Effects of chronic and acute oestrogen treatment on the developing noradrenaline-containing nerves of the rat uterus

Int. J. DevINeuroscience, Vol. 13, No. 8, pp. 791-798, 1995

~)Pergamon

0'736-5"/48(90")~-8

ElsevierScienceLtd Copyright© 1995ISDN Printed in Great Britain. All rightsreserved 0736-5748/95 $9.50+0.00

EFFECTS OF CHRONIC AND ACUTE OESTROGEN TREATMENT ON THE DEVELOPING NORADRENALINE-CONTAINING NERVES OF THE RAT UTERUS M. M. BRAUER,*t$ A. M. CORBACHOt¢ and G. BURNSTOCK§ tDivisi6n Biologia Celular, Instituto de Investigaciones Biol6gicas Clemente Estable, Avenida Italia 3318, Montevideo 11600, Uruguay; ~tSecci6nBioiogia Celular, Facultad de Ciencias, Montevideo, Uruguay; §Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, U.K. (Received 6 March 1995; revised 3 August 1995; accepted 15 August 1995)

Ahslraet--The developing noradrenaline-containing (NA-C) sympathetic nerves of the rat uterus were analyzed following acute and chronic treatment with oestrogen. Histochemical methods were used in association with nerve density measurements and biochemical assays. For comparative purposes, noradrenaline (NA) levels were measured in the urinary bladder and right auricle following chronic oestrogen treatment. Acute treatment was performed by s.c. administration of a single dose of 40 p.g oestradiol cypionate on the 25th day of age. Chronic treatment consisted of four doses of 10 Ixg oestradiol on days 10, 15, 20 and 25 of postnatal development. Both acute- and chronic-treated animals were killed at 28 days of age. The main biochemical findings were the following: (a) both acute and chronic oestrogen treatment increased the weight of the uterine horn, parametrial tissue and uterine cervix; (b) in the uterine horn, the total content of NA was reduced following both oestrogen treatments. However, the degree of reduction was greater after chronic treatment; (c) in the parametrial tissue, the NA levels were reduced only after chronic treatment; (d) in the cervix, the NA total content was increased after both treatments; (e) in the urinary bladder, there was a parallel increase between organ growth and NA content following chronic oestrogen treatment; (e) in the auricle neither the tissue weight nor the total content of NA were changed by chronic estrogen treatment. Histochemical studies showed that: (a) acute treatment with one single dose of oestradiol, provoked a marked reduction in the density of NA-C nerves associated with the myometrial and parametrial smooth muscle, without affecting the innervation of blood vessels; (b) following chronic treatment, the only fibers we were able to recognize were those associated with blood vessels. These fibers were thinner and less intensely fluorescent than in controls. Results are interpreted considering the differential sensitivity of uterine nerves to sex hormones. A possible involvement of oestrogen in changes of noradrenergic innervation of the uterus following puberty is discussed. Key words: uterus, noradrenaline, oestrogen, development.

