In Vitro Contractile Activity of the Mesotubarium Superius from the Rabbit Oviduct in Various Endocrine States*

FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society

Vol. 26, No.3, March 1975 Printed in U.S.A.

IN VITRO CONTRACTILE ACTIVITY OF THE MESOTUBARIUM SUPERIUS FROM THE RABBIT OVIDUCT IN VARIOUS ENDOCRINE STATES* SHERIDAN A. HALBERT, PH.D,t

AND

JOHN T. CONRAD, PH.D.

Department of Obstetrics and Gynecology and Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195

Experimental evidence indicates that a re-evaluation of the role of estrogens in the control of muscular activity in the female reproductive tract is needed. Estradiol has a relatively short lifetime in target tissues, 1 and ovarian E 2 production decreases dramatically several hours prior to ovulation in the rat, 2 the ewe, 3 and the rabbit. 4 This suggests that a significant reduction in smooth muscle estrogen concentration might occur in some mammals at a time when contractile activity of the reproductive organs is normally accelerated. 5 •6 Recent in vivo studies of gamete transport in the oviducts of rabbits indicate that a transient decrease in estrogen concentration might actually increase smooth muscle contractile activity. Boling and Blandau7 •8 observed an increase in motility in the oviducts in response not only to an adequate stimulus for ovulation but also to an acute withdrawal of estrogen. Conflicting evidence has been adduced by other investigators. This study was designed to confirm or deny the hypothesis of Boling and Blandau. 7 •8 We chose the rabbit as an experimental animal because it is a reflex ovulator and it has a reproductive cycle that can be accurately timed and controlled. We Received April 16, 1974. *Supported in part by USPHS training grant 5TIGM00260-13, NIH contract NICHD NO 1-HD3-2788, and The Ford Foundation grant 63-5613. tPresent address: Department of Biological Structure, University ofW ashington, Seattle, Washington 98195.

chose to measure the contractile activity of the mesotubarium superius (MTS) of the rabbit oviduct because its geometrically simple structure would greatly facilitate analysis. Concurrently the interaction of smooth with ovarian steroids could be ex. red, since this simple structure is mod fled by the changing hormonal milieu as much as the hollow and structurally more complex organs of the reproductive tract. We investigated the muscular activity of this membrane in four distinct physiologic states: normal estrous, long-term castrate, short-term (12-hour) castrate, and ovulatory. By considering only those conditions involving endogenous sources of estrogen, physiologic levels of tissue hormone concentration were ensured. The normal estrous and long-term castrate states represent chronic estrogen domination and chronic estrogen deprivation, respectively. The short-term castrate state represents acute estrogen withdrawal, in which we assume that a substantial decrease in smooth muscle estrogen content had occurred by 12 hours after ovariectomy. These relatively straightforward examples are indicative of the role of estrogen in the more complex endocrine state prevailing at the time of ovulation.

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MATERIALS AND METHODS

Animal preparation. Most of the New Zealand White female rabbits used had had at least one litter. All ovaries were

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FIG. 1. Diagrammatic representation of the adnexa of the uterine horn of the rabbit. A=ampulla; F=fimbria; FM=free margin of the mesotubarium superius; I= isthmus; MS= mesosalpinx; MTS= mesotubarium superius; O=ovary; and U=uterine horn.

examined microscopically at autopsy for evidence of appropriate follicular development. The animals were fed a restricted but adequate diet to control excessive adipose tissue that might make the dissection of the mesotubaria from the oviducts difficult. Anesthesia was used only at surgical removal of the ovaries. The rabbits were grouped into four well defined physiologic states: Normal estrous.-Seven mature females were isolated for more than 21 days. This procedure creates a constant state of heat in which mature ovarian follicles develop continuously. 9 Long-term castrate.-Ovariectomies were performed on seven estrous rabbits

under halothane anesthesia. The ovaries were removed electrosurgically, and the oviducts, fimbriae, and supporting mesenteries were left intact and undamaged. These animals were killed not earlier than 60 days postovariectomy. Twelve-hour castrate.-Four rabbits were prepared identically to the longterm castrates, except that they were killed 12 hours after ovariectomy. Ovulatory.-Ovulation was induced in nine normal estrous rabbits by means of an intravenous injection of 100 IU of human chorionic gonadotropin (HCG, Follutein, Squibb). Since ovulation occurs approximately ten hours after an HCG injection, the animals were killed between nine and 12 hours after the injection,

