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Adrenergic Responses of the Various Smooth Muscle Layers at the Human Uterotubal Junction*
Vol. 33, No.3, March 1980 Printed in U.SA.
FERTILITY AND STERILITY Copyright c 1980 The American Fertility Society
ADRENERGIC RESPONSES OF THE VARIOUS SMOOTH MUSCLE LAYERS AT THE HUMAN UTEROTUBAL JUNCTION*
LENNART WILHELMSSON, M.D.t BO LINDBLOM, M.D., PH.D.
Department of Obstetrics and Gynecology, Uniuersity of Goteborg, Goteborg, Sweden
The different muscle layers at the human uterotubal junction (UTJ) were mechanically separated, and 1-mm wide muscle strips were isolated and mounted in organ chambers for isometric recording of contractile activity. It was found that the adrenergic neurotransmitter noradrenalin elicited an inhibition of the spontaneous phasic activity in the inner longitudinal and the intermediate circular layers but caused a clear-cut excitatory response in the outer, spiral-shaped (uterine) layer. The excitatory response was totally blocked by treatment with phenoxybenzamine, an o.-adrenoceptor antagonist, whereas the inhibitory effects were abolished after pretreatment with the f3-adrenoceptor antagonist propranolol. The data demonstrate a differentiated distribution of 0.- and f3-adrenoceptors in the smooth musculature at the proximal end of the tubal isthmus. It is suggested that this arrangement might serve an important purpose in the physiologic control of ovum transport in women. Fertil Steril 33:286, 1980
use of very large segments and the differing methodologic approaches in various studies. 3 Three main muscle layers are present in the isthmic region: the inner longitudinal, the intermediate circular, and the outer longitudinal or spiral-shaped uterine layer. 6 - B The inner longitudinal layer is distinct at the uterotubal junction (UTJ) but diminishes gradually toward the distal end of the isthmus and cannot be demonstrated at the proximal end ofthe ampulla. The circular musculature is the most prominent and well-defined of the three layers, whereas the external layer is characterized by a mixed fiber direction, although most of the fibers run parallel to the long axis of the tube. Earlier inv~stigations have not sufficiently considered this anatomical arrangement of the musculature in this particular oviductal region. Recent investigations in our laboratory have demonstrated that these muscle layers not only exhibit different characteristics of spontaneous activity in vitro, but also respond disparately to various prostaglandins. 9 Earlier studies have demonstrated the differentiated contractile effects of prostaglandins E and F on the muscle layers of
An adrenergic sphincter function has been clearly demonstrated at the level of the ampullaryisthmic junction (AIJ) of the rabbit oviduct. 1 , 2 It has been suggested that this muscular mechanism is significant in the regulation of ovum transport in this species. 3 Although a similar adrenergic innervation is present in the human oviductal isthmus,4 it has not as yet been convincingly shown that such a neuromuscular mechanism plays a functional role in ovum transport in women. 3 Studies of human oviductal contractility are impaired both by ethical constraints and by difficulties in designing proper technology for recording tubal motility in vivo. 5 Furthermore, interpretation of earlier in vitro investigations of human oviductal contractility is difficult because of the
Received July 9, 1979; revised October 18, 1979; accepted October 22, 1979. *Supported by grants from the Goteborg Medical Society and the Faculty of Medicine, University of Goteborg. tReprint requests: Lennart Wilhelmsson, M.D., Department of Obstetrics and Gynecology, University of Goteborg, Sahlgrenska Sjukhuset, S-413 45 Goteborg, Sweden.