S o m e p r e v i o u s s t u d i e s o f o u r g r o u p 2-4 h a v e s h o w n t h a t t h e r a t u t e r u s is i n n e r v a t e d , f r o m t h e e a r l y stages of postnatal development, by noradrenaline-containing (NA-C) nerves which are associated w i t h b l o o d vessels a n d m y o m e t r i a l a n d p a r a m e t r i a l s m o o t h muscle. T h e i n n e r v a t i o n a s s o c i a t e d with the non-vascular smooth muscle shows a progressive increase between the infantile and prepubertal p e r i o d s , w h i c h is a c c o m p a n i e d b y a g r a d u a l i n c r e a s e in t h e t o t a l c o n t e n t a n d c o n c e n t r a t i o n o f noradrenafine (NA). F o l l o w i n g t h e first o e s t r u s at p u b e r t y , t h e r e is a m a r k e d r e d u c t i o n in t h e d e n s i t y o f N A - C n e r v e s a s s o c i a t e d w i t h t h e m y o m e t r i a l a n d p a r a m e t r i a l s m o o t h muscle, which is f o l l o w e d b y a f u r t h e r reduction between the peripubertal and young adult period. Conversely, the growth pattern of N A - C n e r v e s o f u t e r i n e i n t r a m u r a l b l o o d vessels a n d t h e u t e r i n e a r t e r y d o e s n o t c h a n g e at p u b e r t y o r in t h e y o u n g a d u l t a n d fully a d u l t stages. 2-4,6 T h e t o t a l c o n t e n t o f N A in t h e u t e r i n e h o r n a n d p a r a m e t r i a l tissue d o e s n o t c h a n g e d u r i n g p u b e r t y ; h o w e v e r , since t h e w e i g h t o f t h e s e u t e r i n e r e g i o n s i n c r e a s e s m a r k e d l y , a fall in t h e c o n c e n t r a t i o n o f N A is o b s e r v e d . O n t h e o t h e r h a n d , in t h e u t e r i n e cervix t h e r e is a p a r a l l e l i n c r e a s e b e t w e e n o r g a n w e i g h t a n d N A c o n t e n t . 2,3 A m o r p h o m e t r i c s t u d y 2 s h o w e d t h a t s m o o t h m u s c l e cells in t h e u t e r i n e h o r n a n d p a r a m e t r i a l tissue i n c r e a s e b o t h in size a n d n u m b e r d u r i n g t h e p e r i p u b e r t a l t r a n s i t i o n . This c h a n g e w o u l d provoke a "spatial dilution" of non-vascular NA-C nerves, modifying irreversibly the neuromus-

*Towhom all correspondence should be addressed. Abbrevations: NA, noradrenaline; NA-C, noradrenaline-containing.

791

792

M.M. Brauer et al.

cular relationship. In this context it is important to analyze what prevents the NA-C nerves from developing in parallel with the myometrial and parametrial smooth muscle, while sensory nerves of the uterus and NA-C nerves of the cervix and blood vessels seem to develop parallel with their target smooth muscle. 2-4,6 Considering that the N A - C nerves of the uterus are susceptible to s e x h o r m o n e s 15,16,23,31 and that puberty is associated with an important endocrine reorganization, 20 it is possible that changes in the non-vascular innervation of the uterus could be related with the increase in oestrogen and progesterone occurring at puberty. In the present study, as a first approach to test the possibility that NA-C nerves are sensitive to sex hormones during development, the noradrenergic innervation of the rat uterus has been analyzed histochemically and biochemically, following acute and chronic administration of oestrogen during development. High doses of oestrogen were employed to block ovulation and ovarian production of progesterone. For comparative purposes NA levels have been measured in the urinary bladder and right auricle following chronic oestrogen treatment. Part of these results has been published in abstract form. 5

EXPERIMENTAL PROCEDURES Animals and oestrogen treatment

Thirty-five female Wistar-derived albino rats from the I.I.B.C.E. colony (Montevideo, Uruguay) were used in this study. Animals were housed under constant conditions of temperature and illumination and provided with food and water ad libitum. Oestrogen treatment was performed with 13-oestradio117-cypionate, diluted to appropriate doses with peanut oil. Control animals were injected only with the vehicle. Acute treatment. Ten females were injected s.c. with one dose of 40 I~g oestradiol on the 25th day of life and killed at 28 days of age. Chronic treatment. Twelve females were injected s.c. with four doses of 10 Ixg oestradiol on days 10, 15, 20 and 25 of postnatal development and killed at 28 days of age. Animals were killed by cervical dislocation. Tissues were removed and placed in cold phosphatebuffered saline solution for dissection. Tissues for biochemical assay of NA were stored at -70°C until analysis. Biochemical assay for noradrenaline

NA levels were measured in the isolated whole uterine horn, parametrial tissue and uterine cervix by high performance liquid chromatography with electrochemical detection, as previously reported. 2-4,13 For comparative purposes the content of NA was measured in the urinary bladder and right auricle following chronic oestrogen treatment. NA levels have been expressed as NA total content/organ. Histochemistry and nerve density measurement

