Review: neuropathology of acute phase encephalitis lethargica: a review of cases from the epidemic period.

INTRODUCTION: Encephalitis lethargica (EL), an epidemic disease of the early 20th century, has continued to be diagnosed sporadically since that time, including a report of 20 new cases in 2004. Many of the recent case reports state that the primary neuropathology of acute EL consists of inflammatory changes and lesions within the midbrain, basal ganglia and substantia nigra. However, the neuropathology of acute EL cases from the epidemic period was actually much more widespread. METHODS: In order to characterize the neuropathology of acute phase EL, we developed a database of EL pathology based on 112 cases from the years 1915 to 1940, of which most died within 2 weeks of EL onset. RESULTS: Our analysis revealed that cortical damage was prevalent in 75% of the 112 cases; damage to the meninges and brainstem occurred in approximately half of the cases; and the substantia nigra was damaged in only 13% of these acute cases. We also found that after 1921, damage to cranial nerve nuclei was not reported. An analysis of the neuropathology and clinical symptoms revealed little correlation. CONCLUSIONS: Based on these findings, putative modern cases of acute EL with MRI/CT indicated lesions confined solely to the midbrain, brainstem, and/or basal ganglia should not be considered, consistent with that reported during epidemic period.

Effects of a single trace mineral injection at beginning of fixed-time AI treatment regimen on reproductive function and antioxidant response of grazing Nellore cows.

Two experiments evaluated the effects of injectable trace minerals (ITM) administered 11 d before artificial insemination (AI) on body weight (BW), body condition score (BCS), ovarian structures, pregnancy rate, and antioxidant response of Nellore cows. In Experiment 1, 20 multiparous cows were assigned to one of two treatments: subcutaneous injection (6 mL/cow; 11 d before AI) of saline solution or ITM (60, 10, 5, and 15 mg/mL of Zn, Mn, Se and Cu, respectively) and BW, BCS, ovarian structures and blood were evaluated. In Experiment 2, 1,144 multiparous cows were assigned to same treatments described in Experiment 1 and pregnancy rate on d 30 was evaluated. In Experiment 1, ITM did not affect (P ≥  0.23) BW, dominant follicle size, ovulation rate, and plasma concentrations of haptoglobin, ceruloplasmin and progesterone (P4). The ITM treatment tended to increase (P =  0.06) cow BCS and reduce (P ≤  0.06) corpus luteum (CL) diameter and volume. Furthermore, ITM treatment tended to increase (P =  0.06) plasma concentrations of SOD and increased (P =  0.007) GSH-Px compared with saline injection. In Experiment 2, ITM treatment tended (P =  0.06) to increase pregnancy rate of cows with BCS ≤ 5.0 but not cows with BCS > 5.0 (P =  0.99). The ITM treatment did not alter BW, plasma P4, and acute phase response, but enhanced plasma concentrations of antioxidant enzymes, and tended to enhance BCS and pregnancy rates to AI of cows with BCS ≤ 5.0, even though there was a smaller corpus luteum size.

Exogenous glucagon effects on health and reproductive performance of lactating dairy cows with mild fatty liver.

Severe fatty liver, a metabolic disease of dairy cows in early lactation, results in decreased health and reproductive performance, but can be alleviated by treatment with i.v. injections of glucagon. Mild fatty liver in cows effects on health and reproductive performance were determined by treatment with 14-day s.c. injections of glucagon at 7.5 or 15 mg/day. Multiparous Holstein cows (n=32) were grouped into Normal and Susceptible based on liver triacylglycerol concentrations (>1% liver tissue biopsy wet weight) at day 8 postpartum (day 0=day of parturition). Susceptible cows (n=24) were assigned randomly to three groups and s.c. injected with 0mg glucagon [60 ml 0.15M NaCl] [n=8] (same for Normal cows), 2.5 mg glucagon, or 5 mg glucagon every 8 h for 14 days, beginning day 8 postpartum. Mild fatty liver resulted in an increased number of days with elevated body temperature during the injection period, an increased incidence of mastitis after glucagon treatment, increased days to first estrus and insemination, increased days before conception occurred, and decreased conception rate. In cows with mild fatty liver, glucagon (15 mg/day) decreased the number of days with elevated body temperature and the incidence of mastitis after hormone treatment. From these results, we suggest that mild fatty liver is detrimental to health and reproduction of dairy cows and, furthermore, that exogenous glucagon decreases some of these detrimental effects.

