Estrogen-induced collagen reorientation correlates with sympathetic denervation of the rat myometrium.

Estrogen inhibits the growth and causes the degeneration (pruning) of sympathetic nerves supplying the rat myometrium. Previous cryoculture studies evidenced that substrate-bound signals contribute to diminish the ability of the estrogenized myometrium to support sympathetic nerve growth. Using electron microscopy, here we examined neurite-substrate interactions in myometrial cryocultures, observing that neurites grew associated to collagen fibrils present in the surface of the underlying cryosection. In addition, we assessed quantitatively the effects of estrogen on myometrial collagen organization in situ, using ovariectomized rats treated with estrogen and immature females undergoing puberty. Under low estrogen levels, most collagen fibrils were oriented in parallel to the muscle long axis (83% and 85%, respectively). Following estrogen treatment, 89% of fibrils was oriented perpendicularly to the muscle main axis; while after puberty, 57% of fibrils acquired this orientation. Immunohistochemistry combined with histology revealed that the vast majority of fine sympathetic nerve fibers supplying the myometrium courses within the areas where collagen realignment was observed. Finally, to assess whether depending on their orientation collagen fibrils can promote or inhibit neurite outgrowth, we employed cryocultures, now using as substrate tissue sections of rat-tail tendon. We observed that neurites grew extensively in the direction of the parallel-aligned collagen fibrils in the tendon main axis but were inhibited to grow perpendicularly to this axis. Collectively, these findings support the hypothesis that collagen reorientation may be one of the factors contributing to diminish the neuritogenic capacity of the estrogen-primed myometrial substrate.

Expression of beta-adrenergic receptors in the rat uterus: effects of puberty and oestrogen treatment during prepubertal development.

The expression of beta-adrenoceptors in the rat uterus has been analysed during the peripubertal transition and following acute and chronic oestradiol treatment during prepubertal development. The distribution and density of beta-adrenoceptors was assessed autoradiographically on cryostat tissue sections using [3H]-dihydroalprenolol ([3H]-DHA). Binding sites were localised in all ages and experimental situations examined and showed the following intensity of labelling: endometrial epithelium > longitudinal muscle layer > circular myometrial layer > endometrial stroma. Competition experiments with the selective antagonists ICI 118,551 and atenolol, showed that most of the beta-adrenoceptors in the uterus belong to the beta(2) receptor subclass. In prepubertal animals, the density of [3H]-DHA binding sites was extremely low. Following puberty the density of binding sites showed a generalised increase. Acute administration of oestradiol at the end of the prepubertal period provoked an increase in the density of [3H]-DHA binding sites in all uterine regions, but the levels of labelling were lower than in peripubertal animals at proestrus and oestrus. Following chronic oestrogen treatment during postnatal development, oestradiol increased further the density of [3H]-DHA binding sites. Results are discussed considering both the endocrine and neural changes accompanying puberty and oestradiol treatment.

Development and maturation of noradrenaline-containing nerves of the rat uterine artery. Effects of acute and chronic oestrogen treatment.

The development and maturation of noradrenaline-containing nerves of the rat uterine artery was investigated, histochemically and biochemically, at seven different postnatal age-stages and following acute and chronic treatment with oestradiol. Morphological changes in the vessel were quantitatively evaluated on Toluidine Blue-stained semithin sections and low magnification electronmicrographs. In summary, the uterine artery is innervated at birth; the adult pattern of innervation is established at two weeks of age; the innervation density increases progressively between the infantile and prepubertal periods, accompanying proliferation and growth of smooth muscle cells in the tunica media; changes in the innervation are followed by an increase in the tissue concentration of noradrenaline and neither the endocrine changes characterizing puberty nor acute or chronic treatment with oestradiol have an effect on the pattern of development of the uterine artery and associated noradrenaline-containing nerves. Results are interpreted considering the differential susceptibility of urinogenital organs to sex hormones.

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

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 micrograms oestradiol cypionate on the 25th day of age. Chronic treatment consisted of four doses of 10 micrograms 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.

Maturational changes in sympathetic and sensory innervation of the rat uterus: effects of neonatal capsaicin treatment.