NA-C nerves were demonstrated in the uterine horn and parametrial tissue by the glyoxylic acid method performed on cryostat sections and whole-mount stretch preparations, as previously reported. 2--4,7,9Preparations were examined with a Nikon Microphot FX microscope equipped with epifluorescence and fitted with the appropriate filters. Micrographs were taken with Kodak Tri-X Pan, 400 ASA film. The density of NA-C non-vascular nerve fibers was estimated on whole-mount stretch preparations of the uterine horn longitudinal smooth muscle layer and in the parametrial tissue, by using an ocular scale set perpendicular to the organ long axis, as previously reported. 2-4 The density of perivascular nerve fibers was estimated on whole-mount stretch preparations of the parametrial blood vessels. 2-4 The innervation intercept density (the number of fibers which intercept the ocular scale), has been calculated as the mean number of fibers/mm. Because the density of NA-C nerves was extremely sparse after chronic oestrogen treatment, quantitative studies were not attempted.

Effects of oestrogen on developing uterine noradrenergic nerves

793

Statistical analysis Results have been given as the mean_+S.E.M. Data were compared using the Kruskal-Wallis and Wilcoxon non-parametric tests. 18 A level of probability of P<-0.05 was taken to indicate statistical significance. Mater&Is [3-oestradio117-cypionate ([3-oestradio117-cyclopentylpropionate) was supplied by Laboratorios K6ning (Argentina). Glyoxylic acid monohydrate and peanut oil were obtained from Sigma Chemical Co. (U.S.A.).

RESULTS Animals Chronic treatment with oestrogen did not modify animal growth rate [weight of animals at 28 days of age, control: 53.5+1.3 g (n-13); treated: 52.5+1.0 g (n=12)]. Chronic-treated animals showed vaginal canalization following the second injection of hormone (between 15 and 20 days of age). From this moment on vaginal smears showed the constant presence of cornified cells (oestrus). 14 Similarly, animals treated with a single dose of oestrogen at 25 days of age showed--at the time of sacrifice--canalized vagina and cornified cells in the vaginal smear. Neither chronic- nor acute-treated animals showed signs of ovulation (haemorrhagic bodies or corpus lutei in the ovaries or ova in the Fallopian tubes). Biochemistry The wet weight of the different uterine regions was increased after acute and chronic oestrogen treatment (Figs 1-3). Acute treatment with a single dose of 40 p.g of oestrogen gave different responses in the biochemical profile of the three analyzed uterine regions. The total content of NA was reduced in the uterine horn (Fig. 1) and unchanged in the parametrial tissue (Fig. 2). In the cervix, there was a parallel increase between organ growth and total NA content (Fig. 3). Chronic treatment with oestrogen resulted in a reduction in the total content of NA of the uterine horn and parametrial tissue. The degree of reduction in the horn was greater than that observed after acute treatment. In the cervix, NA levels were increased, although to a lesser extent than in acute-treated animals. In the urinary bladder, there was a parallel increase between NA content and organ growth (Table 1). In the auricle neither the weight nor the content of NA were changed (Table 1). Histochemistry and nerve density measurement Results of the histochemical studies are shown in Figs 4-11 and the numerical data have been summarized in Table 2. In controls, whole-mount preparations of the uterine horn (Fig. 4) and parametrial tissue (Fig. 5) showed a rich noradrenergic plexus associated with blood vessels and myometrial and parametrial smooth muscle. Cryostat sections showed NA-C nerve fibers associated with the circular muscle layer and blood vessels located in the connective septum separating the two myometrial layers (Fig. 10). Following administration of one dose of 40 Ixg oestrogen the density of NA-C nerve fibers associated with the myometrial and parametrial smooth muscle was markedly reduced (Figs 6 and 7, Table 2). The pattern and density of innervation of the blood vessels was unchanged (Figs 6 and 7, Table 2). Whole-mount preparations and cryostat sections of the uterine horn and parametrial tissue from chronic-treated oestrogen animals showed that most of the recognized nerve fibers were those associated with blood vessels as perivascular and paravascular fibers (Figs 8, 9 and 11). Although the innervation pattern of blood vessels was similar to controls, fibers were thinner and less intensely fluorescent.