Lack of effects of atrazine on estrogen-responsive organs and circulating hormone concentrations in sexually immature female Japanese quail (Coturnix coturnix japonica).

The widely used herbicide, atrazine, has been reported to exhibit reproductive toxicity in rats and amphibians. The present studies investigate toxicity of atrazine in Japanese quail and its ability to influence reproduction in sexually immature females. Atrazine was administered in the diet at concentrations from 0.001 to 1000 ppm (approximately 109 mg kg-1 per day) or systemically via daily subcutaneous injections (1 and 10 mg kg-1) or Silastic implants. Atrazine did not cause overt toxicity in sexually immature female quail (no effects on change in body weight, feed intake, mortality or on circulating concentrations of the stress hormone, corticosterone). It was hypothesized that if atrazine were to have estrogenic activity or to enhance endogenous estrogen production, there would be marked increases in the weights of estrogen sensitive tissues including the oviduct, the liver and the ovary together with changes in gonadotropin secretion. However, atrazine had no effect on either liver or ovary weights. Atrazine in the diet increased oviduct weights at 0.1 and 1 ppm in some studies. These effects were not consistently observed and were not significant when data from studies were combined. Systemic administration of atrazine had no effect on oviduct weights. Dietary (concentrations from 0.001 to 1000 ppm) and systemically administered atrazine had no effect on circulating concentrations of luteinizing hormone (LH). The present studies provide evidence for a lack of general or reproductive toxicity of atrazine in birds.

Lack of estrogenic or antiestrogenic actions of soy isoflavones in an avian model: the Japanese quail.

Isoflavones are soy compounds that possess weak estrogenic and antiestrogenic activities. In addition, phytochemicals, including isoflavones, may play a role in regulating seasonal reproductive cycles. As soy is a common constituent in poultry diets, the effect of these compounds on the reproductive system of production birds may be of concern. The present study examined the putative effects of soy isoflavones supplemented into the diet at 1 and 5% using endpoints of growth and reproduction in the Japanese quail. Isoflavones did not exert an effect on growth, feed intake, growth:feed, or the weight of the estrogen-sensitive immature oviduct in female quail. Furthermore, isoflavones did not influence the growth of the oviduct stimulated by exogenous estradiol. Similarly, isoflavones did not influence growth, feed intake, or growth:feed in male quail. However, isoflavones at 1%, but not 5%, in the diet reduced photoperiod-induced testis development 40% vs. control. In contrast, isoflavones did not influence testis regression stimulated by exogenous estradiol in sexually maturing male quail. The present results suggest that isoflavones may exert modest endocrine disruptor-like effects on reproduction in male, but not female, quail.

Evaluation of corn furan fatty acid putative endocrine disruptors on reproductive performance in adult female chickens.

Based on evidence from rodent models, it was hypothesized that furan fatty acids found in corn would inhibit reproduction in the laying hen. An isomeric mixture of furan fatty acids [9, (12)-oxy-10,13-dihydroxystearic acid and 10, (13)-oxy-9,12-dihydroxystearic acid] was administered for a period of 3 wk via the diet (1 and 3 ppm) at levels greater than those in corn to 20-wk-old pullets. There were no overt indications of acute or chronic toxicity (no effects on mortality, feed intake, or average daily gain). Similarly, there was no dose-dependent effect on reproductive parameters [egg production, egg weight, shell thickness, ovarian weight, number or weight of large yolky preovulatory follicles, and number of small yellow follicles (4-8 mm in diameter)]. The present data do not suggest that furan fatty acids are a cause of concern to the poultry industry.

Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase.

Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO2 and Mg2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. Incubation of the carboxylase with potassium [14C] cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO2 binding sites on the enzyme.