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and evidence of ovulation or its imminence was obtained from microscopic examination of the ovarian follicles. Tissue preparation. The anatomy of the mesotubarium superius of the rabbit has been described by Westman 10 and Halbert.11 The diagrammatic representation in Figure 1 depicts the relationship of the MTS to other structures in the reproductive tract. Although often referred to as simply "tubal mesentery," the MTS contains a thin sheet of smooth muscle between the double serosal membranes and thus not only supports the oviduct but also contributes to its mobility. MTS from a sexually mature rabbit contains a substantial thickening of the muscle layer of its free margin. This strip may be 0.2 to 0.3 mm thick and 2 to 3 mm wide; it is characterized by a uniformly parallel orientation of muscle fibers and a sparsity of connective tissue. One end of the free margin terminates at the cornu near the uterotubal junction where smooth muscle becomes continuous with the outer longitudinal layer of myometrium. The opposite end terminates at the fimbria but the thick muscle strip may extend past the fimbria 1 em or more toward the major arch of the ampulla. After the rabbits were stunned and bled, the reproductive tracts were removed and the mesotubaria dissected from the fallopian tubes. Appropriate pieces of tissue were subdivided into strips 1.5 to 3 mm wide and 10 to 20 mm long, and fine threads were tied to the ends of each strip. The tissue usually contracted vigorously in response to dissection, and each strip was allowed to relax completely before it was gently stretched to determine the passive "rest length." Muscle strips were mounted vertically in glass tubes, fixed to glass loops at the bottom, and connected at the top to Endevco force probes (model 8107-2). Each tube was filled with a mammalian Ringer's solution containing 154.0 mM NaCl, 5.6 mM KCl, 0.8 mM MgC1 2, 2.1

mM CaC12, 5.5 mM dextrose, 3.5 mM tris-Cl, and tris base to adjust the pH to 7.3. This solution was aerated with 98% 02, 2% C02. The tubes were immersed in a large bath with temperature regulated at 37 ± 0.2"C. The signal from the force transducer was conditioned by a carrier preamplifier (Sanborn model 350llOOC), and records of isometric tension versus time were obtained from a Sanborn 7700 series chart recorder. After a 15-minute initial equilibration period, the muscle was elongated until passive tension began to increase sharply; this established an operating length at which peak active tensio~ occurred (approximately 50% greater ·than the "rest length" dimension determined in the cold dissection solution). At the end of each experiment the tissue between the thread ties was blotted by a standard procedure to remove excess water and weighed to the nearest 0.1 mg on an analytical balance. This weight and the "rest length" of the muscle strip were used to calculate cross-sectional areas. Recording was routinely continued for at least one hour after the contraction pattern had stabilized, and data for analysis were obtained from 10- to 30-minute segments of this prolonged period of stabilized activity. The duration of both the total recording period and the segment used for analysis depended on the complexity of the contraction pattern. RESULTS

General observations. After an equilibration period of 30 to 60 minutes at 37"C, isolated strips of MTS contracted spontaneously with essentially unaltered patterns for as long as eight hours. The contraction pattern and amplitude were unaltered when we administered tetrodotoxin (TTX) at 5x10·6 gm/ml, indicating that the origin of this activity is probably myogenic in nature. TTX at this concentration was sufficient to eliminate nervous influences 12 but not the spon-

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••• FIG. 2. Spontaneous contractile activity from strips of mesotubarium isolated from different rabbits. Note the variety of patterns in different muscle preparations ranging from periodic, prolonged, tetanic contractions (top) to short, twitchlike contractions (bottom). (Mammalian Ringer's solution at 37° C.)