794

M.M. B r a u e r et al.

1

Uterine horn

80

(12) #

70

60 (•)

50 40 30 20 113)

10

(121

0 Control

Acute

Chronic

Wet weight (mg) NA content (ng)

3

Parametrial tissue

Uterine cervix

25

(11)

(9) #

I

(13)

r

(10)

20

~'~

15

(9)

(tl) (10) ~?:~¢';~ i

10

(9)

(o) 1131 (e) J ~W#d

Control

Acute

Control

Chronic

I I

I Wet weight (mg)

Acute

Chronic

J Wet weight (mg) NA content (ng)

NA content (ng)

Figs 1-3. Biochemical determinations of noradrenaline total content (ng) and organ wet weight (mg) of three uterine regions (horn, parametrial tissue and cervix) in controls and following acute and chronic oestrogen treatment. Mean+S.E.M. (n) Data compared by the Kruskal-Wallis non-parametric test. * = Significant difference with control, #= significant difference between treatments. For all comparisons P--0.001.

Effects of oestrogen on developing uterine noradrenergic nerves

795

Table 1. Summary of the wet weight (rag), NA content (ng) and NA concentration (ng/g wet weight tissue) of the urinary bladder (UB) and right auricle (RA), in controls and following chronic oestrogen treatment Tissue U B Control UB Treated R A Control R A Treated

Wet weight

NA content

NA concentration

21.6+-1.1 (n=4) 33.0--- 1.4 (n=4)* 15.4-+3.0

4.7-+0.5 (n=4) 8.8---0.9 (n=4)* 25.1-+4.0

220.5+--26.0 (n=4) 267.0-+27.0 (n=a)ns 1716.0-+188.0

(n=5)

(.=5)

13.4+--1.5 (n=4)ns

21.6-+2.3 (n=4) ns

(n=5) 1613.0+-61.0 (n=4)ns

Results are expressed as the mean - S.E.M. (n). Mean values compared by the Wilcoxon non-parametric test. ns = non-significant, * P-<0.05.

Table 2. Quantitative assessment of the innervation density of noradrenaline-containing nerve fibers in controls and following acute oestrogen treatment. The intercept density, expressed as the n u m b e r of fibers/mm, of non-vascular nerve fibers associated with the longitudinal smooth muscle layer of the uterine horn and parametrial tissue and of the perivascular nerve fibers associated with the parametrial blood vessels was measured Tissue

Number of fibers/mm

Uterine non-vascular Parametrial non-vascular Parametrial vessels

Control Treated Control Treated Control Treated

137-+10 68-+4 60-+5 31-+3 64--- 1 79-+3

(n=5) (n=5)* (n=5) (n=4)* (n=4) (n=4) ns

Results are expressed as the mean - S.E.M. (n). Mean values compared by the Wilcoxon non-parametric test. ns = non-significant, *P<-0.05.

DISCUSSION Several studies have shown that the uterus is supplied by NA-C nerves which are associated with blood vessels and myometrial smooth muscle.26 The innervation of vessels is rich and shows a similar density in all the analyzed mammalian species. However, the innervation of myometrium shows a considerable species variation. 10,15,16,21,22,24,26,28 The sympathetic innervation of the uterus consists of classical long noradrenergic fibers, originating in the lumbar and mesenteric ganglia, and short noradrenergic fibers whose cell bodies are in the paracervical ganglia, located at the celvico-vaginal junction. It is believed that long NA fibers are associated with blood vessels, whereas short fibers mainly innervate the myometrium. 15,16,26 Nevertheless, this concept has been recently reviewed and a mixed innervation of short and long noradrenergic fibers has been proposed for the rat uterine blood vessels. 27 Short and long noradrenergic fibers differ from one another in their physiological properties. For instance, short NA-C nerve fibers are a target for sex steroids. The terminal noradrenergic plexus and the metabolism of NA is changed in response to the hormonal state of the animal as well as during pregnancy. 1,15,16,23,26,33 Factors controlling the differential sensitivity of short noradrenergic neurons to sex steroids have not been ellucidated, although it could be related to a different pattern of expression of the sex hormone receptors in neurons of the paracervical ganglia. The relative sensitivity of nerve fibers to sex hormones shows regional variations, being higher in the uterine horn than in the cervix. 23,26 At present, this difference is not easy to explain, since it has been established that in the guinea pig, 23 the cervix receives about half of its noradrenergic innervation from the paracervical ganglia, by way of short noradrenergic neurons. The remaining

796

M.M. B r a u e r et al.

