Role of the superior ovarian nerve in the regulation of follicular development and steroidogenesis in the morning of diestrus 1.

PURPOSE: Little is known about the role of the superior ovarian nerve (SON) in follicular development during the estrus cycle. The aim of the present study was to analyze the role of neural signals arriving through the SON at the ovaries in the regulation of follicular development and ovarian steroid secretion in diestrus 1 of cyclic rats. METHODS: Cyclic rats were subjected to left, right, or bilateral SON sectioning or to unilateral or bilateral laparotomy at diestrus 1 at 11:00 h. Animals were sacrificed 24 h after surgery. RESULTS: Compared to laparotomized animals, unilateral SON sectioning decreased the number of preovulatory follicles, while bilateral SON sectioning resulted in a decreased number of atretic preantral follicles. An important observation was the presence of invaginations in the follicular wall of large antral and preovulatory follicles in animals with denervation. Furthermore, left SON sectioning increased progesterone levels but decreased testosterone levels, which are effects that were not observed in animals that were subjected to right denervation. CONCLUSIONS: At 11:00 h of diestrus 1, the SON was found to stimulate follicle development, possibly via neural signals, such as noradrenaline and/or vasoactive intestinal peptide, and this stimulation induced the formation of follicle-stimulating hormone receptors. The role of the SON in the regulation of ovarian steroid secretion is asymmetric: the left SON inhibits the regulation of progesterone and stimulates testosterone secretion, and the right nerve does not participate in these processes.

Long-term estradiol-17ß exposure decreases the cholinergic innervation pattern of the pig ovary.

Elevated levels of endogenous estrogens in the course of pathological states of ovaries, as well as xenoestrogens, may lead to hyperestrogenism. It has previously been demonstrated that long-term estradiol-17ß (E2) administration in adult gilts affected the population of sympathetic intraovarian nerve fibers. The aim of this study has been to determine the effect of long-term E2 exposure on the cholinergic innervation pattern of porcine ovaries. Intraovarian distribution and the density of nerve fibers immunoreactive (IR) to vesicular acetylocholine transporter (VAChT) and/or neuronal isoform of nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) were determined. From day 4 of the first estrous cycle to day 20 of the second studied cycle, experimental gilts were intramuscularly injected with E2, while control gilts received corn oil. The ovaries were then collected and processed for double-labelling immunofluorescence. After E2 administration, the total number of fibers IR to VAChT, nNOS and VIP decreased significantly. The numbers of VAChT-, nNOS- and VIP-IR fibers within the ground plexus were significantly lower, while they were significantly higher around small or medium tertiary follicles. In the E2-affected ovaries, the numbers of nNOS- and VIP-IR fibers were significantly higher near secondary follicles and VAChT-IR in the vicinity of medullar blood vessels. In turn, around the latter structures there were significantly lowered populations of nNOS- and VIP-IR nerve fibers. These results suggest that the elevated E2 levels that occur during pathological states may affect the cholinergic innervation pattern of ovaries and their function(s).

Long-term treatment with testosterone alters ovary innervation in adult pigs.

BACKGROUND: Intraovarian distribution and density of nerve fibres immunoreactive (IR) to protein gene product 9.5 (PGP 9.5) and containing dopamine-ß-hydroxylase (DßH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) were determined. METHODS: From day 4 of the first oestrous cycle to day 20 of the second studied cycle, experimental gilts (n = 3) were injected with testosterone (T), while control gilts (n = 3) received corn oil. RESULTS: After T administration the numbers of fibres IR to PGP 9.5 and fibres IR to DßH, NPY and SOM were decreased. Fewer PGP 9.5- and DßH-IR terminals were observed within the ground plexus and around arteries and medullar veins, and medium tertiary follicles, and DßH-IR terminals in the vicinity of small tertiary follicles. T decreased the density of NPY-IR fibres in the medullar part of the ground plexus, and SOM-IR in the cortical part of the ground plexus. CONCLUSIONS: The obtained data show that long-term T treatment of gilts decreases the total number of intraovarian fibres, including sympathetic ones. These results suggest that elevated T levels that occur during pathological states may affect the innervation pattern of ovaries, and their function(s).

