effect, RMI-12,936 is capable of interrupting early pregnancy when given soon after mating in the rat. The safety and efficacy of this compound as a post-coital contraceptive deserves further investigation.

Incidence of polyspermy in mouse eggs fertilized in vivo and in vitro after administration of progesterone and oestradiol

Female mice, induced to superovulate, were injected subcutaneously with progesterone or oestradiol near the time when hCG was given. The incidence of polyspermy in first-cleavage embryos following mating or in-vitro fertilization was then determined. There were no detectable differences in the incidence or degree of polyspermy between treated and control in either the in-vitro or in-vivo groups, although the mean incidence of polyspermy was higher in vitro than in vivo. Furthermore, there was no detectable acceleration of egg transport after administration of either hormone.

Mechanism of antifertility action of low doses of alpha-chlorohydrin in the male rat.

Summary. The minimal effective daily oral dose of α-chlorohydrin in the male Sprague-Dawley rat to prevent fertilization in the female following mating was found to be 2·5 mg/kg/day. The time to take effect and the time of recovery after withdrawal decreased and increased, respectively, as the dose was increased. With doses of up to 10 mg/kg/day, the sterility could not be accounted for on the basis of changes in the histology or weight of the male reproductive organs or accessory glands. The motility of spermatozoa recovered from the female tract the morning after mating was inhibited and, although normal numbers were recovered from the uterus, very few had entered the oviducts. When semen was collected from the uterus of a female 3 to 4 hr after mating with a treated male and transferred directly into the ovarian bursa of an unmated female, the spermatozoa were capable of fertilization. Semen from untreated males, transferred in the same fashion, caused acceleration of egg transport in the oviducts which was not observed with the semen from treated males. Inhibition of sperm transport is assumed to be a major factor in prevention of fertilization and may, at least in part, be mediated through a change in the constituents of semen of treated males. The disparate effect on egg transport, implying effects on oviducal musculature, suggests the involvement of prostaglandins or similar smooth muscle stimulants in the mechanism.

Egg transport through the ampullae of the oviducts of rabbits under various experimental conditions.

Muscle contractility of the ampulla was minimal, and the egg transport was slower in normal, unstimulated, estrous rabbits at a time when estrogens in the plasma and target tissues are presumed to be at a relatively high level. There is an increasing body of data indicating a significant increase in the secretion of progestins in the ovaries as ovulation approaches. We suggest that the increase in vigor and orderly programming of muscle contraction as well as the acceleration of egg transport at the time of ovulation may be related to an increase in the secretion of progestins and a fall in the level of estrogens.

Studies on the antifertility mechanisms of low doses of chlormadinone acetate using egg transfer in the rabbit.

Fertilization, transport, development, and transfer of eggs was studied in rabbits treated with chlormadinone acetate (CA). Mature female rabbits were treated orally for three consecutive days with either 0.09 mg/kg/day of CA in an aqueous suspending medium or with the vehicle alone. On the last day of dosing (≡ Day 0 of pregnancy) all animals were artificially inseminated (AI) and were induced to ovulate with HCG. Recipients for egg transfer were made pseudopregnant by inducing ovulation but omitting AI. Egg recoveries on Days 2, 4, and 6 of pregnancy showed that CA interfered with fertilization, accelerated the rate of egg transport by Day 2, and interfered with blastocyst expansion by Day 4. When Day 2 eggs were flushed from the oviducts of treated donor rabbits and transferred into the oviducts of untreated recipient rabbits on Day 2 of pseudopregnancy normal implantation occurred. Day 2 transfers from untreated donors to treated recipients produced a marked reduction in the number of implantations. When Day 6 blastocysts were recovered from the uteri of treated donors and transferred into the uteri of untreated recipients on Day 6 of pseudopregnancy no viable fetuses developed. Day 6 transfers from untreated donors to treated recipients resulted in normal fetal development. The results indicate a complex antifertility effect of CA in the rabbit. A dose of 0.09 mg/kg, administered on three consecutive days before ovulation interferes with fertilization, causes acceleration of egg transport and produces ovicidal effects between Days 2 and 6 of pregnancy.

Species differences in egg transport in response to exogenous estrogen.

AbstractThe effect of exogenous estrogen on tubal transport of ova was determined in the guinea pig, hamster, mouse, rabbit and rat. The animals were given a single injection of estradiol cyclopentylpropionate (ECP) shortly after mating. The dose of ECP required to interrupt pregnancy in 80% or more of the animals was as follows: guinea pig (10 μg); hamster (25 μg); mouse (1 μg); rabbit (50 μg); rat (10 μg). Acceleration of egg transport through the oviduct occurred after the following doses of ECP: guinea pig (50–100 μg); hamster (100 μg); mouse (1 μg and above); rabbit (25 μg); rat (10 μg and above). Hence, the amount of estrogen which accelerates egg transport in the guinea pig and hamster is considerably higher than the dose which interrupts pregnancy.Retentionof ova for longer than the normal period of tubal passage (tube‐locking) resulted from the following doses of ECP: guinea pig (250 μg); hamster (250 μg); mouse (1 μg); rabbit (100 μg); rat (no dose). In the species in which ova were tubelocked, the majority of eggs were located at the ampullary‐isthmic junction rather than the utero‐tubal region of the oviduct.Tube‐locking of ova was never observed in the rat; ECP always caused premature entry of eggs into the uterus and eventual expulsion per vaginam. For example, eggs passed through the cervix by 12 hours after the administration of 250 μg ECP at day 1 of pregnancy.