Ginkgo,fennel, and flaxseed can affect hormone release by porcine ovarian cells and modulate the effect of toluene

Experimental studies have documented the toxic effects of toluene on the mammalian female reproductive processes. The aim of this in vitro study was to examine the potential of functional food plant extracts, namely, of ginkgo, fennel, and flaxseed, in modifying the toluene-induced effects on ovarian hormone release. Porcine granulosa cells were incubated with ginkgo, fennel, or flaxseed extracts (0, 1, 10, or 100 µg/mL) and/or toluene (10 µg/mL). Enzyme immunoassays were used in order to measure the release of progesterone (P), oxytocin (OT), and prostaglandin F (PGF) in the culture media. Toluene suppressed the release of P and enhanced the release of OT and PGF. All tested plant extracts reduced P and increased OT release, while the PGF output was found inhibited by ginkgo and stimulated by fennel and flaxseed. When the cells were incubated with toluene and each one of the plant extracts, toluene was able to prevent their action on P release, as well as those of fennel and flaxseed on OT and PGF release. Moreover, ginkgo enhanced but fennel or flaxseed prevented the toluene-induced effects on OT and PGF release. These observations (i) document novel aspects of the toluene-induced toxicity; (ii) demonstrate the direct influence of ginkgo, fennel, and flaxseed extracts on the ovarian secretory activity; (iii) inform our understanding of the interrelationship between toluene and the tested plant extracts with regard to their effects on ovarian hormone release; (iiii) demonstrate the ability of fennel and flaxseed to prevent adverse effect of toluene on ovarian hormones.     

Prostaglandin F2 alpha and oxytocin interactions in ovarian and uterine function

The oxytocin-neurophysin gene is expressed in several nontraditional sites within the endocrine system. In the ovary its expression in the corpora lutea is initiated by ovulation. Ovarian oxytocin concentrations reach maximal levels around day 11 of luteal cycle and fall to a nadir at estrus. PGF2 alpha has the capacity to release oxytocin from the corpus luteum, and oxytocin in turn releases PGF2 alpha from the uterine endometrium or decidua. This positive feedback loop between the ovary and the uterus ensures the completion of luteolysis in species that depend on the presence of the uterus for the termination of luteal lifespan. Immunization against oxytocin has been shown to disrupt this loop, resulting in much-prolonged luteal cycles. In primates and other species in which luteal life span is independent of the uterus, an oxytocin PGF2 alpha interaction may take place within the ovary itself. At parturition a related interaction takes place which ensures the expulsion of the fetus and placenta in an orderly manner. Oxytocin of both pituitary and ovarian origin reaches the uterus via its blood supply and binds to two types of receptors: one on myometrial cells, the occupation of which initiates contractions, and the other on decidual cells, the occupation of which initiates prostaglandin generation. This prostaglandin diffuses into the adjacent myometrium and augments the oxytocin-induced contractions. In conjunction with a direct softening effect by prostaglandins on the cervix the augmented contractions achieve the force needed to dilate the cervix and expel the fetus. An additional source of oxytocin during labor may be the placenta, another non-traditional site for the occurrence of oxytocin.     

Interaction between paraventricular nucleus extracts and median eminence extracts on the formation of melanocyte stimulating hormone-release-inhibiting factor

Median eminence extracts (ME) incubated with oxytocin produced the formation of a melanocyte-stimulating hormone-release-inhibiting factor (MSH-R-IF). When extracts of paraventricular nucleus (PVN) were added to that system and incubated together no formation of MSH-R-IF was detected. The effect of PVN extracts is due not to the inactivation of MSH-R-IF, and is proportional to the amount of extract used. PVN extracts were effective in preventing the formation of MSH-R-IF when 150 mU oxytocin were used as substrate but not when 1000 mU were used, suggesting that the mechanism by which the blocking effect is exerted is similar to that of two enzymes competing for the same substrate. It is postulated that MSH-R-IF formation depends of two hypothalamic agents of antagonistic influence acting on oxytocin.     