Control of male and female fertility by the netrin axon guidance genes.

The netrin axon guidance genes have previously been implicated in fertility in C. elegans and in vertebrates. Here we show that adult Drosophila lacking both netrin genes, NetA and NetB, have fertility defects in both sexes together with an inability to fly and reduced viability. NetAB females produce fertilized eggs at a much lower rate than wild type. Oocyte development and ovarian innervation are unaffected in NetAB females, and the reproductive tract appears normal. A small gene, hog, that resides in an intron of NetB does not contribute to the NetAB phenotype. Restoring endogenous NetB expression rescues egg-laying, but additional genetic manipulations, such as restoration of netrin midline expression and inhibition of cell death have no effect on fertility. NetAB males induce reduced egg-laying in wild type females and display mirror movements of their wings during courtship. Measurement of courtship parameters revealed no difference compared to wild type males. Transgenic manipulations failed to rescue male fertility and mirror movements. Additional genetic manipulations, such as removal of the enabled gene, a known suppressor of the NetAB embryonic CNS phenotype, did not improve the behavioral defects. The ability to fly was rescued by inhibition of neuronal cell death and pan-neural NetA expression. Based on our results we hypothesize that the adult fertility defects of NetAB mutants are due to ovulation defects in females and a failure to properly transfer sperm proteins in males, and are likely to involve multiple neural circuits.

Sympathetic nerve activity in normal and cystic follicles from isolated bovine ovary: local effect of beta-adrenergic stimulation on steroid secretion.

Cystic ovarian disease (COD) is an important cause of abnormal estrous behavior and infertility in dairy cows. COD is mainly observed in high-yielding dairy cows during the first months post-partum, a period of high stress. We have previously reported that, in lower mammals, stress induces a cystic condition similar to the polycystic ovary syndrome in humans and that stress is a definitive component in the human pathology. To know if COD in cows is also associated with high sympathetic activity, we studied isolated small antral (5 mm), preovulatory (10 mm) and cystic follicles (25 mm). Cystic follicles which present an area 600 fold greater compared with preovulatory follicles has only 10 times less concentration of NE as compared with small antral and preovulatory follicles but they had 10 times more NE in follicular fluid, suggesting a high efflux of neurotransmitter from the cyst wall. This suggestion was reinforced by the high basal release of recently taken-up 3H-NE found in cystic follicles. While lower levels of beta-adrenergic receptor were found in cystic follicles, there was a heightened response to the beta-adrenergic agonist isoproterenol and to hCG, as measured by testosterone secretion. There was however an unexpected capacity of the ovary in vitro to produce cortisol and to secrete it in response to hCG but not to isoproterenol. These data suggest that, during COD, the bovine ovary is under high sympathetic nerve activity that in addition to an increased response to hCG in cortisol secretion could participate in COD development.

Neonatal superior ovarian nerve transection inhibits follicle development by enhancing follicular atresia and suppressing granulosa cell proliferation in rats.

The ovarian sympathetic nerves participate in the regulation of mammalian ovarian function, but it is still not known whether the neonatal ovarian sympathetic nerve is involved in follicular development and related mechanisms. In the present study, the superior ovarian nerve (SON) of the neonatal rat was transected on postnatal day (PD) 2, and follicle development, ovarian hormone secretion, ovulation rate, granulosa cell proliferation and apoptosis were analysed on PD 30 and PD 90. The results demonstrate that SON transection decreases follicle number and size, reduces ovulation induced by gonadotrophin and enhances follicular atresia. Bromodeoxyuridine (BrdU) and cleaved caspase-3 immunohistochemistry staining provide evidence that SON transection inhibits granulosa cell proliferation and promotes granulosa cell apoptosis. In addition, SON transection increases serum oestradiol levels, but has no influence on serum progesterone levels. These results suggest that the sympathetic nerve supply to the ovaries is important in regulating follicle development and ovary function. These results are critical for further understanding of the neuroendocrine regulation of ovary development and function, although the mechanism needs to be elucidated in future studies.

