Circular versus longitudinal myometrial contractile response to selective tachykinin receptor agonists in rat.

This study compared the nature and magnitude of the contractile response produced in vitro by selective NK1, NK2 and NK3 tachykinin receptor agonists in circularly and longitudinally oriented strips of myometrium from ovariectomised and ovariectomised oestrogen-treated rats. The nature of the responses produced upon stimulation of the tachykinin receptors varied between the different myometrial preparations and the hormonal environment from which the tissue was taken. Variations included: (i) sustained contraction until washout of agonist; (ii) biphasic contraction until washout of agonist; and (iii) monophasic contraction. The major differences in magnitude of contractions were seen in preparations from oestrogen-treated animals in which responses to stimulation of all tachykinin receptors were reduced in comparison to preparations from non-oestrogen treated animals. Furthermore, the responses in circularly oriented myometrium preparations from oestrogen-treated animals were all markedly reduced compared to responses in longitudinally oriented myometrium preparations. These results suggest that the tachykinin receptors in longitudinally and circularly oriented myometrial layers are differentially regulated, especially in tissue isolated from an oestrogen-dominated environment.

Hormonal variation of rat uterine contractile responsiveness to selective neurokinin receptor agonists.

Regulated uterine contractions are important in many reproductive functions such as sperm transport and embryo positioning during implantation. The role of classical neurotransmitters including acetylcholine and norepinephrine in regulating myometrial contractility has been well studied; however, the peripheral role of sensory neurotransmitters such as the neurokinins is less clear. The major neurokinins are substance P, neurokinin A, and neurokinin B, which predominantly activate neurokinin receptors (NK-Rs) 1, 2, and 3, respectively. This study utilized selective receptor agonists to examine the role of NK-Rs in uterine contractility. Uterine tissues, obtained from the major stages of the rat estrous cycle, were stimulated with selective NK-R agonists. Addition of each agonist resulted in a significant contractile response. However, the magnitude and nature of the response were dependent upon the stage of the estrous cycle, with responses to all agonists being significantly decreased in tissue from proestrus and estrus. Furthermore, the nature of NK3-R-mediated contraction was different in tissue from proestrus and estrus compared to metestrus and diestrus. The hormonal dependence of NK-R-mediated contractility was then examined in the ovariectomized estrogen-supplemented rat model. These studies confirmed that the magnitude and nature of uterine contractility in response to NK-R activation depend upon the hormonal environment.

Differentiation-dependent expression of gelatinase B/matrix metalloproteinase-9 in trophoblast cells.

The purpose of this study was to evaluate the Rcho-1 trophoblast culture system as a model for studying trophoblast invasion and to examine stage-specific expression of enzyme(s) potentially participating in rat trophoblast giant cell invasive behavior. The invasive behavior of the differentiating Rcho-1 trophoblast cells was demonstrated using Matrigel invasion chambers. Gelatin zymography and Western blot analysis of conditioned medium from differentiating Rcho-1 trophoblast cell cultures and rat ectoplacental cone outgrowths revealed a differentiation-dependent increase in gelatinase B/matrix metalloproteinase (MMP-9). Nothern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of Rcho-1 trophoblast or ectoplacental cone cells also showed increasing expression of MMP-9 accompanying cell differentiation. Rcho-1 trophoblast cells stably transfected with MMP-9 promoter/luciferase reporter constructs exhibited a differentiation-dependent increase in MMP-9 promoter activation. In conclusion, trophoblast giant cell differentiation is characterized by transcriptional activation of the MMP-9 gene and appearance of the invasive phenotype.

Inhibitory and stimulatory effect of oestrogens upon ovarian 17 alpha-hydroxylase/C17,20-lyase in immature hypophysectomized rats treated with gonadotrophin.

A study was designed to compare the effects of exogenous and endogenous oestrogens upon the expression of steroid 17 alpha-hydroxylase/C17,20-lyase (CYP17) in the immature hypophysectomized rat ovary. C17,20-lyase activity was measured ex vivo using [21-14C]progesterone, while serum concentrations of androstenedione indicated in vivo activity. Immunocytochemistry was used to localize the enzyme within the ovary. Activity of CYP17 increased dramatically and remained high in thecal and interstitial cells after injection of equine chorionic gonadotrophin, even though serum oestradiol (OE2) levels exceeded 2 nmol/l. Production of comparable serum levels by the use of Silastic capsules containing OE2 greatly suppressed but did not stop, expression of the enzyme induced by repeated doses of human chorionic gonadotrophin (hCG). Subcutaneous implantation of Silastic capsules (1 cm) containing diethylstilboestrol (DES) stimulated follicular growth and increased ovarian weight by 67%, 5 days later. Injection of 50 IU hCG at various times after removal of the implant produced time-dependent changes in CYP17 activity and serum androstenedione levels, when measured 30 h later. Although the initial effect of removal of DES was an increase in CYP17 activity, delaying injection of hCG resulted in a reduced response. The results indicated that: (1) endogenous oestrogens do not inhibit CYP17 expression; (2) exogenous oestrogens only reduce the number of thecal/interstitial cells expressing CYP17 when they are exposed to hCG; (3) pretreatment with oestrogen removes the ovarian interstitial but not the thecal cell expression of CYP17 in response to hCG; and (4) oestrogens can be stimulatory for CYP17 expression in thecal cells.