taneous activity of certain visceral smooth muscles. 13 Spontaneous activity persisted even when the tissue was refrigerated for as long as five days, a period during which autonomic nerve influences would have been eliminated. 14

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The contractile behavior of each MTS was characterized by durability and persistence of both amplitude and pattern of contractions throughout a wide variety of experimental conditions. A number of times we compared strips dissected from one MTS and stored at 5°C for varying periods up to 48 hours; neither the patterns nor intensities of contractions were substantially affected. Anoxia decreased contraction amplitude without changing the established pattern; eventually activity ceased but this was readily reversible. Although the pattern of contractions was especially sensitive to rapid changes in temperature, such behavior was predictable and a return to 37° C generally resulted in a resumption of the original pattern. Rapid accommodation to length changes, combined with a broad lengthtension curve, 11 allowed the MTS to be relatively insensitive to muscle length. Although the MTS from each animal was characterized predominantly by one particular contraction pattern, the character of spontaneous activity varied dramatically when comparing MTS preparations from different rabbits (Fig. 2). The control exerted on the smooth muscle by the particular hormonal constitution of the animal may have caused this. Indeed, a comparison of in vitro spontaneous contractions in MTS from ovariectomized, estrous, and ovulatory rabbits emphasized the wide variety of behavior that may be found in this tissue. Normal estrous state. Contractile activity of estrous MTS frequently resembled· that of pregnant myometrium. Phasic tetanic contractions were separated by quiet periods of nearly equal duration and were repeated with a frequency of about one per minute (top, Fig. 2). However, one third of the estrous MTS preparations displayed contractions of more complex configuration, usually characterized by the addition of oscillations on the trailing edge of tetanic contractions (Fig. 3B). These latter patterns appeared to be

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A

FIG. 3. Isometric contraction records from mesotubaria excised from rabbits in different physiologic states. Note the variety of activity in MTS from different animals within each physiologic state: (A) Long-term castrate. (JJ) Normal estrous. (C) Ovulatory. (j)) 12-hr castrate. (Spontaneous activity in mammalian Ringer's solution at 37' C.)

characteristic of the individual rabbit and were not induced by procedures such as storing for several hours at room temperature, storing overnight at 5o C, or prolonged exposure in the muscle bath at 37o C. Long-term castrate state. MTS from long-term castrate rabbits generally displayed continuous, low-level, oscillatory activity. Weak tetanic contractions pre-

dominated in about one quarter of the preparations (Fig. 3A). Twelve-hour castrate state. A dramatic increase in contractile activity was observed in the 12-hour castrate MTS. Both isometric tension and average frequency of contractions were substantially greater than those of the estrous control group. In about one half of these preparations, prolonged tetanic contractions of variable

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duration were followed by long trains of high frequency oscillations (top, Fig. 3D). This configuration resembled that observed in many estrous MTS preparations except that in the 12-hour castrate MTS the oscillations were more pronounced. About one quarter of these muscle strips had patterns similar to those in the middle record of Figure 3D; they resembled the contractions most frequently observed in estrous MTS, except that the estrous MTS developed an average of 50% less tension. Sometimes short duration, high frequency, pulsatile, twitch-like contractions prevailed. Quiet periods, occasionally of several minutes duration, occurred at random intervals. The 12-hour castrate patterns were, in general, more complex and occurred with greater variety than those of the other states, and consequently required longer periods of observation for adequate analysis of the activity. Ovulatory state. Contractions in ovulatory MTS were more vigorous than those in the estrous group but not as strong as those in the 12-hour castrate group. Prolonged tetanic contractions were noticeably absent, and only limited degrees of temporal summation of tension were observed. Instead, short, pulsatile contractions dominated the records (Fig. 3C), and in the extreme, they resembled single twitches (bottom, Fig. 2). Comparison of endocrine states. The wide variety of MTS contraction patterns observed in the different groups suggested the need for a method of analysis that

