Interaction of NPY and VIP in regulation of myometrial blood flow and mechanical activity

The occurrence, molecular characteristics and biological function of neuropeptide Y (NPY) has been studied in the female genital tract of non-pregnant rabbits. NPY immunoreactivity was demonstrated throughout the genital tract. Maximum concentrations were found in the salpinx (fallopian tube), 570 pmol/g (median) lower within the uterine body (1.5 pmol/g), cervix (2.8 pmol/g) and vagina (3.6 pmol/g). In vitro, NPY had a dose-dependent stimulatory effect on non-vascular smooth muscle (ED50 10(-9) mol/l) as studied by myometrial tension recordings. In vivo, NPY (50 pmol/min.kg) induced a dose-related, non-adrenergic and non-cholinergic decrease in myometrial blood flow. Small C-terminal (NPY31-36) or N-terminal (NPY1-16) fragments of NPY had no effect on myometrial blood flow. NPY was found to interact with the smooth muscle effect of VIP; the presence of VIP (10(-8) mol/l) counteracted the contraction elicited by NPY (10(-8) mol/l) returning the response to control value. VIP and NPY displayed a similar physiological antagonism on myometrial blood flow. There was a clear difference in the response to VIP and NPY as the effect of NPY on myometrial blood flow first appeared after a lag period of 2 minutes whereas the effect of VIP was almost instantaneous. It is concluded that NPY and VIP may interact in the local nervous control of genital functions.     

Peptide-immunoreactive nerves in the mammalian female genital tract

Summary Vasoactive intestinal polypeptide, substance P, neuropeptide Y and peptide histidine isoleucine immunoreactivities have been demonstrated in the female genitalia of rat, cat, mouse and guinea-pig using immunocytochemistry and radioimmunoassay. They were localized to nerves. Each type of immunoreactive nerve showed a distinct pattern of distribution, though all were associated to some degree with blood vessels and smooth muscle. Vasoactive intestinal polypeptide-immunoreactive and neuropeptide Y-immunoreactive nerves were the most abundant. Higher concentrations of peptides were detected in the female genitalia of the mouse than those of the other species studied. Vasoactive intestinal polypeptide-immunoreactive nerves were particularly concentrated in the cervix (89.1±17.2 pmol/g, mean±S.E.M.) and the uterus (57.4±14.8 pmol/g) of the mouse, while neuropeptide Y immunoreactivity was more abundant in the Fallopian tube of the mouse (31.6±11.8 pmol/g) and the vagina of the rat (38.6±4.8 pmol/g) than in other regions. Separate populations of ganglion cells in the paracervical ganglia were found to contain vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities. Peptide histidine isoleucine-immunoreactive and vasoactive intestinal polypeptide-immunoreactive nerves were similarly distributed, but the former were much less frequent. Substance P-immunoreactive nerves were seen mainly beneath the epithelium of the vagina and were, in general, more numerous in the guinea-pig than in other species. The significance of these peptide-immunoreactive nerves in the female genital organ remains to be determined.Dr. Wang is on leave from The Institute of Acupuncture, The Academy of Chinese Traditional Medicine, Peking, China.     

Distribution of subgroups of neuropeptide Y-immunoreactive and noradrenergic nerves in the female rat uterine cervix

Summary Nerves in the uterine cervix of the rat were examined with regard to co-existence of markers for noradrenaline and neuropeptide Y, and differential tissue innervation by nerves containing different combinations of these markers. Immunohistochemical labeling of single and adjacent serial cryostat sections, and double labeling was employed. Some animals were treated with the noradrenergic neurotoxin, 6-hydroxydopamine. In control animals neuropeptide Y-immunoreactive fibers were numerous in the myometrium and around arteries; noradrenergic fibers were few in the myometrium and moderate in number around arteries. Myometrial neuropeptide Y-immunoreactive fibers were not decreased, but apparently increased, in 6-hydroxydopamine-treated rats; in contrast, perivascular neuropeptide Y-immunoreactive fibers were markedly reduced, but not totally absent. Noradrenergic fibers were absent in the myometrium and around arteries following 6-hydroxydopamine treatment. Labeling of adjacent sections and double labeling revealed coincident labeling of markers for neuropeptide Y and noradrenaline in perivascular, but not myometrial, nerves. We concluded that most myometrial neuropeptide Y-immunoreactive nerves did not contain noradrenaline since they were not sensitive to 6-hydroxydopamine and did not stain doubly; however, perivascular neuropeptide Y-immunoreactive fibers which degenerated after 6-hydroxydopamine treatment and did label doubly must co-store noradrenaline. Some neuropeptide Y-immunoreactive perivascular fibers may contain neuropeptide Y but not noradrenaline. Thus, it appears there is a differential innervation of tissues in the cervix by neuropeptide Y/noradrenergic nerves; this could reflect a differential regulation of tissues innervated by these nerves.     