Conformational changes associated with the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase.

Crystalline ribulose-1,5-bisphosphate carboxylase-oxygenase (3-phospho-D-glycerate carboxy-lyase (dimerising), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is partially inactivated by cold treatment and fully reactivated by simple heating in the absence of sulfhydryl reagents and effectors. Since the reversible cold inactivation of this bifunctional enzyme does not involve a gross change in the association state of subunits, a subtle conformational change induced by low temperatures was implicated (Chollet, R. and Anderson, L.L. (1976) Arch. Biochem. Biophys. 176, 344-351). Chemical modification of the cold-inactivated and heat-reactivated enzymes by 5,5'-dithiobis-(2-nitrobenzoate) and p-mercuribenzoate at 25 degrees C revealed no difference in the number of free -SH groups per mol protein. However, the reactivity of the sulfhydryl residues on the inactivated protein was considerably greater than that of the reactivated enzyme. Pretreatment of the two proteins with sodium dodecyl sulfate completely abolished the difference in -SH reactivity, indicating its dependence on protein conformation. Both the cold-inactivated and heat-reactivated enzymes enhanced the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) and caused a blue shift of the emission maximum from 510 to 472 nm. When the inactivated enzyme was reactivated by heating, the increase in catalytic activity was closely paralleled by a concomitant decrease in the fluorescence intensity of the ANS - protein complex at 25 degrees C. Fluorescence titration experiments revealed that the decrease in fluorescence intensity accompanying heat reactivation of the inactivated enzyme was due to a reduction in the number of hydrophobic sites available for ANS binding rather than to a change in the dissociation constant of the ANS - protein complex. These results indicate that the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase is associated with a reversible change in the conformation of the protein. This cold-induced conformational change resluts in a greater exposure of sulfhydryl groups and hydrophobic regions to the external environment and is closely paralleled by changes in the catalytic activity of the protein. By analogy to other oligomeric enzymes also subject to reversible cold inactivation, perhaps low temperatures induce a partial dissociation of the octameric structure of the hydrophobic catalytic subunits, but complete dissociation is arrested in some unknown manner by the small hydrophilic subunits.

Early effect of myo-inositol deficiency on phosphatidylinositol metabolism in rat liver.

Young rats (100 g) were fed either a myo-inositol-deficient or supplemented (control) diet for up to 14 days following a 12 h fast. At various times during this period animals were killed, livers were removed, and a microsomal fraction was prepared and assayed for CDPdiacylglycerol inositol transferase activity and for phosphatidylinositol-inositol exchange activity. Within 2 days after beginning the regimen, rats consuming the deficient diet had a 40% lower activity of the transferase than rats consuming the control diet. This difference was maintained throughout the feeding period and developed simultaneously with the accumulation of triacylglycerol in the deficient livers. In contrast, the specific activity of the exchange enzyme was unchanged by feeding the deficient diet.

The anterior pituitary gland: lessons from livestock.

There has been extensive research of the anterior pituitary gland of livestock and poultry due to the economic (agricultural) importance of physiological processes controlled by it including reproduction, growth, lactation and stress. Moreover, farm animals can be biomedical models or useful in evolutionary/ecological research. There are for multiple sites of control of the secretion of anterior pituitary hormones. These include the potential for independent control of proliferation, differentiation, de-differentiation and/or inter-conversion cell death, expression and translation, post-translational modification (potentially generating multiple isoforms with potentially different biological activities), release with or without a specific binding protein and intra-cellular catabolism (proteolysis) of pituitary hormones. Multiple hypothalamic hypophysiotropic peptides (which may also be produced peripherally, e.g. ghrelin) influence the secretion of the anterior pituitary hormones. There is also feedback for hormones from the target endocrine glands. These control mechanisms show broadly a consistency across species and life stages; however, there are some marked differences. Examples from growth hormone, prolactin, follicle stimulating hormone and luteinizing hormone will be considered. In addition, attention will be focused on areas that have been neglected including the role of stellate cells, multiple sub-types of the major adenohypophyseal cells, functional zonation within the anterior pituitary and the role of multiple secretagogues for single hormones.