Figs 4-9: NA-C nerve fibers demonstrated by the glyoxylic acid method in whole-mount stretch preparations of the rat uterine horn (Figs 4, 6 and 8) and parametrial tissue (Figs 5, 7 and 9). Figures 4 and 5: four-week-old control; Figs 6 and 7: following acute oestrogen treatment; Figs 8 and 9: following chronic oestrogen treatment. Thick arrow=blood vessels; thin arrows=isolated non-vascular nerve fibers. Figs 10 and 11. Transverse cryostat sections of the rat uterine horn processed for the fluorescence histochemical demonstration of NA-C nerve fibers. Figure 10: four-week-old control; Fig. 11: following chronic oestrogen treatment. Thick arrows=perivascular nerve fibers in the connective septum separating the * circular and "~ longitudinal myometrial layers. Thin arrows=non-vascular nerve fibers, E= endometrium. Calibration bar=80 ~m.

Effects of oestrogen on developing uterine noradrenergic nerves

797

half is supplied by postganglionic fibers in the hypogastric nerves. In the uterine horn, only one-third of the noradrenergic innervation comes from the paracervical ganglia. Another third comes from the inferior mesenteric ganglion, via the hypogastric nerves. The tubal end of the uterine horn receives the remaining third via the costo-uterine ligament. This pathway of innervation is not affected by the endocrine state of the animal or by pregnancy. The presence in the adult rat uterus of a substantial contribution of short noradrenergic fibers still remain uncertain. Some authors 1-4,24,3° have described a moderately dense NA plexus supplying the rat myometrium whereas others have claimed that in this species most--if not all--noradrenergic fibers are perivascular. 10,12,19 One-third to one-half of the neurons in the paracervical ganglia have been described as noradrenergic; 11A2 however, only a few or no NA-C neurons have been recognized. 1,1°,25 Reports on the effects of ovariectomy and exogenous oestrogen administration to rats are also conflicting. Falk et al. 8 reported that neither ovariectomy nor ovariectomy plus oestrogen administration alter the total content of NA in the rat uterus. Conversely, Rudzik et al. 29 showed reduced NA levels following ovariectomy, whereas McKercher et al. 17 reported lowered NA levels following oestrogen administration to intact adult rats. Our results in the developing rat seem to coincide with those reported by McKercher et al. 17 in the adult animal, in that they show a reduction in the content of NA of the uterine horn following oestrogen treatment. These authors, however, did not interpret this reduction in NA levels as an effect on the system of myometrial short noradrenergic fibers, but as an effect of oestrogens on the innervation of blood vessels. They associate this effect with the hyperaemia and oedema accompanying proestrus. This contention seems to be supported by the fact that in other species, such as the guinea pig and rabbit, oestrogens increase rather than decrease the content of NA of the uterus. 8,15,16,26,3132 Our histochemical results on chronic oestrogen-treated animals showed that most NA-C fibers were associated with blood vessels. Although visualization of myometrial fibers in whole-mount preparations was complicated by the increased thickness of tissues, cryostat sections point to confirm this observation. In this context, two possible explanations could be considered for the reduction in NA-C fibers associated with the myometrium following chronic exposure to oestrogen: (a) that the development of non-vascular NA-C nerves is inhibited; or (b) that NA levels in fibers are reduced and cannot be detected by the histochemical methods used in this study. Although the oestrogen doses employed for chronic treatment were not physiological, results obtained in the urinary bladder, auricle and the uterine cervix exclude a generalized effect of the hormone on all noradrenergic nerves. In general, acute treatment with oestrogens mimicked the effects of puberty in the different uterine regions. Histochemical preparations showed a 'spatial dilution' of non-vascular NA-C nerves of the uterine horn and parametrial tissue, without affecting the innervation of blood vessels. Biochemical assays point to confirm the differential sensibility of uterine regions to sex steroid.23,26 Although results reported in this paper do not prove that, in the developing rat, noradrenergic nerves associated with the myometrium belong to the system of short NA fibers; they do show a differential response to oestrogens by nerves associated with the vascular and non-vascular smooth muscle. In addition, these results suggest a possible role of oestrogens in the changes observed in the sympathetic innervation of the uterine myometrium following puberty.2 Acknowledgements--The authors would like to thank the Neurochemistry Division of the I.I.B.C.E. for the HPLC facilities. This study was partially supported by CSIC, Universidad de la Reptiblica, Uruguay.