280
Vol. 33, No.3
Per cent of 100 max stimulation
TABLE 1. Cycle Phase and Age of Patients No. of patients
Follicular Luteal
4 6
Mean
Range
80
46.2 41.0
35-51 36--48
80
the AIJ .10 Furthermore, a recent investigation has indicated an unequal distribution of adrenoceptors in the circular and longitudinal layers of the AIJ. 11 It thus seems clear that the various smooth muscle layers of the human oviductal isthmus possess specific physiologic and pharmacologic characteristics. To achieve a more complete picture of the contractile function of the tubal isthmus it is necessary also to elucidate the adrenoceptor responses of the different layers of smooth muscle at the proximal borderline of the isthmic segment, i.e., the UTJ. This preliminary report describes the influence of the adrenergic neurotransmitter noradrenalin (NA) on this muscle structure and the modification of its effects by a- and ~ adrenoceptor blockade. MATERIALS AND METHODS
Patients. Oviductal segments were obtained from 10 women of fertile age with regular menstrual cycles who were undergoing sterilization. The tissue was immediately placed in ice-cold buffer and transported to the laboratory. Muscle strips (length 4 mm, cross-sectional area 1 to 1.5 sq mm) were dissected from various muscle layers and suspended in 50-ml organ chambers for isometric recording of the contractile activity at 37 0 C. Further methodologic details have been
U
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Age
Cycle phase
A
281
ADRENERGIC RESPONSES AT THE HUMAN UTEROTUBAL JUNCTION
~
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illUllllliWl1JJ t
~
~_\
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,
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,1/
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.
.
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8
Per cent 01 max inhibition
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FIG. 2. An example of concentration-effect curves for NA on the inner longitudinal and the intermediate circular layer before ( - ) and after (- - -) treatment with propranolol (12 f.LMJ. The inhibitory effects are abolished by propranolol and, in the case of the inner longitudinal layer, even changed to a marked excitatory response (compare Fig. 18).
describedY Included in the study were subjects in the follicular phase as well as the luteal phase (Table 1). Buffer and Chemicals. Oxygenated KrebsRinger bicarbonate buffer solution was used for the dissection procedure and fortified with 10.0 mM d-glucose for the contractility experiments. Ethy lenediaminetetraacetic acid (EDT A) was added to the buffer solution to give a final concentration of 0.026 mM. The following drugs were used: Noradrenalin bitartrate (Nordrenalin, Apoteksbolaget Ltd., Stockholm, Sweden), propranolol chloride (lnderal, ICI Ltd., Manchester, United Kingdom), and EDTA disodium calcium salt (Sigma Chemical Co., St. Louis, Mo.). RESULTS
L
t
t
FIG.!' A, Control. Maximal effect ofNA (induced by 1.0 f.LM) on the three separate smooth muscle layers of the UTJ. B, I3-Blockade. The effect of propranolol (12 f.LM) on the responses to NA (1.0 f.LM) of the same specimens as in A. U, Uterine spiral-shaped layer; C, intermediate circular layer; L, inner longitudinal layer. Vertical calibration, 4 mN; horizontal calibration, 3 minutes.
The three types of preparations exhibited the typical contractile patterns previously described, i.e, a comparatively low frequency and long duration of contractions in strips from the uterine layer· and a high contraction frequency and short duration of individual contractions in specimens from the intermediate circular and inner longitudinal layers. 9.
WILHELMSSON AND LINDBLOM March 1980 282 dinallayer. The absence of such an effect in the The adminsitration of NA in concentrations of circular musculature illustrates the relative lack 0.01 to 10 ILM caused an inhibition of spontaneous of a-adrenoceptors in this layer. The present study contractile activity in preparations from the indid not reveal any qualitative differences in adretermediate circular layer and the inner longitunoceptor responses during the menstrual cycle. dinal layer. This response lasted throughout the Nevertheless, at present the possibility cannot be exposure period, but after repetitive rinsing the initial activity reappeared. In the specimens excluded that the "balance" between a- and 13adrenoceptor sensitivity in this region is prone to a obtained from the outer spiral-shaped layer, the addition ofNA (0.01 to 10 ILM) induced a clear-cut hormonal influence, as was suggested by Moawad excitatory response with increased amplitude and et al. 13 An