Physiology of ghrelin and related peptides.

Growth hormone (GH) released from pituitary under direct control of hypothalamic releasing (i.e., GHRH) and inhibiting (i.e., sst or SRIF) hormones is an anabolic hormone that regulates metabolism of proteins, fats, sugars and minerals in mammals. Cyril Bowers' discovery of GH-releasing peptide (GHRP-6) was followed by a search for synthetic peptide and nonpeptide GH-secretagogues (GHSs) that stimulate GH release, as well as a receptor(s) unique from GHRH receptor. GHRH and GHSs operate through distinct G protein-coupled receptors to release GH. Signal transduction pathways activated by GHS increase intracellular Ca2+ concentration in somatotrophs, whereas GHRH increases cAMP. Isolation and characterization of ghrelin, the natural ligand for GHS receptor, has opened a new era of understanding to physiology of anabolism, feeding behavior, and nutritional homeostasis for GH secretion and gastrointestinal motility through gut-brain interactions. Other peptide hormones (i.e., motilin, TRH, PACAP, GnRH, leptin, FMRF amide, galanin, NPY, NPW) from gut, brain and other tissues also play a role in modulating GH secretion in livestock and lower vertebrate species. Physiological processes, such as neurotransmission, and secretion of hormones or enzymes, require fusion of secretory vesicles at the cell plasma membrane and expulsion of vesicular contents. This process for GH release from porcine somatotrophs was revealed by atomic force microscopy (AFM), transmission electron microscopy (TEM) and immunohistochemical distribution of the cells in pituitary during stages of development.

Acute decrease in progesterone and increase in estrogen secretion caused by relaxin during late pregnancy in beef heifers.

Purified porcine relaxin (3000 U/mg) was administered im (RLX-IM; 1 mg; n = 2) and in the cervical os (RLX-OS; 1 mg; n = 2) on day 273 (approximately 10 days before parturition normally occurs) of gestation to determine the profiles of immunoreactive relaxin and its effects on progesterone, estrone (E1), and 17 beta-estradiol (17 beta-E2) secretion in peripheral blood plasma of beef heifers. Controls received either 0.01 M PBS (1 ml, im; n = 2) or 0.01 M gel-PBS (gel; 1 ml, os; n = 2) in cervical os. One relaxin-treated (im) heifer calved at 4 h and 36 min after treatment; thus, data from this heifer were not included in subsequent analysis. Relaxin-treated heifers showed an acute elevation in relaxin, a precipitous decrease in progesterone, and a significant (P less than 0.05) elevation of E1 and 17 beta-E2. Plasma relaxin levels were 4.95, 1.5, and 0.24 ng/ml at 0.5 h in RLX-IM, RLX-OS, and control animals, respectively. Peripheral plasma relaxin peaked between 23-31 ng/ml 1-2.5 h before returning to less than 0.5 ng/ml 5-12 h after treatment. Relaxin administration accounted for 70%, 73%, and 58% of the progesterone, E1, and 17 beta-E2 variability between treatments, respectively. An abrupt decrease (P less than 0.01) in progesterone preceded the rises (P less than 0.05) in E1 and 17 beta-E2 at 1.5, 2-2.5, and 2-3.5 h, respectively. Maximum progesterone deviations from the pretreatment mean concentration were -5.43, -3.05, and -0.92 ng/ml for RLX-IM, RLX-OS, and controls. Progesterone rebounded from 36% to 61% and 62% to 79% of respective pretreatment means for RLX-IM and RLX-OS. Peak elevation of E1 was 407.3, 306.5, and 71.5 pg/ml and that of 17 beta-E2 was 82.2, 35.8, and 7.8 ng/ml for RLX-IM, RLX-OS, and controls, respectively. These results provide strong evidence that a pharmacological dosage of relaxin induces an acute depression of progesterone secretion beginning within 90 min in beef heifers during late pregnancy. We suggest that these early and marked luteolytic effects of relaxin on progesterone secretion in cattle could be by direct or indirect actions via mechanisms that are yet unknown.