Superior ovarian nerve (SON) transection leads to stunted follicular maturation: a histomorphologic and morphometric analysis in the rat model.

Histomorphologic and morphometric effects of peripubertal superior ovarian nerve (SON) transection were evaluated during adult life in rats. Twenty Sprague-Dawley rats were randomly divided into two groups: the SON-transected (SON-t) group (n = 10) and the sham-operated control group (n = 10). Total follicle development was not influenced; however, follicular maturation was seriously stunted by the procedure, as evidenced by significantly thinner theca interna and follicular wall.

Evidence for a potential role of neuropeptide Y in ovine corpus luteum function.

Neuropeptide Y (NPY) is a neurohormone that is typically associated with food intake, but it has also been reported to affect the production of progesterone from luteal tissue in vitro. However, NPY has not been previously immunolocalized in the ovine ovary or in the corpus luteum (CL) of any species, and the effects of this neurohormone on luteal function in vivo are not known. Thus, we performed fluorescent immunohistochemistry (IHC) to localize NPY in the ovine ovary and used avidin-biotin immunocytochemistry (ICC) to further define the intracellular localization within follicles and the CL. We then infused NPY directly into the arterial supply of the autotransplanted ovaries of sheep to determine the in vivo effect of exogenous NPY on ovarian blood flow and on the luteal secretion rate of progesterone and oxytocin. Immunohistochemistry revealed that the NPY antigen was localized to cells within the follicles and CL, in the nerve fibers of the ovarian stroma, and in the vessels of the ovarian hilus. In the follicle, the NPY antigen was localized to nerves and vessels within the theca interna layer, and strong staining was observed in the granulosal cells of antral follicles. In the CL, NPY was localized in large luteal cells and in the vascular pericytes and/or endothelial cells of blood vessels, found dispersed throughout the gland and within the luteal capsule. In vivo incremental infusions of NPY at 1, 10, 100, and 1,000 ng/min, each for a 30-min period, into the arterial supply of the transplanted ovary of sheep bearing a CL 11 d of age increased (P< or =0.05) ovarian blood flow. The intra-arterial infusions of NPY also increased (P< or =0.05) in a dose-dependent manner the secretion rate of oxytocin, which was positively correlated (P< or =0.05) with the observed increase in ovarian blood flow. The infusions of NPY had a minimal effect on the secretion rate of progesterone, and similar intra-arterial infusions of NPY into sheep with ovarian transplants bearing a CL over 30 d of age had no significant effect on ovarian blood flow or on the secretion rate of progesterone. These results suggest that NPY acts on the luteal vascular system and the large luteal cells to rapidly stimulate blood flow and the secretion of oxytocin, respectively, which collectively implies a putative role for NPY during the process of luteolysis when increasing amounts of oxytocin are secreted from the ovine CL in response to uterine pulses of prostaglandin F2alpha.

An immunohistochemical study of ovarian innervation in the emu (Dromaius novaehollandiae).

The present study investigated the distribution of nerves in the ovary of the emu. The neuronal markers, protein gene product 9.5, neurofilament protein and neuron specific enolase demonstrated the constituents of the extrinsic and intrinsic ovarian neural systems. The extrinsic neural system was composed of ganglia in the ovarian stalk, as well as nerve bundles, which were distributed throughout the ovary. Isolated neuronal cell bodies, in the medulla and cortex, formed the intrinsic neural system. An interesting finding of the study was the presence of nerve bundles, circumscribed by lymphocytes, in the ovarian stalk. The findings of the study indicate that the distribution of nerve fibres and neuronal cell bodies in the emu ovary is similar, but not identical to that of the domestic fowl and ostrich.