increased l3-adrenoceptor sensitivity frequency of contractions (Fig. lA). induced by increased plasma levels of progesterAfter treatment with the a-adrenoceptor antagone by the newly formed corpus luteum may in fact onist phenoxybenzamine (0.1 ILM) the excitatory be expedient for the passage of the fertilized ovum effects ofNA administration were completely abolthrough the isthmus of the fallopian tube. ished. Acknowledgment. Thanks are expressed to Dr. Anders NorPretreatment for 20 minutes with propranolol strom for histologic examinations. (12 ILM) reversed the inhibitory effect ofNA on the inner longitudinal layer to an excitatory response REFERENCES and abolished the inhibitory response to NA ofthe 1. Brundin J: A functional block in the isthmus of the rabbit intermediate circular musculature (Fig. lB). Figfallopian tube. Acta Physiol Scand 60:295, 1964 ure 2 illustrates the change in contractile force of 2. Howe GR, Black DL: Autonomic nervous system and oviduct function in the rabbit. I. Hormones and contraction. J the two types of specimens induced by various conReprod Fertil 33:425, 1973 centrations ofNA before and after treatment with 3. Paton DM, Widdicombe JH, Rheaume DE, Johns A: The propranolol. The excitatory effect of NA on strips role of the adrenergic innervation of the oviduct in the from the outer longitudinal layer was further augregulation of mammalian ovum transport. Pharmacol Rev mented after treatment with propranolol. 29:67,1978
DISCUSSION
Earlier in vivo studies have shown that the human oviductal isthmus may react with both excitation and inhibition to adrenergic stimulation produced by intravenously administered catecholamines. 12 Such in vivo methods, however, are hampered by difficulties in determining the localization of an intraluminal recording device during long-term experiments, and the presence of a probe within the oviduct may indeed interfere with various local regulatory mechanisms (e.g., release of neurotransmittors and prostaglandins). In addition, the narrow lumen of the UTJ limits the possibility of using intraluminal techniques for recording contractile activity in this region. The present in vitro model has offered the possibility of elucidating the adrenergic responses of multidirectionally arranged smooth musculature at the UTJ of the human female. It was shown that a-adrenoceptor-mediated excitatory responses were confirmed to the outer longitudinallspiralshaped layer, whereas l3-adrenoceptor-mediated inhibitory effects were predominantly found in specimens from the intermediate circular and inner longitudinal layers. After l3-blockade, however, an a-adrenoceptor-mediated excitatory response could be demonstrated in the inner longitu-
4. Brundin J, Wirsen C: Adrenergic nerve terminals in the human fallopian tube examined by fluorescence microscopy. Acta Physiol Scand 61:505, 1964 5. Blandau RJ, Boling JL, Halbert S, Verdugo P: Methods for studying oviductal physiology. Gynecol Invest 6:123,1975 6. Lisa JR, Gioia JD, Rubin IC: Observations on the interstitial portion of the fallopian tube. Surg Obstet Gynecol 99:159,1954 7. David A, Czernobilsky B: A comparative histological study of the uterotubal junction in the rabbit, rhesus monkey and human female. Am J Obstet Gynecol101:417, 1968 8. Daniel EE, Posey VA, Paton DM: A structural analysis of the myogenic control systems of the human fallopian tube. Am J Obstet Gynecol 121:1054, 1975 9. Wilhelmsson L, Lindblom B, Wiqvist N: The human uterotubal junction: contractile patterns of different smooth muscle layers and influence of prostaglandins E 2 , F 2Ot, and 12 in vitro. Fertil Steril 32:303, 1979 10. Lindblom B, Hamberger L, Wiqvist N: Differentiated contractile effects of prostaglandins E and F on the smooth muscle activity of the human oviduct. Fertil Steril30:553, 1978 11. Lindblom B, Ljung B, Hamberger L: Adrenergic and novel nonadrenergic neuronal mechanisms in the control of smooth muscle activity in the human oviduct. Acta Physiol Scand 106:215, 1979 12. Coutinho EM, Maia H, Filho JA: Response of the human fallopian tube to adrenergic stimulation. Fertil Steril 21:590, 1970 13. Moawad AH, Hedqvist P, Kim MH: Correlation of plasma estrogens and progesterone levels with the in vitro adrenergic responses in the isthmus of the human oviduct. In Ovum Transport and Fertility Regulation, Edited by MJK Harper, CJ Pauerstein, CE Adams, EM Coutinho, HB Croxatto, DN Paton. Copenhagen, Scriptor, 1976, p 276
FERTILITY AND STERILITY Copyright c 1980 The American Fertility Society
ADRENERGIC RESPONSES OF THE VARIOUS SMOOTH MUSCLE LAYERS AT THE HUMAN UTEROTUBAL JUNCTION*
LENNART WILHELMSSON, M.D.t BO LINDBLOM, M.D., PH.D.