would enable a meaningful comparison of endocrine states. The method used was based on the most important function of the MTS, ie, to pull the fimbria across the surface of the ovary to facilitate ovum transport. 15•16 Accordingly, the parameters of greatest importance are the frequency of contractions and the amount of shortening per contraction, since the product of these two parameters gives a quantity indicative of the increased area of contact between fimbria and ovary. The amount of MTS shortening in vivo is determined by the amount of force the smooth muscle develops against opposing structures. However, the easiest method of evaluating the ability to shorten in vitro was to measure isometric tension. Therefore, in this study the product of the frequency and average amplitude of isometric contractions has been used to quantitate MTS contractile activity in vitro. To simplify the analysis in those cases where the contraction pattern was extremely complex, small fluctuations in isometric tension (<0.25 Tmax) that were not likely to contribute significantly to MTS function as defined here were not included in the analysis. This product (fxT) was normalized to a unit cross-sectional area and expressed in units of kg/cm2-minute. Table 1 contains a summary of the contraction data for each of the four physiologic states. Quantitative comparisons of contractile activity in various endocrine states were made by the rank-sum test. This non-

TABLE 1. Summary of Data from in Vitro Measurements of MTS Spontaneous Isometric Contractions• Muscle parameter

Maximum tension Tmax/A (kg/cm2) _bverage tension T/A (kg/cm2-contraction) Contraction frequency f (contractions/min) "Activity" fxT/A (kg/cm2 -min) •Mean±SD.

Long-term castrate (n=ll)

Normal estrous (n=15)

Ovulatory (n=17)

12-hr castrate (n=l4)

0.08±0.06

0.20±0.14

0.30±0.10

0.53±0.15

0.05±0.05

0.15±0.10

0.21±0.10

0.29±0.14

2.9 ±1.7

2.3 ±2.1

2.3 ±1.0

4.4 ±2.5

0.14±0.14

0.25±0.20

0.44±0.20

1.04±0.47

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1.711 1.110

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I

711"

c:'

e

...

1.00

~ II-'

.711

-.

mean tad

1.211 110" 211"

.110 .211 0

L.T.C. (noll)

o.

(n•l7)

12 hr. C. (n•l4)

FIG. 4. Graphic representation of a summary of mesotubarium contractile activity. The product fxT/A is used as a measure of the level of activity where f=contraction frequency,T=mean amplitude (tension) of isometric contractions, and A=crosssectional area of the muscle strip. LTC= long-term castrate, NE=normal estrous, O=ovulatory, 12-hr, C=12-hr castrate, and n=total number of muscle strips.

parametric method was chosen primarily because of the skewed distribution of activity, particularly within the long-term castrate and estrous states, and the resulting lack of power of the more traditional methods based on normal or closely related "t" distributions. With fxT/A as a measure of contractile activity, long-term castrate MTS was found to be less active than the normal estrous MTS (J>< 0.05), the ovulatory more active than the estrous (P
Our experimental evidence demonstrates that the endocrine state of the

March 1975

rabbit significantly influences the spontaneous in vitro contractile response of its mesotubaria. Furthermore, it indicates that acute withdrawal of estrogen is a potent contractile stimulus and that spontaneous activity may be inhibited by a continual secretion of estrogen by the ovaries. The concept of estrogen withdrawal as a stimulator of female reproductive muscle has been controversial. Boling5 observed that conflicting evidence regarding estrogen influence is due, in part, to the uncertainty of duration of the direct effects of this steroid on the reproductive tract. He stressed the evidence that estrogens have a relatively short lifetime in the target tissues1 and that in past experiments the effects of acute estrogen withdrawal might have been attributed erroneously to the direct influence of estrogen. The importance of the concept that the life-span and physiologic effect of estrogen in the target organ are short cannot be overemphasized. Certainly, there is ample evidence of transient changes in the steroid environment during the important, initial sequence of events leading to conception. In several animal species,2·4· 17 a dramatic decrease in ovarian estrogen production has been observed to precede ovulation by several hours. This rapid change in estrogen output is likely causally related to the change in muscular activity that occurs in the reproductive tract about the time of ovulation5 and it is probably an important factor in the normal processes leading to conception. The data collected by Boling5 •6 and Boling and Blandau 7 • 8 point to acute estrogen withdrawal as a primary factor in the enhancement of muscular activity in the female reproductive tract. This interpretation is supported by their measurements of ampullary egg transport time and observations of contractions of the adnexal structures under a variety