Role of stress and sympathetic innervation in the development of polycystic ovary syndrome.

This article presents a review of the role of the sympathetic activity in ovarian pathologies affecting reproductive function. We provide a succinct outline of the findings of our group in this area. The participation of stress as an etiological factor for ovarian pathologies throughout animal models and data in patients with polycystic ovary syndrome give strong support for participation of sympathetic nerves in the ovary function both in normal and pathological status.

Neurotrophic and cell-cell dependent control of early follicular development.

Neurotrophins (NTs) and their receptors play an essential role in the differentiation and survival of defined neuronal populations of the central and peripheral nervous systems. Their actions, however, do not appear to be limited to the nervous system, as both NTs and their receptors have been found in non neuronal cells, including cells of the endocrine system. At least four of the five known neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), and their receptors (p75 NTR, trkA, trkB and trkC) are present in the developing ovary. Using mice carrying null mutations of the genes encoding neurotrophins (NGF, NT-4, BDNF) or the receptor that mediates the actions of NT-4 and BDNF (trkB), we have obtained initial results consistent with the notion that neurotrophins are required for the growth of primordial follicles. NGF-deficient mice show a decreased formation of both primary and secondary preantral follicles. Null mutation of the NT-4 gene failed to affect either folliculogenesis or follicular development. However, formation of primary and secondary follicles was compromised in mice carrying a null mutation of both the NT-4 and BDNF genes, suggesting compensation of function by BDNF in NT-4 knockouts. Support for this concept is provided by the similar deficiency in follicular growth observed in animals carrying a null mutation of the gene encoding trkB, the receptors mediating NT-4 and BDNF actions. Initial experiments, using differential display, to isolate genes that may be involved in the process of folliculogenesis and/or early follicular development, resulted in the isolation of a recently identified cell adhesion molecule and a novel transcription factor originally shown to induce cell transformation. It thus appears that formation and development of mammalian follicles requires the concerted action of genes originally thought to be only involved in cell differentiation/survival of neuronal cells, and genes that may control the growth, differentiation, and cell-cell interactions of somatic and germ cells in the ovary.

Dorsal rhizotomy does not alter rat estrous cycles or ovarian compensatory responses.

Rats were subjected to right side dorsal rhizotomy of spinal nerves T12-L2 and ipsi- or contralateral unilateral ovariectomy at estrus (day 1). Estrous cycles were recorded daily, and on day 15 the remaining ovary was removed and weighed. Antral follicles were counted in representative day 1 and day 15 ovaries. Dorsal rhizotomy did not affect estrous cycles during the period after surgery. Also dorsal rhizotomy did not alter ipsilateral ovarian hypertrophy or follicular activation following unilateral ovariectomy. Thus, although the major portion of the ovarian sensory innervation is via the lesioned segments, interruption of these segments centrally does not alter compensatory ovarian responses subsequent to unilateral ovariectomy.

GABA suppresses stimulation-induced release of [3H]-noradrenaline from sympathetic nerve fibres in bovine ovarian follicles.

1 Strips from the bovine ovarian follicle wall were incubated in Krebs-Ringer solution containing [3H]-noradrenaline in order to saturate sympathetic nerve fibres with radiolabelled transmitter. This allowed the study of field stimulation-evoked transmitter release. 80.3 +/- 3.9% of the tritium released upon stimulation (10 Hz, pulse duration 1 ms, 10 V between the electrodes) was noradrenaline. 2 The stimulated release of tritium was totally blocked in calcium-free, EGTA (1 mM) containing medium or by 1 microM tetrodotoxin. Chemical sympathectomy (6-hydroxydopamine treatment) in vitro reduced the tritium content of the strip by 85%. The neuronal amine uptake blocker desipramine (0.6 microM) was almost equally effective in inhibiting the incorporation of tritium. The extraneuronal amine uptake blocker normetanephrine (10 microM) reduced the tritium content by 30%. Together, the results suggest that the electrically evoked release of tritium reflects the release of [3H]-noradrenaline from sympathetic nerve fibres. 3 gamma-Aminobutyric acid (GABA) concentration-dependently reduced the electrically evoked tritium release. Also the GABAB-receptor agonist baclofen (30 microM) reduced the stimulated tritium release whereas muscimol (100 microM), a GABAA-receptor agonist, failed to affect the release. 4 The selective GABAA-receptor antagonist bicuculline (3 and 100 microM) did not block the effect of GABA, while 3-amino-1-propanesulphonic acid (3-APA), a blocker of GABAB-receptors reversed the inhibitory effect of GABA. The results suggest that neuronal GABAB-receptors are involved in the GABA-evoked suppression of stimulated noradrenaline release.