Peripheral insulin administration attenuates the increase in neuropeptide Y concentrations in the hypothalamic arcuate nucleus of fasted rats

Fasting increases neuropeptide Y (NPY) concentrations in the arcuate nucleus (ARC), its site of synthesis, and in other regions of the rat hypothalamus. Neuropeptide Y is a potent central orexigenic agent and may therefore stimulate appetite during fasting. We tested the hypothesis that low plasma insulin levels stimulate ARC levels of NPY in fasted rats. Compared with freely fed controls (n = 8), rats fasted for 72 h (n = 8) showed significantly lower plasma insulin levels (28.9 ± 1.6 vs. 52.6 ± 5.7 pmol/l; p < 0.001) and higher ARC NPY concentrations (14.2 ± 1.8 vs. 8.4 ± 2.2 fmol/μg protein; p < 0.001). Fasted rats treated with subcutaneous insulin (5 U/kg/day; n = 10), which nearly normalized plasma insulin (46.6 ± 2.8 pmol/l), showed intermediate ARC NPY levels (11.2 ± 1.4 fmol/μg protein; p < 0.01 vs. controls and untreated fasted rats). Insulin administered peripherally, therefore, attenuates fasting-induced NPY increases in the ARC, supporting the hypothesis that hypoinsulinemia stimulates hypothalamic NPY.     

An immunohistochemical study of the catecholamine synthesizing enzymes and neuropeptides in the female guinea-pig uterus and vagina

Summary The uterus and vagina of the guinea pig have been examined, region by region, for acetylcholinesterase, tyrosine hydroxylase, dopamine -hydroxylase and aromatic amino acid decarboxylase activity, as well as for the neuropeptides, neuropeptide Y, vasoactive intestinal peptide, substance P, enkephalin and somatostatin. No acetylcholinesterase activity was localized in the uterus, though it was present in associated paracervical ganglion tissues. Of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine -hydroxylase activity was found virtually throughout the reproductive tract, whereas aromatic amino acid decarboxylase activity was restricted in its distribution. Neuropeptide distribution was quite varied. Neuropeptide Y was found throughout the endometrium/submucosa but only in the muscularis of the vagina and not in the myometrium. Substance P was localized in the vagina and uterine horn, though not the body of the uterus. Vasoactive intestinal peptide was present in all regions of the endometrium/submucosa, but not in the myometrium of the uterine horn. Enkephalin and somatostatin were not localized in any part of the reproductive tract examined, apart from paracervical ganglion tissues. The types and significance of the nerves supplying the reproductive tract are discussed.     

Projections of the guinea-pig paracervical ganglion to pelvic viscera

Summary The uterine cervix, urinary bladder and rectum of guinea pigs were injected with Fast Blue dye for retrograde transport studies. Dye-laden neuronal perikarya were detected for each viscus in the paracervical ganglion. These same perikarya also exhibited immunoreactivities for tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine -hydroxylase, neuropeptide Y, or vasoactive intestinal peptide, though the perikarya projecting to the urinary bladder did not exhibit immunoreactivity for aromatic amino acid decarboxylase. The results of this study indicate that the guinea-pig paracervical ganglion projects to viscera in addition to the uterus, and that the ganglion contains a range of immunoreactivities related to adrenergic and non-adrenergic neurotransmitters.     