·~

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of hormonal conditions in anesthetized rabbits. Faster egg transport and more vigorous muscular contractions were noted in response to withdrawal or inhibition of estrogen as well as in response to ovulation induced ten to 12 hours after HCG administration. They concluded that a decrease in estrogen, in addition to a preovulatory increase in progesterone, might explain increased muscle activity during the time of ovum transport in the ampullary portion of the oviduct. In recent years other investigators have noted at about the time of ovulation increased motility of the reproductive tract that may be attributed to estrogen withdrawal. Salomy and Harper 18 measured intraluminal pressure in the isthmus of the oviducts of rabbits implanted with balloon-tipped catheters and found an increase in the amplitude and frequency of contractions for up to 24 hours after an ovulation-inducing injection of HCG. Fuchs 19 noted an increase in contractions of the cornua with intrauterine balloons in conscious, unrestricted rabbits which began a few hours after either mating or luteinizing hormone (LH) injection, and lasted for at least 24 hours. This activity also increased after bilateral ovariectomy. Hilliard and Eaton 4 interpreted differently the role of estrogen in the activity changes occurring at ovulation. They measured a transient surge and a subsequent cessation of ovarian E 2 production in the first five hours after coitus. They suggested that a surge of E 2 could explain the increased activity on the day of mating but that the withdrawal of estrogen is more probably associated with the delayed quiescent phase of muscular activity occurring on the following days. On the other hand, Shaikh and Harper 17 failed to observe a significant increase in ovarian estrogen production after mating or HCG injection, contradicting the findings of Hilliard and Eaton4 and pointing to estrogen withdrawal as a

likely cause of increased activity at the time of ovulation in the rabbit. The data on MTS contractile activity presented in this paper support the concept that estrogen withdrawal contributes to elevated reproductive smooth muscle activity. The increase in MTS activity that resulted from acute removal of the endogenous estrogen source resembled in many respects that resulting from induced ovulation. Our analysis indicates, however, a more dramatic response of the smooth muscle to estrogen withdrawal than to the more complex changes in the endocrine environment associated with ovulation. It is suggested that increased activity induced by the cessation of estrogen production may be modified by the surge ofprogestins occurring in the ovulatory process.

SUMMARY

We investigated the influence of estrogen on the contractile activity of smooth muscle in the nonpregnant female reproductive tract, using as a model isolated strips of mesotubarium superius (MTS) removed from the rabbit oviduct. We studied four physiologic states in which muscular activity was influenced to varying degrees by endogenous estrogen. Normal estrous (estrogen dominated) rabbits were used as controls. We observed decreased activity in MTS from rabbits chronically deprived of estrogen (60 days postovariectomy). However, a dramatic increase in activity occurred in response to acute withdrawal of ovarian function (12 hours postovariectomy). An increase in activity also occurred at the time of ovulation. We concluded that acute withdrawal of endogenous estrogen is a potent stimulus to reproductive smooth muscle in the female rabbit and that it might contribute to increased muscular activity at ovulation.

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16. Blandau RJ: Normal egg transport through the oviducts of mammals. In Pathways to Conception. Edited by J Sherman. Springfield, CC Thomas, 1971, p 92 17. Shaikh AA, Harper MJK: Ovarian steroid secretion in estrous, mated and HCG-treated rabbits, determined by concurrent cannulation of both ovarian veins. Bioi Reprod 7:387, 1972 18. Salomy M, Harper MJK: Cyclical changes of oviduct motility in rabbits. Bioi Reprod 4: 185, 1971 19. Fuchs A-R: Uterine activity during and after mating in the rabbit. Fertil Steril 23:915, 1972