Department of Obstetrics and Gynecology, Uniuersity of Goteborg, Goteborg, Sweden
The different muscle layers at the human uterotubal junction (UTJ) were mechanically separated, and 1-mm wide muscle strips were isolated and mounted in organ chambers for isometric recording of contractile activity. It was found that the adrenergic neurotransmitter noradrenalin elicited an inhibition of the spontaneous phasic activity in the inner longitudinal and the intermediate circular layers but caused a clear-cut excitatory response in the outer, spiral-shaped (uterine) layer. The excitatory response was totally blocked by treatment with phenoxybenzamine, an o.-adrenoceptor antagonist, whereas the inhibitory effects were abolished after pretreatment with the f3-adrenoceptor antagonist propranolol. The data demonstrate a differentiated distribution of 0.- and f3-adrenoceptors in the smooth musculature at the proximal end of the tubal isthmus. It is suggested that this arrangement might serve an important purpose in the physiologic control of ovum transport in women. Fertil Steril 33:286, 1980
use of very large segments and the differing methodologic approaches in various studies. 3 Three main muscle layers are present in the isthmic region: the inner longitudinal, the intermediate circular, and the outer longitudinal or spiral-shaped uterine layer. 6 - B The inner longitudinal layer is distinct at the uterotubal junction (UTJ) but diminishes gradually toward the distal end of the isthmus and cannot be demonstrated at the proximal end ofthe ampulla. The circular musculature is the most prominent and well-defined of the three layers, whereas the external layer is characterized by a mixed fiber direction, although most of the fibers run parallel to the long axis of the tube. Earlier inv~stigations have not sufficiently considered this anatomical arrangement of the musculature in this particular oviductal region. Recent investigations in our laboratory have demonstrated that these muscle layers not only exhibit different characteristics of spontaneous activity in vitro, but also respond disparately to various prostaglandins. 9 Earlier studies have demonstrated the differentiated contractile effects of prostaglandins E and F on the muscle layers of
An adrenergic sphincter function has been clearly demonstrated at the level of the ampullaryisthmic junction (AIJ) of the rabbit oviduct. 1 , 2 It has been suggested that this muscular mechanism is significant in the regulation of ovum transport in this species. 3 Although a similar adrenergic innervation is present in the human oviductal isthmus,4 it has not as yet been convincingly shown that such a neuromuscular mechanism plays a functional role in ovum transport in women. 3 Studies of human oviductal contractility are impaired both by ethical constraints and by difficulties in designing proper technology for recording tubal motility in vivo. 5 Furthermore, interpretation of earlier in vitro investigations of human oviductal contractility is difficult because of the
Received July 9, 1979; revised October 18, 1979; accepted October 22, 1979. *Supported by grants from the Goteborg Medical Society and the Faculty of Medicine, University of Goteborg. tReprint requests: Lennart Wilhelmsson, M.D., Department of Obstetrics and Gynecology, University of Goteborg, Sahlgrenska Sjukhuset, S-413 45 Goteborg, Sweden.