Distribution and localization of neurokinin A-like immunoreactivity and neurokinin B-like immunoreactivity in rat peripheral tissue

Using specific radioimmunoassays and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of these peptides in rat peripheral tissues were studied. NKA-like immunoreactivity (NKA-LI) was present in highest levels of 15.7–23.9 pmol/g wet wt. and NKB-like immunoreactivity (NKB-LI) was in levels of 0.33–0.67 pmol/g wet wt., throughout the gastrointestinal tract involving stomach, duodenum, jejunum, ileum and colon. Immunocytochemical analysis of gastrointestinal tract revealed that NKA-LI and NKB-LI localized in ganglia of both the submucosal and myenteric plexuses as well as varicose neurons in the mucosa and the muscle layer of the small and large intestine. On the other hand, high levels of NKB-LI were observed in oesophagus (0.83 ± 0.08 pmol/g wet wt.), adrenal (1.02 ± 0.21), head of pancreas (0.73 ± 0.06) and kidney (0.98 ± 0.05).

The present study shows the difference of localization of NKA-LI and NKB-LI in peripheral tissues and suggests that NKB may have some physiological role differing from that of NKA in peripheral tissues.     

The effect of seminal sperm concentration on sperm transport in the reproductive tract of female rabbits treated with progesterone or diethylstilbestrol

Forty-two mature Baladi female rabbits were used in a randomized 3x2 factorial experiment to determine the effects of three treatments (control, progesterone injection: 2 mg/doe and DES injection: 0.1 mg/doe) and two semen sperm cell concentrations (1x10(6) and 60x10(6) sperm/0.25 ml semen on sperm transport and distribution in the female reproductive tract. The injections were given for three consecutive days after which rabbits were injected with 5 IU HCG and inseminated with 0.25 ml semen. The does were sacrificed 10 hrs after insemination and the sperm were recovered and counted from the oviducts, uterine horns, cervices and vagina. Total spermatozoa recovered was high when rabbits were inseminated with 60x10(6) sperm as compared to those inseminated with 1x10(6) sperm. When rabbits were injected with progesterone or DES, the number of sperm recovered relative to the total number of sperm inseminated was high in rabbits inseminated with 1x10(6) sperm, in comparison to those inseminated with 6x10(6) sperm. The number of sperm recovered was highest from cervix which was followed by vagina, uterus and oviducts. DES increased the number of the total sperm recovered while progesterone decreased the number as compared to control. This trend was also observed within the different segments of the reproductive tract and with groups inseminated with 1x10(6) or 60x10(6) sperm/0.25 ml semen. The effect of DES was more obvious with does inseminated with low sperm numbers. Significant correlation coefficients were found between the sperm numbers recovered in the uterus and oviducts and in the cervix and uterus of all groups of rabbits.     

Influence of pregnancy and sex steroids on concentration, motor effect and receptor binding of VIP in the rabbit female genital tract

The influence of sex steroid and pregnancy on the tissue concentration, uterine motor effect and receptor binding of VIP has been studied in the female genital tract of pregnant rabbits and oophorectomized rabbits during progesterone and/or oestrogen substitution. The concentration of immunoreactive VIP was high in the vagina and cervix, and lower in the uterine body of both pregnant and non-pregnant rabbits. A significant decrease in the VIP concentration (pmol/g wet weight) of the uterine body was observed toward term of pregnancy. The total uterine content of VIP, however, seems unchanged. Treatment of oophorectomized rabbits with ovarian steroids had no effect on the VIP concentration. The sensitivity for and potency of VIP on the relaxation of uterine muscle was significantly higher in oophorectomized rabbits treated with a combination of progesterone and oestrogen than in control rabbits. No difference was observed between non-pregnant and pregnant rabbits. The degradation and binding affinity for 125I-labelled VIP was highest in oophorectomized rabbits substituted with both oestrogen and progesterone. In the pregnant rabbits, the amount of receptors was decreased near term. In conclusion, sex steroids are able to influence the motor effect of VIP at the receptor level, but have no effect on the VIP concentration in the female genital tract.     