Relaxin and progesterone secretion as affected by luteinizing hormone and prolactin after hysterectomy in the pig.

Plasma levels of relaxin and progesterone in hysterectomized and pregnant gilts were determined from days 100-120 to evaluate the effects of purified porcine (p) LH and pPRL on the secretory activity of the aging corpora lutea. Gilts were bred on the second observed estrus or were hysterectomized between 6-8 days after estrus (estrus = day 0) and were assigned randomly to one of three treatment groups; saline-treated control, im injections of pLH, and iv injections of pPRL from days 110-120. In control, pLH-treated, and pPRL-treated animals, average gestation lengths were 114 +/- 0.8, 116 +/- 1.9, and 115 +/- 0.5 days (+/- SE), respectively. The relaxin level in mated gilts on day 100 was less than 2 ng/ml; it began to increase after day 110 and peaked in control animals on day 113 (66 ng/ml), whereas in pLH- and pPRL-treated animals, prepartum peak values were greater (P less than 0.01) and occurred on days 113 (104 ng/ml) and 114 (117 ng/ml), respectively. Relaxin dropped to basal levels (less than 1 ng/ml) by day 115 in controls and by day 116 in both pLH- and pPRL-treated gilts. Although pLH and pPRL treatments markedly accentuated peak relaxin secretion, they did not significantly accelerate or delay parturition or delay the abrupt demise of the corpora lutea immediately postpartum. In hysterectomized gilts, relaxin began to increase after day 110, peaked in control animals on day 113 (27 ng/ml), and decreased abruptly thereafter to less than 4 ng/ml. In contrast, pLH caused an immediate release of relaxin on day 111 (23 ng/ml) and sustained elevated levels (P less than 0.01) of relaxin until day 118, but the original corpora lutea regressed. Relaxin in pPRL-treated animals increased steadily after day 110, reaching peak values by day 115 (29 ng/ml), and remained consistently elevated (P less than 0.01) until day 120. Progesterone secretion was maintained in the pPRL-treated hysterectomized gilts from days 110-120 by the original corpora lutea and with no luteinization of follicles or formation of new corpora lutea. It is evident from this study that administration of pPRL starting on day 110 enhanced and prolonged the preprogramed release of relaxin and maintained progesterone secretion by aging corpora lutea in hysterectomized animals until day 120.

Divergent effects of antiprogesterone, RU 486, on progesterone, relaxin, and prolactin secretion in pregnant and hysterectomized pigs with aging corpora lutea.

Porcine corpora lutea produce progesterone and relaxin during pregnancy and after hysterectomy. Peak amounts of relaxin are released into peripheral blood in both pregnant and hysterectomized animals on about day 113 (estrus = day 0 and term = 114), and this release coincides with an abrupt decrease in the progesterone concentration. RU 486, a progesterone receptor antagonist, was used to investigate the effects of interruption of progesterone binding to its receptor on luteal function and gonadotropin secretion of pigs with aging corpora lutea. RU 486 was administered orally to hysterectomized gilts (surgery on day 8) once a day (0800 h) on days 111-115 at two dosages (group 1, 2 mg/kg BW; group 2, 4 mg/kg BW). During 5 days of RU 486 treatment, plasma progesterone concentrations in both treated groups were markedly elevated (32 and 37 ng/ml for groups 1 and 2) compared with 22 ng/ml in the controls (group 3; P less than 0.01). PRL concentrations increased in both groups (9 and 13 ng/ml) and differed significantly from those of the controls (3 ng/ml) (P less than 0.04). RU 486 treatment delayed the time of relaxin peak to days 116.1 and 117.0 in groups 1 and 2 compared with day 114.1 in the controls (P less than 0.01). Pregnant gilts received RU 486 orally once a day (0800 h) at 4 mg/kg BW beginning on day 111 until parturition occurred. Parturition was induced on day 112.7 after only two RU 486 treatments compared with day 114.7 in the control group (P less than 0.01). Progesterone decreased abruptly from a pretreatment mean of 11 to less than 0.6 ng/ml during the 2 days that RU 486 was given compared with a shift from 12 to 6 ng/ml during the same period in the controls (P less than 0.01). The time of the relaxin peak was advanced to day 112.1 in RU 486-treated gilts compared with day 113.9 in the controls (P less than 0.01). Results from this study provide strong evidence that the antagonistic effect of RU 486 on progesterone receptor results in an abrupt increase in PRL and progesterone secretion in hysterectomized gilts with aging corpora lutea. In marked contrast with hysterectomized animals, the acute luteolytic effects of RU 486 depend on the presence of the uterus and/or conceptuses in the pig. Disruption of the regulatory loop of progesterone secretion by RU 486 alters the ability of corpora lutea to produce and release peak quantities of relaxin.