Effects of the progesterone antagonist RU486 on myometrial activity in vivo in early pregnant and pseudopregnant rats.

Myometrial activity in vivo was quantified by video-laparoscopy in early pregnant rats given doses of RU486 which caused embryo abortion and blocked the action of progesterone on the vagina. All treatments diminished the frequency of circular contractions and abolished the curling movements of the uterus which are characteristic of pregnant, pseudopregnant and progestin-treated rats. The effects of RU486 on circular contractions were similar in pseudopregnant rats, i.e. they were not a consequence of embryo abortion. These results support the thesis that increased myometrial circular contractions in early pregnancy and pseudopregnancy are induced by increasing levels of progesterone. Effects of RU486 on longitudinal contractions were more complex: the highest dose inhibited longitudinal contractions on Day 5 of pregnancy and pseudopregnancy, but increased their frequency on Day 6. The acute inhibition of longitudinal contractions by RU486 was unexpected and the mechanism remains to be elucidated. The later increase in the frequency of longitudinal contractions appears to be due to antagonism of progesterone by RU486. The frequency of caudal longitudinal contractions on Day 6 in mated rats given RU486 was similar to that in unmated oestrous rats, but the frequency of cranial longitudinal contractions was significantly higher. These results support the hypothesis that stimuli received during copulation may have long-term effects on myometrial activity, by increasing pacemaker activity at the cervix.

Pace-maker activity in the myometrium of the oestrous rat: in vivo studies using video-laparoscopy.

Female rats had one oviduct, or the cranial tip of one or both uterine horns, lesioned by coagulation, or separated from the remainder of the uterus. After recovery and return to oestrous cycles, myometrial activity at oestrus was analysed by video-laparoscopy. Lesioning the oviduct had no effect on myometrial activity. Coagulating the cranial tip of one horn initially reduced the frequency of ipsilateral longitudinal contractions propagating caudally, but this returned to normal after 14 days. Separating the cranial tip of the uterus had permanent effects on myometrial activity. Separation of one tip reduced the frequency of ipsilateral longitudinal contractions propagating caudally, had no effect on ipsilateral contractions propagating cranially, but reduced the frequency of contralateral contractions propagating cranially. The effect of a lesion near one uterotubal junction on contractions originating contralaterally near the cervix results from communication between uterine horns at the cervical junction; arrival of a caudally propagating contraction in one horn frequently generates a cranially propagating contraction in the other. Separating both uterine tips reduced the frequency of longitudinal contractions propagating in both directions. We conclude that, at oestrus, most spontaneous myometrial contractions are generated by pace-makers in the cranial tip of each uterine horn. The pace-makers are close to the uterotubal junction and regenerate after destruction by coagulation. When their influence is permanently removed, new pace-makers do not develop in myometrium caudal to the lesion. We conclude that most myometrial cells do not exhibit spontaneous pace-maker activity in vivo.

In vivo myometrial activity in the rat during the oestrous cycle: studies with a novel technique of video laparoscopy.

Previous techniques of recording myometrial activity in vivo gave limited information about the nature of contractions, and disrupted normal reproductive events. To overcome these drawbacks we developed a new in vivo method of video laparoscopy (VL). This involves positioning a laparoscope in the abdomen of anaesthetized rats to view the caudal ends of both uterine horns. Myometrial activity is recorded by video camera onto video tape. Myometrial contractions are classified according to the muscle layers involved, the interaction between layers and the direction of propagation. Experiments with intrauterine balloons and electromyography (EMG) in conscious and ketamine/xylazine anaesthetized rats showed that this anaesthetic does not have major effects on myometrial activity. To validate the VL method, recordings were obtained throughout the oestrous cycle and compared with results obtained with EMG in conscious rats. The frequency and pattern of activity were similar with both techniques although more information was obtained from VL. Frequency of contractions was highest in oestrus and dioestrus and lowest in pro-oestrus, when contractions occurred in groups separated by quiescent intervals. At all stages, longitudinal contractions propagating towards the cervix predominated. Circular muscle activity was only seen at oestrus and dioestrus; that at oestrus consisted of weak peristalses, that at dioestrus was more complex. A major advantage of VL is that it does not interfere with the course of the oestrous cycle, pseudopregnancy or early pregnancy.