Ultrasonographic evaluation of the pre-pubertal development of the reproductive tract in beef heifers

To study the development of the reproductive tract in heifers, the ovaries, uterus, cervix and vagina were examined by transrectal ultrasonography every 2 weeks, from 2 to 60 weeks after birth. First ovulation occurred at 63.7 +/- 1.1 weeks of age. Ovarian dimensions increased rapidly from 2 to 14 weeks of age, and increased again after 34 weeks of age (P<0.05). The size of the largest ovarian follicles increased from 8 to 14 weeks of age, from 38 to 42 weeks of age, and finally from 52 to 60 weeks of age (P<0.05). The number of follicles > or =3 mm in diameter tended to increase from 6 to 14 weeks of age (P<0.10) and increased significantly from 6 to 60 weeks of age (P<0.05). Mean numerical pixel values of the ovarian images decreased from 4 to 26 weeks of age, and then rose to 44 weeks of age (P<0.05). Diameter of the uterine body, cervix and vagina increased from 2 to 20-24 weeks of age, and again after 32 weeks of age (P<0.05). Mean numerical pixel values for the uterus and vagina decreased initially (uterus: 4-8 weeks and vagina: 6-22 weeks of age) and then increased (uterus: 14-42 weeks and vagina: 22-32 weeks of age; P<0.05). Pixel heterogeneity showed a consistent peak at 20-22 weeks of age for the uterus, cervix and vagina (P<0.05). In summary, in the heifer calf, the marked growth of the reproductive tract in the first few months of age, and prior to first ovulation, reflects phases of increased ovarian follicle (> or =3 mm in diameter) numbers and size. Ultrasonographic image analysis revealed patterns of numerical pixel values and heterogeneity that may be useful in determining important stages of growth and differentiation of the reproductive system.     

Comparative distribution of neuropeptide tyrosine-,vasoactive intestinal polypeptide-, substance P-immunoreactive,acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat

Summary Location, distribution and density of nerve fibers immunoreactive to neuropeptide tyrosine, vasoactive intestinal polypeptide and substance P were studied in the reproductive tract of the female rat and compared with acetylcholinesterase-positive (cholinergic) and noradrenergic nerves. Plexuses of all types of fibers were present in the vagina, uterine cervix, uterine horn and oviduct. In the tubular reproductive organs all of these types of nerve fibers appeared to innervate vascular and non-vascular smooth muscle and nearly all types of fibers formed plexuses subjacent to the epithelium lining the organs. Individual fibers of all classes appeared to innervate fascicles of smooth muscle in the mesometrium of the uterine horn. A few acetylcholinesterase-positive and substance P-immunoreactive fibers were present in the ovary but no vasoactive intestinal polypeptide-immunoreactive nerves were observed. Noradrenergic and neuropeptide tyrosine-immunoreactive nerves were numerous in the ovary where they were seen in the interstitial gland tissue and associated with follicles and blood vessels. It is suggested that these nerves may influence hemodynamic events and non-vascular smooth muscle in such functions as transport of sperm and ova and parturition. Substance P-immunoreactive nerve fibers are likely to be sensory fibers that could have roles in neurohormonal reflexes.     

Inhibition of human gastric lipase by intraduodenal fat involves glucagon-like peptide-1 and cholecystokinin

Seven healthy volunteers were intubated with two double lumen nasogastric tubes, one in the stomach, the other in the duodenum. This system allows simultaneous sampling of gastric juice and separate intraduodenal perfusion with a dietary fat (fish oil, 1269 kJ). Gastrin-17 was infused i.v. at a rate of 40 pmol/kg/h throughout the study. Gastric lipase was measured at 15-min intervals as activity (tributyrin) and as immunoreactivity (ELISA). Infusion of gastrin-17 resulted in a stable increase in the plasma concentration from a basal concentration of 8.3±0.8 pmol/l to 41.4±4.2 pmol/l. Perfusion with fat reduced gastric lipase activity from 24.2±5.3 to 7.2±2.5 kU/l (P<0.05), and immunoreactivity from 0.7±0.1 to 0.42±0.1 mg/l (P<0.05). After termination of fat perfusion, gastric lipase secretion increased again, though not reaching preinhibitory concentrations. During the intraduodenal perfusion with fat the plasma concentrations of glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) increased from 6.9±0.5 to 15.1±1.5 pmol/l (P<0.05) and from 1.2±0.4 to 3.8±0.9 pmol/l (P<0.05). This study reveals a negative effect of fat in the duodenum on gastric lipase secretion. This effect may be mediated by GLP-1 and/or CCK.     