280
Vol. 33, No.3
Per cent of 100 max stimulation
TABLE 1. Cycle Phase and Age of Patients No. of patients
Follicular Luteal
4 6
Mean
Range
80
46.2 41.0
35-51 36--48
80
the AIJ .10 Furthermore, a recent investigation has indicated an unequal distribution of adrenoceptors in the circular and longitudinal layers of the AIJ. 11 It thus seems clear that the various smooth muscle layers of the human oviductal isthmus possess specific physiologic and pharmacologic characteristics. To achieve a more complete picture of the contractile function of the tubal isthmus it is necessary also to elucidate the adrenoceptor responses of the different layers of smooth muscle at the proximal borderline of the isthmic segment, i.e., the UTJ. This preliminary report describes the influence of the adrenergic neurotransmitter noradrenalin (NA) on this muscle structure and the modification of its effects by a- and ~ adrenoceptor blockade. MATERIALS AND METHODS
Patients. Oviductal segments were obtained from 10 women of fertile age with regular menstrual cycles who were undergoing sterilization. The tissue was immediately placed in ice-cold buffer and transported to the laboratory. Muscle strips (length 4 mm, cross-sectional area 1 to 1.5 sq mm) were dissected from various muscle layers and suspended in 50-ml organ chambers for isometric recording of the contractile activity at 37 0 C. Further methodologic details have been
U
C
,,
,, ,,
Age
Cycle phase
A
281
ADRENERGIC RESPONSES AT THE HUMAN UTEROTUBAL JUNCTION
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~_\
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,, ,,
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~----------~--------~-------
:s;:
-40
-80
-80
·100
,t
.
.
9
8
Per cent 01 max inhibition
7
6
~og
(NAI M
FIG. 2. An example of concentration-effect curves for NA on the inner longitudinal and the intermediate circular layer before ( - ) and after (- - -) treatment with propranolol (12 f.LMJ. The inhibitory effects are abolished by propranolol and, in the case of the inner longitudinal layer, even changed to a marked excitatory response (compare Fig. 18).
describedY Included in the study were subjects in the follicular phase as well as the luteal phase (Table 1). Buffer and Chemicals. Oxygenated KrebsRinger bicarbonate buffer solution was used for the dissection procedure and fortified with 10.0 mM d-glucose for the contractility experiments. Ethy lenediaminetetraacetic acid (EDT A) was added to the buffer solution to give a final concentration of 0.026 mM. The following drugs were used: Noradrenalin bitartrate (Nordrenalin, Apoteksbolaget Ltd., Stockholm, Sweden), propranolol chloride (lnderal, ICI Ltd., Manchester, United Kingdom), and EDTA disodium calcium salt (Sigma Chemical Co., St. Louis, Mo.). RESULTS
L
t
t
FIG.!' A, Control. Maximal effect ofNA (induced by 1.0 f.LM) on the three separate smooth muscle layers of the UTJ. B, I3-Blockade. The effect of propranolol (12 f.LM) on the responses to NA (1.0 f.LM) of the same specimens as in A. U, Uterine spiral-shaped layer; C, intermediate circular layer; L, inner longitudinal layer. Vertical calibration, 4 mN; horizontal calibration, 3 minutes.
The three types of preparations exhibited the typical contractile patterns previously described, i.e, a comparatively low frequency and long duration of contractions in strips from the uterine layer· and a high contraction frequency and short duration of individual contractions in specimens from the intermediate circular and inner longitudinal layers. 9.
WILHELMSSON AND LINDBLOM March 1980 282 dinallayer. The absence of such an effect in the The adminsitration of NA in concentrations of circular musculature illustrates the relative lack 0.01 to 10 ILM caused an inhibition of spontaneous of a-adrenoceptors in this layer. The present study contractile activity in preparations from the indid not reveal any qualitative differences in adretermediate circular layer and the inner longitunoceptor responses during the menstrual cycle. dinal layer. This response lasted throughout the Nevertheless, at present the possibility cannot be exposure period, but after repetitive rinsing the initial activity reappeared. In the specimens excluded that the "balance" between a- and 13adrenoceptor sensitivity in this region is prone to a obtained from the outer spiral-shaped layer, the addition ofNA (0.01 to 10 ILM) induced a clear-cut hormonal influence, as was suggested by Moawad excitatory response with increased