Characterization of presynaptic 5-HT receptors on adrenergic nerves supplying the bovine ovarian follicle.

1. The effects of 5-hydroxytryptamine (5-HT) on contraction and release of [3H]-noradrenaline were investigated in vitro in bovine ovarian follicle strips. Using available selective agonists and antagonists, an effort was made to characterize the type of receptor mediating the inhibitory effect of 5-HT on neurogenic contraction and release of [3H]-noradrenaline by electrical field stimulation. 2. 5-Hydroxytryptamine inhibited the neurogenic contraction and release of [3H]-noradrenaline evoked by electrical field stimulation in a concentration-dependent manner. Like 5-HT, 5-carboxamidotryptamine (5-CT) and methysergide reduced the transmitter release as well as the neurogenic contraction, whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) failed to inhibit both responses in concentrations up to 0.1 microM. 3. The 5-HT (1 microM)-induced inhibition of contractile responses was more evident during stimulation at low frequencies (4 and 8 Hz) than during high frequency electrical stimulation (16 and 32 Hz). 4. Methiothepin (1 microM) and methysergide (10 microM) significantly antagonized the inhibitory effect of 5-HT on the electrically evoked release of tritium, whereas cyanopindolol, MDL 72222 and ketanserin (all 0.1 microM) were without effect. In addition, ketanserin, MDL 72222, cimetidine, pyrilamine, atropine, propranolol and indomethacin were without effect on the 5-HT-induced inhibition of the neurogenic contraction. 5. It is suggested that 5-HT inhibits the electrically evoked transmitter release from adrenergic nerves in the bovine ovarian follicle wall via prejunctional 5-HT1-like receptors. This was based on the findings that 5-CT was a potent agonist, methiothepin an antagonist and the lack of effect of MDL 72222, cyanopindolol and ketanserin.

Evidence for prejunctional GABAB receptors mediating inhibition of ovarian follicle contraction induced by nerve stimulation.

The motor effects of gamma-aminobutyric acid (GABA) on the bovine ovarian follicle were studied in vitro using strips from follicle walls. Electrical field stimulation of nerves in the preparation, secured by tetrodotoxin blockade, caused a contraction that was almost totally abolished by phentolamine and only slightly affected by atropine. This mainly adrenergic neurogenic response was inhibited by GABA in a dose-dependent way. The GABAA-receptor antagonists, bicuculline and picrotoxin, did not affect the GABA action whereas the GABAB-receptor antagonist, homotaurine, significantly inhibited the GABA effect. The GABAA-receptor agonist, muscimol, did not affect the contractile response while the GABAB-receptor agonist, baclofen, imitated the action of GABA. On the other hand, GABA had no direct contractile or relaxing effect on the follicle strips nor did it interfere with the contractile response induced by noradrenaline or acetylcholine. The findings suggest that activation of prejunctional GABAB receptors inhibits transmitter release from mainly adrenergic nerves associated with the follicle, thereby affecting nerve-mediated tension in the follicle wall.

Evidence for a role of prostaglandins in the adrenergic neuromuscular mechanism of the ovarian follicle wall.