REFERENCES 1. Adham H. and'Schenk E. A. (1969) Autonomic innervation of the rat vagina, cervix and uterus and its cyclic variations. Am. J. Obstet. Gynecol. 1114,508-516. 2. Brauer M. M., Lincoln J., Blundell D. and Corbacho A. (1992) Development of noradrenaline-containing nerves of the rat uterus. J. Autonom. Nerv. Syst. 39, 37-50. 3. Brauer M. M., Lincoln J., Sarner S., Blundell D., Milner P., P~issaro M. and Burnstock G. (1994) Maturational changes in sympathetic and sensory innervation of the rat uterus: effects of neonatal capsaicin treatment. Int. J. devl Neurosci. 12, 157-171. DIt 13-8-B

798

M.M. B r a u e r et al.

4. Brauer M. M., Lincoln J., Milner P., Sarner S., Blundell D., Pfissaro M., Corbacho A. and Burnstock G. (1994) Plasticity of autonomic nerves: differential effects of long-term guanethidine sympathectomy on the sensory innervation of the rat uterus during maturation. Int. J. devl Neurosci. 12, 579-586. 5. Braner M. M., Corbacho A. and Burnstock G. (1994) Effects of chronic and acute oestrogen treatment on the developing noradrenaline-containing (NA-C) nerves of the rat uterus. XIV Congreso Latinoamericano de Farmacolog/a, Santiago, Chile, CL2. 6. Corbacho A., Brauer M. M. and P6rez T. (1994) La inervaci6n noadren6rgica de la arteria uterina de la rata durante el desarrollo y la pubertad. XVIII Congreso Latinoamericano de Ciencias Fisiol6gicas, Montevideo, Uruguay, p. 191. 7. de la Torre J. C. and Surgeon J. W. (1976) A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method. Histochemistry 49, 81-93. 8. Falk B., Gardmark S., Nybell G., Owman Ch., Rosengren E. and Sj6berg N.-O. (1974) Ovarian influence on the content of norepinephrine transmitter in the guinea-pig and rat uterus. Endocrinology 94, 1475-1479. 9. Furness J. B. and Costa M. (1975) The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts. Histochemistry 41, 335-352. 10. Garfield R. E. (1986) Structural studies of innervation of non-pregnant rat uterus. Am. J. Physiol. 251 (Cell Physiol. 20), C41-C54. 11. Inyama C. O., Hacker G. W., Gu J., Dahl D., Bloom S. R. and Polak J. M. (1985) Cytochemical relationships in the paracervical ganglion (Frankenh~user) of rat studied by immunocytochemistry: Neurosci. Lett. 55, 311-316. 12. Kanerva L., Mustonen T. and Ter/iv/iinen H. (1972) Histochemical studies of uterine innervation after neurectomies. Acta Physiol. Scand. 86, 359-365. 13. Keller R., Oke A., Mefford I. et al. (1976) Liquid chromatographic analysis of catecholamines: routine assay for regional brain mapping. Life Sci. 19, 995-1004. 14. Long J. A. and Evans H. M. (1922) The estrous cycle in the rat and associated phenomena. Mem. Univ. Calif. 6. University of California Press, Berkeley. 15. Marshall J. M, (1981) Effects of ovarian steroids and pregnancy on adrenergic nerves of uterus and oviduct. Am. J. Physiol. 240 (Cell Physiol. 2), C165--C174. 16. Marshall J. M. (1970) Adrenergic innervation of the female reproductive tract: anatomy, physiology and pharmacology. Rev. Physiol. Biochem. Pharmacol. 62, 5-67. 