Progesterone secretion as affected by 17 beta-estradiol after hysterectomy in the pig.

Serum levels of progesterone in hysterectomized gilts were determined to evaluate the effects of exogenous 17 beta-estradiol (17 beta-E2) on the secretory activity of aging corpora lutea. Gilts were hysterectomized on day 6 of the estrous cycle and given im injections of 17 beta-E2 to mimic blood levels of endogenous estrogen during normal pregnancy. Serum progesterone was determined every third or sixth day, and estrone and 17 beta-E2 were measured at 12-day intervals from days 42-192. Progesterone decreased (P less than 0.01) after day 104 in hysterectomized gilts given sesame oil. Exogenous estrogen consistently decreased (P less than 0.02) progesterone secretion during an extended period (days 45-108) in aging corpora lutea of hysterectomized gilts. Abrupt decreases in estrone, 17 beta-E2, and progesterone levels occurred after termination of estrogen injections on day 114. The decrease in the secretion of these steroids in hysterectomized gilts was similar to that observed in previous studies at parturition and during the early postpartum period. Estrogen treatment beyond day 114 inhibits follicular growth and suppresses ovulation, but behavioral estrus was induced during the terminal stages of luteal activity. In the absence of the uterus, aging corpora lutea respond to exogenous estrogen by decreased progesterone secretion, which is similar to that observed during normal pregnancy in the pig.

Relaxin production and release after hysterectomy in the pig.

Relaxin secretion by luteal tissue and into peripheral blood was examined in relation to changes in the fine structure of aging porcine corpora lutea. Sequential bleedings every sixth day reveal a similar increase in serum relaxin concentrations from days 6-114 in hysterectomized and pregnant gifts. The span of the estrous cycle is about 21 days, and the length of pregnancy is about 115 days in pigs. Estrone (E1) and 17 beta-estradiol (17 beta-E2) serum levels increase with placental development; after hysterectomy, they are low from days 12-168. In luteal tissue, peak quantities of relaxin and electron-dense granules occur on day 100 in hysterectomized and pregnant gilts. By day 112, luteal tissue relaxin levels and granule populations decrease by about half, while relaxin secretion into peripheral blood approaches peak concentrations about day 114 in hysterectomized as well as pregnant gilts. Between days 114 and 120, serum relaxin decreases by half even though the corpora lutea persist to day 150 in hysterectomized animals. Thereafter, cytoplasmic granules and blood and luteal tissue levels of relaxin decrease gradually to day 150 in hysterectomized gilts; in contrast, they disappear abruptly after parturition. In hysterectomized gilts, relaxin release coincides with diminished populations of granules in corpora lutea, and the initiation of these events seems to be precisely timed. E1 and 17 beta-E2 secretion is unrelated to relaxin secretion in pregnant and hysterectomized gilts. High peripheral blood levels of E1 and 17 beta-E2, primarily of placental origin, or even the presence of the uterus are not required in the release of relaxin at day 114. By using steroidogenic and protein synthetic capabilities of luteal cells as criteria essential to the evolution of reproduction in this species, the results presented here suggest that 114 days rather than 21 days characterize the reproductive cycle, even in the absence of the uterus. These results indicate that the production and, particularly, the release of relaxin into peripheral blood on about day 114 in hysterectomized gilts as well as pregnant animals may be genetically controlled.

Abrupt shifts in relaxin and progesterone secretion by aging luteal cells: luteotropic response in hysterectomized and pregnant pigs.