Sympathetic nerves innervate smooth muscle cells in the theca externa of Graafian follicles and induce contraction of the follicle wall. The interaction of prostaglandins (PGs) with the function of this neuromuscular complex has been elucidated by the use of isolated strips from the protruding part of the wall of bovine ovarian follicles. PGF2 alpha contracts the strips, and it also potentiates the contractile response to electrical field stimulation of the nerves, but it has no effect on noradrenaline-induced contractions. PGE1 and PGE2 have direct relaxatory actions on the strip preparation (previously given an active contraction by carbamylcholine) and in high doses they, therefore, reduce the contractile response induced by noradrenaline. In low doses not affecting the noradrenaline response, PGE1 and PGE2 both decrease the amount of contraction induced by electric nerve stimulation, indicating an inhibitory action of the PGEs on the transmitter release. It is suggested that the role of PGs in the process of ovulation might, at least partly, be mediated by their interactions with smooth muscle cells and their sympathetic innervation in the wall of the Graafian follicle.

Histochemistry and ultrastructure of adrenergic and acetylcholinesterase-containing nerves supplying follicles and endocrine cells in the guinea-pig ovary.

The autonomic nerve supply of the guinea-pig ovary was investigated by a combination of light- and electron microscopy. At the light-microscopic level, adrenergic fibres were identified due to their formaldehyde-induced fluorescence. In addition, the ovary contained acetylcholinesterase-positive fibres. In all parts of the ovary, the adrenergic fibres were most numerous. At the ultrastructural level it was possible to identify the adrenergic nerve terminals with the aid of the false adrenergic transmitter, 5-hydroxy-dopamine. Thus, large numbers of adrenergic terminals, characterized by their content of 50-60 nm, electron-dense synaptic vesicles, were seen within the interstitial gland, where they formed close contacts with the endocrine cells (membrane-to-membrane distance, 20-100 nm). The follicular theca externa was also richly supplied by adrenergic nerves. At this location, close contacts (50-100 nm) were identified between the nerve terminals and the smooth muscle-like cells. Very few adrenergic nerve fibres were present in the theca interna of follicles or in the corpus luteum. Non-adrenergic nerve terminals, characterized by electron-lucent synaptic vesicles of 50-60 nm diameter, were observed together with the adrenergic fibres. They were always present in much lower numbers than the latter. No "p-type" nerves were identified by electron microscopy.

Effect of ovarian sympathectomy on follicular development during compensatory ovarian hypertrophy in the guinea-pig.

Selective ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian bursa on Day 3 of the oestrous cycle (Day 1 = day of oestrus). Control ovaries had the periovarian bursa surgically closed and were injected with solvent. On Day 15 of the cycle, serum was collected for progesterone determination and ovaries were processed for morphometric analysis of follicles. In both control and sympathectomized ovaries remaining after unilateral ovariectomy on Day 3, there was an increase in ovarian weight and an increase in healthy preovulatory follicles (greater than 700 microns diam.). Sympathectomy of the ovary remaining after unilateral ovariectomy increased healthy follicles 510-700 microns diameter but decreased the total number of follicles per ovary. In animals bearing both ovaries, unilateral sympathectomy did not affect any of the characteristics measured. Serum progesterone concentrations were unaffected by any of the treatments. These results indicate that adrenergic nerves play a role in follicular dynamics in ovaries undergoing compensatory hypertrophy but are not necessary for compensatory increases in weight and number of preovulatory follicles.

Contraction of the ovarian follicle induced by local stimulation of its sympathetic nerves.

Strips of bovine ovarian follicle wall, known to contain smooth muscle cells innervated by adrenergic nerves, were dissected out and exposed to transmural electrical stimulation in an organ bath. A frequency-dependent contractile response was obtained with a maximum at 8-16 Hz. The response was abolished in the presence of tetrodotoxin and inhibited by bretylium, phentolamine and reserpine. Thus, stimulation of the sympathetic nerves in the ovarian follicle releases sufficient amounts of norepinephrine to produce a contraction of its wall, an effect mediated by alpha-adrenergic receptors.