amplitude and et al. 13 An increased l3-adrenoceptor sensitivity frequency of contractions (Fig. lA). induced by increased plasma levels of progesterAfter treatment with the a-adrenoceptor antagone by the newly formed corpus luteum may in fact onist phenoxybenzamine (0.1 ILM) the excitatory be expedient for the passage of the fertilized ovum effects ofNA administration were completely abolthrough the isthmus of the fallopian tube. ished. Acknowledgment. Thanks are expressed to Dr. Anders NorPretreatment for 20 minutes with propranolol strom for histologic examinations. (12 ILM) reversed the inhibitory effect ofNA on the inner longitudinal layer to an excitatory response REFERENCES and abolished the inhibitory response to NA ofthe 1. Brundin J: A functional block in the isthmus of the rabbit intermediate circular musculature (Fig. lB). Figfallopian tube. Acta Physiol Scand 60:295, 1964 ure 2 illustrates the change in contractile force of 2. Howe GR, Black DL: Autonomic nervous system and oviduct function in the rabbit. I. Hormones and contraction. J the two types of specimens induced by various conReprod Fertil 33:425, 1973 centrations ofNA before and after treatment with 3. Paton DM, Widdicombe JH, Rheaume DE, Johns A: The propranolol. The excitatory effect of NA on strips role of the adrenergic innervation of the oviduct in the from the outer longitudinal layer was further augregulation of mammalian ovum transport. Pharmacol Rev mented after treatment with propranolol. 29:67,1978
DISCUSSION
Earlier in vivo studies have shown that the human oviductal isthmus may react with both excitation and inhibition to adrenergic stimulation produced by intravenously administered catecholamines. 12 Such in vivo methods, however, are hampered by difficulties in determining the localization of an intraluminal recording device during long-term experiments, and the presence of a probe within the oviduct may indeed interfere with various local regulatory mechanisms (e.g., release of neurotransmittors and prostaglandins). In addition, the narrow lumen of the UTJ limits the possibility of using intraluminal techniques for recording contractile activity in this region. The present in vitro model has offered the possibility of elucidating the adrenergic responses of multidirectionally arranged smooth musculature at the UTJ of the human female. It was shown that a-adrenoceptor-mediated excitatory responses were confirmed to the outer longitudinallspiralshaped layer, whereas l3-adrenoceptor-mediated inhibitory effects were predominantly found in specimens from the intermediate circular and inner longitudinal layers. After l3-blockade, however, an a-adrenoceptor-mediated excitatory response could be demonstrated in the inner longitu-
4. Brundin J, Wirsen C: Adrenergic nerve terminals in the human fallopian tube examined by fluorescence microscopy. Acta Physiol Scand 61:505, 1964 5. Blandau RJ, Boling JL, Halbert S, Verdugo P: Methods for studying oviductal physiology. Gynecol Invest 6:123,1975 6. Lisa JR, Gioia JD, Rubin IC: Observations on the interstitial portion of the fallopian tube. Surg Obstet Gynecol 99:159,1954 7. David A, Czernobilsky B: A comparative histological study of the uterotubal junction in the rabbit, rhesus monkey and human female. Am J Obstet Gynecol101:417, 1968 8. Daniel EE, Posey VA, Paton DM: A structural analysis of the myogenic control systems of the human fallopian tube. Am J Obstet Gynecol 121:1054, 1975 9. Wilhelmsson L, Lindblom B, Wiqvist N: The human uterotubal junction: contractile patterns of different smooth muscle layers and influence of prostaglandins E 2 , F 2Ot, and 12 in vitro. Fertil Steril 32:303, 1979 10. Lindblom B, Hamberger L, Wiqvist N: Differentiated contractile effects of prostaglandins E and F on the smooth muscle activity of the human oviduct. Fertil Steril30:553, 1978 11. Lindblom B, Ljung B, Hamberger L: Adrenergic and novel nonadrenergic neuronal mechanisms in the control of smooth muscle activity in the human oviduct. Acta Physiol Scand 106:215, 1979 12. Coutinho EM, Maia H, Filho JA: Response of the human fallopian tube to adrenergic stimulation. Fertil Steril 21:590, 1970 13. Moawad AH, Hedqvist P, Kim MH: Correlation of plasma estrogens and progesterone levels with the in vitro adrenergic responses in the isthmus of the human oviduct. In Ovum Transport and Fertility Regulation, Edited by MJK Harper, CJ Pauerstein, CE Adams, EM Coutinho, HB Croxatto, DN Paton. Copenhagen, Scriptor, 1976, p 276