Although a prominent role of conceptuses in the hormonal control of normal parturition in the pig is well founded, mechanisms are entrained that cause a timed relaxin release and an abrupt decrease in progesterone secretion in the complete absence of the uterus. This study focused on the steroidogenic and peptide hormone secretory capacities of aging porcine luteal cells in culture on specific days during a narrow window (days 110-116) when abrupt shifts occur in their function. Dispersed luteal cells (3 x 10(5) cells/well) from hysterectomized and pregnant gilts on days 110, 113, and 116 after estrus were cultured for 24 h with porcine (p) LH, pPRL, prostaglandin E1 (PGE1), PGE2, and (Bu)2cAMP. Progesterone and relaxin concentrations in cells preceding culture and in the spent medium were quantified by RIA. Progesterone in cells before culture was less (P less than 0.05) than that released into the medium, while relaxin levels were greater (P less than 0.05) than those in the medium. Relaxin stored in luteal cells of hysterectomized gilts was consistently greater (P less than 0.01) than that during the same days of late pregnancy; however, progesterone was greater (P less than 0.01) in hysterectomized compared with pregnant gilts only on day 116. Relaxin release was greater (P less than 0.01) in hysterectomized compared with pregnant gilts on days 113 and 116, whereas progesterone was greater (P less than 0.05) on days 110 and 116 in hysterectomized compared with pregnant animals. pLH, pPRL, and (Bu)2cAMP increased (P less than 0.05) relaxin secretion in hysterectomized gilts on days 110, 113, and 116, but only on day 110 in pregnant animals. These hormones consistently increased (P less than 0.05) progesterone release only on day 110 in both groups of gilts. PGE1 increased (P less than 0.01) relaxin release in hysterectomized, but not pregnant, gilts; it was not effective in altering progesterone in either group. PGE2 increased the release of both hormones on day 113 in both groups. The percentage of large cells (i.e. greater than 20 microns in diameter) was consistently greater (P less than 0.01) on days 110-116 in hysterectomized compared with pregnant gilts. Both luteal cell relaxin concentrations and the population of electron-dense granules decreased from days 110-116 in pregnant, but not in hysterectomized, gilts; luteal cells from postpartum animals were nearly depleted of granules, and empty vesicles were prominent. The secretory response of the cells to tropic hormone stimulation is preprogrammed from the previous estrus that occurred more than 110 days previously.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of prolactin secretion in the pig: central effects of relaxin and the antiprogesterone RU 486.

Our previous study has shown that oral administration of a potent progesterone antagonist, RU 486, caused a marked elevation of plasma concentrations of both PRL and progesterone in hysterectomized pigs bearing aging corpora lutea. Hysterectomized pigs (hysterectomy performed on day 8; estrus = day 0) were subjected to cranial surgery for chronic placement of a head-mounted stereotaxic apparatus for intracerebroventricular (icv) administration of relaxin (300 U once daily on days 111 and 113; n = 6) and RU 486 (4 mg once daily on days 111, 113, and 115; n = 5) to test whether relaxin and RU 486 exert their actions within the central nervous system and/or pituitary gland to affect PRL and GH secretion. Control pigs (n = 3) received icv injection of vehicle. Intensive blood sampling revealed that icv injection of relaxin on day 111 markedly increased the plasma PRL concentration from 8 to 38 ng/ml within 10 min (P < 0.01). An identical icv injection of relaxin on day 113 caused only a modest increase in PRL, but the overall mean concentration of PRL after relaxin treatment was greater than that before treatment (14 vs. 8 ng/ml; P < 0.05). Intracerebroventricular injection of RU 486 on day 111 greatly elevated plasma PRL. The increase in PRL lasted more than 2 h, with several peak increases of 18-29 ng/ml (P < 0.01). The PRL response to subsequent icv infusion of RU 486 on days 113 and 115 was blunted, but the overall mean concentration of PRL (14 ng/ml) after icv injection of RU 486 remained greater (P < 0.01) than that before treatment (9 ng/ml). In contrast, PRL concentrations in the control group remained unchanged after injection. Plasma concentrations of GH, relaxin, and progesterone were significantly altered in neither hormone- nor vehicle-treated groups during this brief period of sequential blood sampling. This study provides strong evidence that relaxin has a central role in modulating PRL secretion in the pig. In addition, the antagonistic effects on progesterone receptor by RU 486 in the central nervous system and/or pituitary gland caused an abrupt increase in PRL secretion in these hysterectomized gilts.