The plasticity of the sympathetic and sensory innervation of the rat uterus was examined, before and after puberty, in controls and in animals where primary sensory nerves had been destroyed by neonatal capsaicin treatment. Immunohistochemical and histochemical methods were used in association with nerve density measurements and biochemical assays. The main findings were as follows: (1) Puberty was associated with a marked increase in the weight of the uterine horn, uterine cervix and parametrial tissue. This was unaffected by capsaicin treatment. (2) The sympathetic innervation of the uterine horn and parametrial tissue was reduced following puberty as revealed by a decrease in the density of noradrenaline-containing nerves and a marked decrease in the tissue concentration of noradrenaline. Sympathetic nerves supplying the uterine cervix and the blood vessels of the uterus appeared to be unaffected by puberty. (3) In contrast, the sensory supply of the uterus by substance P and calcitonin gene-related peptide-containing nerves increased in parallel with uterine growth during puberty resulting in no change in nerve density and only a slight reduction in peptide concentration. (4) Neonatal capsaicin treatment caused a long-lasting depletion of substance P- and calcitonin gene-related peptide-containing nerves. In the uterine horn and parametrial tissue, capsaicin-resistant calcitonin gene-related peptide, but not substance P, still increased with tissue weight during puberty, indeed, in the uterine horn, the relative increase was greater than in controls. (5) Sensory denervation resulted in an increase in the non-vascular sympathetic supply of the uterus, although there was a regional variation in the time course of the response. Perivascular sympathetic nerves were unaffected by capsaicin treatment. The pattern of change in non-vascular noradrenaline-containing nerves associated with puberty was similar in nature to controls. Thus, there is considerable plasticity in the innervation of the uterus both during puberty and following sensory denervation. A complex pattern of change occurs with differential responses in vascular and nonvascular nerves and in different regions of the uterus. Such differences may be due in part to the different origins of individual nerve populations and/or to their relative sensitivities to sex hormones.

Differential effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in uterine myometrial and perivascular sympathetic nerves.

Previous studies have shown that chronic administration of oestrogen to prepubertal rats reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. The mechanisms underlying these changes are not known. In the present study we have analysed the effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in the rat uterine sympathetic nerves using biochemical and immunohistochemical approaches. Tyrosine hydroxylase activity was evaluated biochemically, by measuring the in vivo accumulation of dihydroxyphenylalanine (DOPA) in the presence of a DOPA-decarboxylase inhibitor. In addition, nerve fibres were visualised immunohistochemically using antibodies against tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DbetaH) and the general marker protein gene product 9.5 (PGP 9.5). After chronic oestrogen treatment, the total content of noradrenaline of the uterine horn was reduced, whereas the total content of DOPA was increased. In controls, TH-immunoreactive, DbetaH-immunoreactive and PGP 9.5-immunoreactive nerve fibres were distributed in both the circular and longitudinal myometrial layers and in the blood vessels of the intran-myometrial region. After chronic oestrogen treatment the only fibres recognised by the three antibodies were those associated with the blood vessels, but no myometrial-associated fibres could be recognised. These results suggest that noradrenaline synthesis is selectively reduced in myometrial-associated uterine sympathetic nerves, but is preserved in perivascular sympathetic nerves. The increased DOPA levels measured after chronic exposure to oestrogen was interpreted as the consequence of the substantial increase in size and number of blood vessels observed in the uterus of oestrogen-treated animals. A possible neurodegenerative effect of oestrogen on myometrial sympathetic fibres is discussed.

Plasticity of autonomic nerves: differential effects of long-term guanethidine sympathectomy on the sensory innervation of the rat uterus during maturation.

The sensory nerves, containing substance P and calcitonin gene-related peptide, and noradrenaline-containing sympathetic nerves of the rat uterus were analyzed following long-term sympathectomy with guanethidine in prepubertal (four weeks), young adult (eight weeks) and fully adult animals (18 weeks). Immunohistochemical and histochemical methods were used in association with nerve density measurements and biochemical assays. The main findings were as follows: (1) long-term guanethidine treatment completely abolished the noradrenergic innervation of the uterine horn and parametrial tissue and markedly reduced the tissue levels of noradrenaline in both regions at the three ages analysed; (2) in the uterine horn guanethidine treatment had no effect on the tissue levels of either calcitonin gene-related peptide or substance P or on the density of calcitonin gene-related peptide-containing nerves, at any of the three ages studied; (3) in the parametrial tissue increased levels of calcitonin gene-related peptide were observed at 8 and 18 weeks of age, together with a significant increase in the density of calcitonin gene-related peptide-containing nerves. Substance P levels showed a transient increase in this tissue at eight weeks. In conclusion, long-term sympathectomy with guanethidine resulted in an increase in calcitonin gene-related peptide and substance P in sensory nerves in the parametrial tissue, but not in the uterine horn. The changes in the parametrial tissue only occurred after puberty. It is suggested that sensory nerves in the uterine horn may be less responsive to sympathetic denervation since loss of sympathetic nerves occurs as part of a normal physiological process during pregnancy in this region.