17. McKercher T. C., Van Orden L. S., Bhatnagar R. K. and Burke J. P. (1973) Estrogen-induced biogenic amine reduction in rat uterus. J. Pharmacol. exp Ther. 185, 514-522. 18. Milton J. S. and Tsokos J. O. (1989) Estadistica para Biologia y Ciencias de la Salud. Interamericana-Mc Graw-Hill. 19. N•rberg K. and Fredriccs•n B. ( • 966) Ce••u•ar distributi•n •f m•n•amines in the uterine and tuba• wa••s •f the rat. Acta Physiol. Scand. (Suppl.) 68, 149. 20. Ojeda S. R. and Urbanski H. F. (1988) Puberty in the rat. In Physiology o f Reproduction (eds Knobil E. et al.), pp. 1699-1738. Raven Press, New York. 21. Owman Ch. and SjOberg N.-O. (1966) Adrenergic nerves in the female genital tract of the rabbit with remarks on cholinesterase-containing structures. Z. Zellforsch. Abt. Histochem. 74, 182-197. 22. Owman Ch., Rosengren E. and Sj6berg N.-O. (1967) Adrenergic innervation of the human female reproductive organs: a histochemical and chemical investigation. Obstet. Gynecol. 30, 763-773. 23. Owman Ch. and Stjernquist M. (1988) Origin, distribution, and functional aspects of aminergic and peptidergic nerves in the male and female genital tracts. In Handbook ofChemicalNeuroanatorny (eds Bj6rklund A., H6kfelt T. and Owman Ch.), pp. 445-544. Elsevier Science, Amsterdam. 24. Papka R. E., Cotton J. P. and Traurig H. H. (1985) Comparative distribution of neuropeptide tyrosine-, vasoactive intestinal polypeptide, substance P-immunoreactive, acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat. Cell Tissue Res. 242, 475-490. 25. Papka R. E., Traurig H. H. and Klenn P. (1987) Paracervical ganglia of the female rat: histochemistry and immunohistochemistry of neurons, SIF cells and nerve terminals. Am. J. Anat. 179, 243-257. 26. Papka R. E. and Traurig H. H. (1992) Autonomic and visceral sensory innervation of the female reproductive system: special reference to neurochemical markers in nerves and ganglionic connections. In Nervous Control o f the Urogenital System (ed. Maggi C. A.), pp. 423-436. Harwood Academic, Switzerland. 27. Proimina F. I. and Rakitskaia V. V. (1990) The source of the adrenergic innervation of the rat uterus. Fiziologicheskii Zhurnal Sssr Imeni l-M-Sechenova 76, 1245-1250. 28. Rosengren E. and Sj6berg N.-O. (1967) The adrenergic nerve supply of the female reproductive tract of the cat. Am. J. Anat. 12L 271-283. 29. Rudzik A. D. and Miller J. W. (1962) The effect of altering the catecholamine content of the uterus on the rate of contractions and the sensitivity of the myometrium to relaxin. J. Pharmacol. exp. Ther. 138, 88-95. 30. Silva D. G. (1966) The ultrastructure of the myometrium of the rat with special reference to the innervation. Anat. Rec.

158, 21-34. 31. Sj6berg N.-O. (1967) The adrenergic transmitter level of guinea pig uterus. Acta Physiol. Scand. (Suppl.) 305, 1-32. 32. Thorbert G., Aim P. and Rosengren E. (1978) Cyclic and steroid-induced changes in adrenergic neurotransmitter level of guinea pig uterus. Acta Obstet. Gynecol. Scand. 57, 45-48. 33. Van Orden D. E., Goodale D. B., Baker H. A., Farley D. B. and Bhatnagar R. K. (1980) Uterine catecholamines and prostaglandins during the estrous cycle of the rat. Endocrinology 106,1650-1654.