Relaxin on induction of parturition in beef heifers.

Purified porcine relaxin (3000 U/mg) was administered into the cervical os of primiparous beef heifers on day 278 of gestation (approximately 5 days before parturition normally occurs) to determine its effects on the induction of parturition, changes in progesterone, estrone (E1), 17 beta-estradiol (17 beta-E2), cervical dilation, and pelvic relaxation. Heifers were assigned randomly to 1 of 3 treatments: relaxin-double (two infusions of 3000 U, 12 h apart; n = 17), relaxin-single (3000 U; n = 14), and PBS-gel vehicle (n = 16). Relaxin induced marked earlier calving (P less than 0.002) than PBS-gel vehicle. The intervals between the administration of relaxin or the PBS-gel vehicle and calving were 2.0, 2.5, and 5.3 days for heifers given relaxin-double, relaxin-single, and PBS-gel vehicle, respectively. The duration of gestation was significantly reduced (P less than 0.002) in relaxin-treated heifers compared with that in control heifers. A precipitous decrease in progesterone (7.1 ng/ml) occurred in peripheral blood plasma within 24 h after relaxin treatment. Coincident with a decline in levels of progesterone, E1 and 17 beta-E2 increased by 1700 and 400 pg/ml, respectively, an increase of 35% compared with the 12% increase in these steroids in control heifers. Mean deviations of cervical dilation increased 643%, 526%, and 11% in heifers given relaxin-double, relaxin-single, and PBS-gel vehicle, respectively. Relaxin induced maximum pelvic opening between 12-36 h after treatment. Although relaxin induced significantly earlier calving, there was no incidence (0 of 31 heifers) of retained placenta. We conclude from this study that purified relaxin administered intracervically to primiparous beef heifers during late pregnancy induced premature parturition. Marked shifts of progesterone, E1, 17 beta-E2, pelvic canal expansion, and cervical relaxation reflect the premature parturition induced by relaxin.

Characterization of the circulating form of porcine relaxin: biological activity and terminal amino acids.

Relaxin is structurally related to insulin, and it induces pregnancy-related changes in the reproductive tract of several mammalian species. Relaxin isolated from the ovaries of pregnant sows has been used for primary structure determination, for much of the biological characterization of the hormone, and for the development of RIAs. Immunoreactive (IR) relaxin is found in peripheral blood during pregnancy in pigs and other species, but it has not been established that the substance identified by RIA is structurally or biologically equivalent to the native ovarian hormone. IR relaxin was, therefore, isolated from peripheral plasma of late pregnant gilts (days 112-114) for bioassay and determination of terminal amino acid residues. IR relaxin was monitored by a specific homologous RIA during fractionation of plasma by gel filtration, cation exchange, and hydrophobic binding to octadecysilica. IR relaxin circulates unbound and is equipotent with ovarian relaxin in the mouse pubic ligament bioassay. Amino acids released from IR relaxin by pyroglutamic aminopeptidase and carboxypeptidase-Y were converted to their 4-dimethylamino-azo-4'-sulfonyl derivatives for identification by HPLC. The B-chain of IR relaxin had an amino-terminal pyroglutamic acid. Amino acids sequentially released from the carboxy-terminal indicated a chain length of 28-30 amino acids, suggesting a heterogeneity reminiscent of that of ovarian relaxin isolated by other methods. Arginine was released from the free amino-terminal by dimethylaminozaobenzene-isothiocyanate degradation, indicating an intact A-chain of 22 amino acids. Blood immunoreactive relaxin in pigs is, therefore, a secreted biologically active form of relaxin with an amino acid composition similar to that of the form stored in the corpus luteum.