Differentiation of smooth muscle in the genital tract of the female mouse and its temporal relation with the development of the Wolffian nerve.

The development of the smooth muscle in the genital tract of the female mouse was studied by light and electron microscopy before and after birth. These studies showed that: a) between 13 days of fetal development and 2 days after birth the cells surrounding the Mullerian duct were undifferentiated and showed a fibroblast-like appearance; b) between 3 and 10 days after birth the cells acquired several characteristics of smooth muscle but they did not seem fully mature; c) between 30 and 180 days after birth the cells acquired a mature appearance; and d) the Wolffian nerve reached the Mullerian duct surrounding tissue before the start of smooth muscle differentiation.

The oestrogenized rat myometrium inhibits organotypic sympathetic reinnervation.

Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

Effects of dexamethasone on estrogen- and pregnancy-induced plasticity in rat uterine sympathetic nerves.

Estrogen and glucocorticoids are known to evoke opposing effects on the uterus. We analyzed the effects of dexamethasone (DEX) on uterine sympathetic denervation elicited by short- and long-term exposure to estrogen of intact prepubertal rats. We also studied the effects of DEX on the physiological degeneration of uterine sympathetic nerves at term pregnancy. Changes in innervation were assessed quantitatively by using computer-assisted methods on uterine cryostat tissue sections stained for tyrosine hydroxylase. At 24 h following treatment of prepubertal rats (25 days of age) with 1 microg or 2.5 microg estrogen, marked increases in uterine size and reductions in the percentage nerve area were observed. Co-administration of DEX (4 mg/kg) attenuated both these short-term estrogen-induced effects. Treatment of 19-day-old rats with a single dose of 25 mug estrogen provoked, at 26 days of age, a 54% reduction in the total nerve area. This reduction was abolished by the co-administration of nine doses of DEX (0.5 mg/kg) at 18-26 days of age. Treatment of rats with the same regime of DEX alone increased the total nerve area by 46% of the control values. Studies of control pregnant rats revealed the unexpected presence of intrauterine nerve fibers at term. Treatment of pregnant rats with six doses of DEX (4 mg/kg) at 16-21 days of age had no effects on the density of uterine sympathetic nerves. These results suggest that DEX has growth-promoting effects on immature uterine sympathetic nerves and may antagonize the degenerative effects elicited by long-term exposure to estrogen.

Morphological differentiation of the growing oocyte of Ctenomys torquatus (Rodentia, Octodontidae).

The ultrastructural changes observed during the growth phase of oocytes of Ctenomys torquatus (Rodentia, Octodontidae) are reported. Interest was particularly centered on the transformation and/or distribution of the components of the endoplasmic reticulum. According to the observations made it is suggested that the endoplasmic reticulum stores some kind of material which may support early stages of development.

Large scale isolation of zonae pellucidae from ovarian oocytes of mice.

A large scale method for the isolation of zonae pellucidae (ZP) from mouse ovaries is described. It involves the squashing of the ovaries on a screen (70 X 70 M mesh) and sedimentation of the material in a discontinuous gradient. It is possible to obtain free ZP in useful quantities in about 1 h's work.

Postnatal development of noradrenaline-containing nerves of the rat uterus.

The postnatal development of noradrenaline (NA)-containing nerves of the rat uterus and its associated blood vessels was investigated using histochemical and biochemical methods. These studies were carried out in conjunction with examination of the morphology of the uterus and the density of blood vessels at the prepubertal, peripubertal and adult stages. It was demonstrated that: (1) the rat uterus is innervated at birth; (2) the innervation of blood vessels develops earlier than that of the myometrium and the density of NA-containing perivascular nerves is not affected by puberty; (3) between birth and day 30 (prepubertal) there was a progressive increase in the innervation of the myometrium which was accompanied by a progressive increase in the total NA content of the organ; (4) at the first oestrus there was a marked increase in the weight of the uterus and isolated myometrial and parametrial tissue. Both muscle cell size and number were also increased. The density of myometrial innervation by NA-containing nerves was markedly reduced, although the total NA content did not change at this stage. This indicates a 'dilution' of myometrial NA-containing nerves in a greater amount of non-neuronal tissue; and (5) between the peripubertal and adult stages there was a further increase in uterine weight together with an increase in the number of smooth muscle cells and a reduction in the density of myometrial NA-containing nerves. Although the density of perivascular nerves was unaffected by puberty, the number of blood vessels supplying the uterus increased during the transition to the adult stage. This was reflected by a significant increase in the total NA content of the uterine horn and of isolated myometrial and parametrial preparations.