Dominant activation of the hedgehog signaling pathway alters development of the female reproductive tract

The role of hedgehog (HH) signaling in reproductive tract development was studied in mice in which a dominant active allele of the signal transducer smoothened (SmoM2) was conditionally expressed in the Müllerian duct and ovary. Mutant females are infertile, primarily because they fail to ovulate. Levels of mRNA for targets of HH signaling, Gli1, Ptch1, and Hhip, were elevated in reproductive tracts of 24-day-old mutant mice, confirming overactivation of HH signaling. The tracts of mutant mice developed abnormally. The uterine luminal epithelium had a simple columnar morphology in control mice, but in mutants contained stratified squamous cells typical of the cervix and vagina. In mutant mice, the number of uterine glands were reduced and the oviducts were not coiled. Expression of genes within the Hox and Wnt families that regulate patterning of the reproductive tract were altered. Hoxa13, which is normally expressed primarily in the vagina and cervix, was expressed at 12-fold higher levels in the uterus of mutant mice compared with controls. Wnt5a, which is required for development of the cervix and vagina and postnatal differentiation of the uterus, was expressed at higher levels in the oviduct and uterus of mutant mice compared with controls. Mating mutant females with fertile or vasectomized males induced a severe inflammatory response in the tract. In summary, overactivation of HH signaling causes aberrant development of the reproductive tract. The phenotype observed could be mediated by ectopic expression of Hoxa13 in the uterus and elevated levels of Wnt5a in the oviducts and uterus.     

Platelet-activating factor in the rabbit uterus during early pregnancy

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.     

Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

High and low responding strains of laboratory opossums differ in sterol 27-hydroxylase and acyl-coenzyme A:cholesterol acyltransferase activities on a high cholesterol diet

Two partially inbred strains of laboratory opossums exhibit extremely high or low levels of VLDL+LDL cholesterol concentrations, respectively, when challenged with a high cholesterol and high fat diet. The present studies were conducted to determine whether the high and low responding strains differ in activities of important enzymes that have been shown to affect lipemic responsiveness to diet. We measured plasma 27-hydroxycholesterol and hepatic activities of 27-hydroxylase and 7-hydroxylase in high and low responding opossums while consuming the basal diet and cholesterol-enriched diets. Plasma 27-hydroxycholesterol concentration and 27-hydroxylase activity in liver did not differ between groups on the basal diet, but both were significantly higher in low responders than in high responders on the cholesterol-enriched diet with unsaturated fat (10.79 ± 0.56 in low vs. 7.31 ± 0.50 μg/dl in high responders for 27-hydroxycholesterol and 14.14 ± 0.79 in low vs. 10.07 ± 0.80 pmol/mg protein/min in high responders for 27-hydroxylase activity). On the other hand, 7-hydroxylase activity was significantly higher in high responding opossums (75.72 ± 6.81 pmol/mg protein/min) than in low responding opossums (51.39 ± 6.18 pmol/mg protein/min) on the basal diet, but it did not differ on the high cholesterol and high fat diet. We measured hepatic ACAT and extrahepatic hepatic 27-hydroxylase activities in high and low responding opossums on the cholesterol enriched diet. Hepatic ACAT activity was significantly higher in high responding opossums (137.00 ± 18.33 pmol/mg protein/min) than in low responding opossums (47.67 ± 2.71 pmol/mg protein/min), whereas extrahepatic 27-hydroxylase activity was higher in low responding opossums (33.00 ± 2.10 pmol/mg protein/min in lungs and 3.69 ± 0.20 in kidneys) than in high responding opossums (21.17 ± 1.54 pmol/mg protein/min in lungs and 2.82 ± 0.31 in kidneys). We also compared the composition of bile between high and low responders. The concentration of taurine conjugates of cholic acid in bile of both groups was similar, but concentration of taurine conjugates of chenodeoxycholic acid in bile of low responding animals was higher than in high responding animals (124.9 ± 17.3 in low vs. 59.2 ± 13.2 μmol/ml in high responders). The results of these studies suggest two enzymes may affect the lipemic response to diet in laboratory opossums: sterol 27-hydroxylase and ACAT. Each of these enzymes may influence diet-induced hyperlipidemia at a different step of lipoprotein metabolism.     

A sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide Y

The development of a new enzyme immunoassay for neuropeptide Y (NPY) is reported. Two monoclonal antibodies directed against distinct epitopes of NPY are used, one as a capture antibody (NPY02) and the other one as an indicator antibody (NPY05), this latter antibody being labeled with alkaline phosphatase. The assay calibration curve was performed over concentrations of 1 to 250 pM in a NPY-free plasma. The intra-assay coefficient of variation (CV) ranged from 0.025 to 11.9%, whereas the interassay CV was comprised between 5 and 12%. The limit of detection of this assay was 1 pM (100 amol/well). Neuropeptide Y levels are related to sampling conditions; basal concentrations of NPY with low SEM are found when less than 1.2 ml of blood is taken in EDTA tubes, the sample is centrifuged at 4°C, and immediately frozen. Unanesthetized spontaneously hypertensive rats exhibited higher NPY plasma concentrations than normotensive Wistar-Kyoto controls (53 ± 7 pM and 25 ± 2 pM, respectively, mean ± SEM, p < 0.01). Plasma NPY levels are similar in 16- and 36-week-old animals. In conclusion, this technique makes it possible to assay a large number of samples within 24 h without requiring radioactivity.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/10⁵ cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10⁵ cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

Cervical patency during non-ovulatory and ovulatory estrus cycles in domestic cats

The cervix functions as a barrier to spermatozoa. Vaginal artificial insemination in cats is, therefore, likely to be successful only at the period of estrus when the cervix is open. This study aimed to define the period of cervical patency in cats in both non-ovulatory and ovulatory estrus cycles. A total of 15 reproductive cycles were studied in six cats during the estrous stage. Cervical patency was monitored with the cats under sedation, by infusing 2 mL of Iohexol contrast medium via a 3.5 French tomcat catheter into the cranial vagina during estrus. Day one of estrus was defined as the first day the cats showed estrous behavior. Non-ovulatory cycles were characterized by a serum progesterone concentration on days 11-15 that was below 5 nmol/L and a normal interestrus interval of 7-14 days. Ovulatory cycles were characterized by a serum progesterone concentration on days 11-15 that was above 5 nmol/L and an interestrus interval that exceeded 30 days. The cervix was considered to be open when the contrast medium was seen to enter the uterus, and to be closed when the contrast medium remained in the vagina. Blood samples were collected at each examination and were assayed for estradiol-17beta and progesterone concentrations. The cervix was open on the first day of standing estrus at a mean estradiol-17beta serum concentration of 87.4+/-21.8 pmol/L (range 14 to >or=180 pmol/L) and closed at an estradiol concentration of 47.1+/-12.4 pmol/L (range 4 to >or=180 pmol/L). In the ovulatory cycles the cervix was closed at a progesterone concentration of 9.8+/-4.4 nmol/L (range 0.6-28.4 nmol/L). There was no difference in the duration of cervical patency in non-ovulatory and ovulatory cycles (5.5+/-1.2 days and 5.2+/-0.5 days, respectively) (p>0.05). The higher overall mean concentrations of estradiol-17beta seen in the ovulatory cycles than in the non-ovulatory cycles, indicate that a high level of estradiol is necessary for induction of ovulation. Ovulation in 60% of unmated females in this study indicates that the techniques used for evaluation of cyclus stage and cervical opening have the potential to induce ovulation in the cat. This study demonstrates that cervical patency is not influenced by the occurrence of ovulation, but is due to individual variations between cats.     

The primary structure of peptide Y (PY) of the spiny dogfish, Squalus acanthias: immunocytochemical localisation and isolation from the pancreas.

1. Endocrine cells within islets, exocrine parenchyma and ductal epithelium in the pancreas of the spiny dogfish, Squalus acanthias, were immunostained with an antiserum to the C-terminal region of mammalian neuropeptide Y (NPY). 2. Radioimmunoassay of pancreatic extracts with the same antiserum detected immunoreactivity in the dorsal lobe (338 pmol/g) and ventral lobe (433 pmol/g). Reverse phase HPLC analysis of both extracts resolved a single immunoreactive peptide. 3. The primary structure of the isolated peptide was established as: YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY.NH2. 4. Peptide Y (PY) from Squalus acanthias is identical in primary structure to an NPY-related peptide isolated from the pancreas of Scyliorhinus canicula and has a 31/36 residue homology with porcine NPY. The 5 substitutions are highly-conservative.