Is the antiprogestin RU486 luteolytic in rats?

Subcutaneous injections of RU486 on Day 1, Day 4 or Days 3 and 4 of pregnancy in rats induced abortion of the embryo and transient vaginal cornification. Nevertheless, most corpora lutea appeared to be functional at autopsy on Day 12. The same treatments in pseudopregnant rats also produced transient vaginal cornification, but did not terminate pseudopregnancy. This suggests that transient antagonism of progesterone by RU486 does not terminate function of the corpus luteum in rats and that positive feedback by progesterone is not essential for continuing luteal function in rats.

Postcopulatory myometrial activity in the rat as seen by video-laparoscopy.

Myometrial activity was analysed by video-laparoscopy in unmated and mated oestrous female rats. In the former, most contractions propagated caudally and were predominantly longitudinal, with circular contractions limited to weak peristalsis. Mating had dramatic effects on activity, inducing a high frequency of strong peristaltic and non-peristaltic circular contractions propagating both cranially and caudally. Activity remained elevated for up to 10 h, decreasing to control levels as uterine contents were lost when the vaginal plug disintegrated. Mechanical stimulation of the cervix, sufficient to induce pseudopregnancy, increased the frequency of weak peristalsis but did not induce myometrial activity comparable to that seen after mating. Treatment of mated females with indomethacin returned myometrial activity towards control levels, suggesting that myometrial stimulation involved prostanoid production. After mating to intact males the uterus took on a characteristic blue colour suggestive of hypoxia. This too was abolished by indomethacin treatment. Removal of accessory reproductive glands from males showed that induction of myometrial activity and the colour change both required constituents from the vas deferens, seminal vesicles and coagulating glands. Males lacking seminal vesicles or coagulating glands were infertile. The prolonged myometrial activity and putative hypoxia induced by seminal constituents may be important in survival and selection of sperm.

Effects of medroxyprogesterone acetate (MPA) on myometrial activity in unmated rats: a study in vivo using video-laparoscopy.

Unmated female rats received medroxyprogesterone acetate (MPA) on the day of oestrus. Myometrial activity in vivo was quantified by video-laparoscopy 24-96 h later. Changes induced by MPA resembled those in early pregnancy/pseudopregnancy, but were not as extreme. There was a significant increase in circular contractions, followed by a decline, as in mated rats. Increases in circular contractions propagating caudally were as large as in mated rats, but increases in those propagating cranially were much smaller and qualitatively different. MPA induced only small increases in the frequency of longitudinal contractions propagating cranially, whereas large increases occurred in pregnancy/pseudopregnancy. We conclude that in early pregnancy/pseudopregnancy, increased circular activity is induced by rising progesterone levels. However, progesterone alone does not account for the increased contractions propagating cranially in mated rats. We suggest that these are induced by increased pacemaker activity in the region of the cervix, possibly activated by stimuli received during copulation.

In vivo myometrial activity during early pregnancy and pseudopregnancy in the rat.

Video-laparoscopic studies in early pregnant and pseudopregnant rats showed large changes in frequency, direction of propagation and nature of myometrial contractions. Day 2 patterns of activity were essentially the same as in unmated animals at the equivalent stage of the cycle. From Days 3 to 5 there was a large increase in longitudinal and circular contractions propagating towards the oviduct, circular contractions making the greatest contribution. This circular activity may be important in retaining and spacing embryos. Circular contractions propagating towards the cervix showed smaller increases and there was a transient diminution in the frequency of longitudinal contractions in this direction on Day 5. In pregnant rats, the frequency of discrete contractions declined on Days 6-7. However, circular tone appeared to be increased and uteri showed dramatic twisting and curling, apparently due to resistance to the shortening imposed by longitudinal contractions. None of the major changes in activity appeared to be caused by embryos, because they were seen in pseudopregnant rats and, after embryo implantation, in both horns of unilaterally pregnant rats. The earliest divergence from the activity patterns of unmated rats occurred when progesterone levels first increased significantly above those of the undisturbed oestrous cycle, suggesting that progesterone has a major influence on myometrial activity. The complexity of the changes in activity raises questions about other regulatory factors, particularly in regard to coordination between the circular and longitudinal muscle layers. Anomalous results from pregnant, unilaterally pregnant, and pseudopregnant animals on Day 7 suggested that embryos exert systemic effects on myometrial activity.