In oculo transplants of myometrium from postpartum guinea pigs fail to support sympathetic reinnervation.

Sympathetic nerves to the enlarged fetus-containing region of the uterus undergo degenerative changes during late pregnancy and show slow regrowth after parturition. It is not known whether this unusual response of sympathetic nerves to smooth muscle hypertrophy is due to the sensitivity of short adrenergic neurons to hormonal changes, or whether the nerves respond to changes in the neurotrophic capacity of the target. We have investigated this question using in oculo transplantation. Small pieces of myometrium from the uterine horn of virgin guinea pigs, or from the region previously occupied by the placenta and fetus in postpartum guinea pigs, were transplanted into the anterior eye chamber. After 3 wk in oculo, the pattern of reinnervation of the transplants was assessed on whole mount stretch preparations stained for tyrosine hydroxylase. The histology of the transplants was examined in toluidine blue-stained semithin sections. Myometrial transplants from virgin donors and uterine artery transplants from both virgin and postpartum donors became organotypically reinnervated by sympathetic fibres from the host iris. In contrast, sympathetic nerves did not reinnervate myometrial transplants from postpartum donors, although they approached the transplants and became distributed in the surrounding connective tissue. All transplanted tissues showed a normal histological appearance. Both the myometrium and uterine artery from postpartum donors retained a hypertrophic appearance after 3 wk in oculo. We interpret these results to indicate that the degeneration of sympathetic nerves in late pregnancy, as well as their slow regrowth to the uterus after delivery, may be due to changes in uterine smooth muscle rather than a particular sensitivity of short adrenergic neurons to hormonal changes.

Development of the innervation of the urogenital complex in the fetal mouse.

The developing urogenital complex of the fetal mouse was studied by means of silver impregnation and electron microscopy. These studies showed that: 1) the mesonephric field is innervated during prenatal stages (Wolffian nerve); 2) nerve penetration precedes the differentiation of the gonads and related ducts; and 3) the Wolffian nerve arises during the earliest stages from the first pair of abdominal rami communicantes. The identity between the fetal Wolffian nerve and the nerve of the suspensory ligament (higher pathway) of the adult is discussed.

Effects of chronic oestrogen treatment are not selective for uterine noradrenaline-containing sympathetic nerves: a transplantation study.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.

Differential effects of oestrogen on developing and mature uterine sympathetic nerves.

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.

The role of NGF in pregnancy-induced degeneration and regeneration of sympathetic nerves in the guinea pig uterus.

In the guinea pig, pregnancy is associated with a generalised depletion of noradrenaline in uterine sympathetic nerves and, in the areas of the uterus surrounding the foetus, by a complete degeneration of sympathetic nerve fibres. These pregnancy-induced changes have been interpreted as a selective effect of placental hormones on the system of short sympathetic fibres arising from the paracervical ganglia. An alternative explanation is that pregnancy affects the neurotrophic capacity of the uterus. We measured NGF-protein levels in the guinea pig uterine horn, tubal end and cervix at early pregnancy, late pregnancy and early postpartum, using a two-site enzyme-linked immunosorbent assay. For comparative purposes the distribution and relative density of noradrenaline-containing sympathetic nerve fibres were assessed histochemically, and tissue levels of noradrenaline were measured biochemically, using high-performance liquid chromatography with electrochemical detection. In all the uterine regions analysed, NGF-protein levels showed a decline at term pregnancy, but in no case was this change statistically significant. After delivery, NGF-protein levels showed a marked increase in the cervix as well as in both the fertile and empty horns. These results suggest that alterations in NGF-protein do not account for the impairment of uterine sympathetic innervation during pregnancy, but may contribute to their recovery after delivery.

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA.

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.