Prostaglandin F2 alpha-stimulated release of ovarian oxytocin in the sheep in vivo: threshold and dose dependency

To determine the threshold of prostaglandin F2 alpha (PGF2 alpha)-stimulated oxytocin secretion from the ovine corpus luteum, low levels of PGF2 alpha (5-100 pg/min) were infused into the ovarian arterial blood supply of sheep with ovarian autotransplants. PGF2 alpha was infused for six sequential 10-min periods at hourly intervals, 6, 12, or 24 days after estrus (n = 3 for each day). Each cycle day was studied during a separate cycle. Oxytocin and progesterone in ovarian venous and carotid arterial plasma was measured by radioimmunoassay, and secretion rates were determined (venous-arterial concentration x plasma flow). In animals treated on Day 6, 5 pg/min PGF2 alpha caused a significant release of oxytocin (p less than 0.01), whereas in animals treated on Day 12, this threshold was 40 pg/min (p less than 0.05). In animals treated on Day 24, the threshold for oxytocin release was greater than 100 pg/min. PGF2 alpha did not significantly change ovarian blood flow or progesterone secretion rate on any day (p greater than 0.05). To determine residual luteal oxytocin after each threshold experiment, 5 mg PGF2 alpha was given i.m. to all animals. Significantly more oxytocin was released by Day 6 than by Day 12 and Day 24 corpora lutea, and by Day 12 than by Day 24 corpora lutea (1.2 micrograms, 0.7 microgram, and 0.3 microgram, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxytocin secretion induced by osmotic stimulation in rats during the estrous cycle and after ovariectomy and hormone replacement therapy

Hyperosmolality is a potent stimulus for the secretion of oxytocin. Oxytocinergic neurons are modulated by estrogen and oxytocin secretion in rats varies according to the phase of the estrous cycle, with higher activity during proestrus. We investigated the oxytocin secretion induced by an osmotic stimulus (0.5 M NaCl) in female rats. Plasma oxytocin and the oxytocin contents in the neurohypophysis and the paraventricular and supraoptic nuclei were determined during the morning (8-9 h) and afternoon (17-18 h) of the estrous cycle and after ovariectomy followed or not by hormone replacement. Plasma oxytocin peaked in control animals during proestrus. Oxytocin content decreased in the paraventricular and supraoptic nuclei during proestrus and estrus compared to diestrus and increased in the neurohypophysis during proestrus morning. No significant difference was observed in the oxytocin content of the neurohypophysis, nuclei or plasma between ovariectomized animals and ovariectomized animals treated with estrogen or estrogen plus progesterone. Therefore, any ovarian factor other than estrogen or progesterone seems to play a direct or indirect role in the increase in oxytocin secretion. The osmotic stimulus caused an increase in plasma oxytocin throughout the estrous cycle. A reduction in oxytocin content during diestrus and an increase during proestrus were observed in the paraventricular nuclei. In ovariectomized animals, the treatment with estrogen potentiated the response of oxytocin to the osmotic stimulus, with the response being even stronger in the case of estrogen plus progesterone. In conclusion, the ovarian steroids estrogen plus progesterone could modulate the osmoreceptor mechanisms related to oxytocin secretion.     

Enhanced secretion of oxytocin from bovine granulosa cells treated with adrenal steroids

Bovine granulosa cells were exposed in vitro to various adrenal steroids (cortisol, cortisone, corticosterone, aldosterone; 1 mumol/l), in the presence and absence of stimulation by ascorbic acid (0.5 mmol/l), to determine the possible effects of these hormones on ovarian oxytocin and progesterone secretion. Only cortisol produced a consistent stimulation of the cells; the response was dose-related over the range 0.01 to 1.0 mumol/l and was greatly enhanced in the presence of ascorbate. The secretion of oxytocin was stimulated to a greater extent and with more consistency than was that of progesterone. Although the secretion of oxytocin could be stimulated by cortisol on the day of treatment, the cells also showed a delayed and persistent response to exposure earlier in the culture. It is concluded that cortisol may directly stimulate the secretion of ovarian oxytocin in the cow and that granulosa cells may respond in such a way as to smooth out the effects of short-term fluctuations in cortisol concentration.     

Effects of arterial infusions of adrenalin and acetylcholine on luteal secretion of progesterone and oxytocin in goats

The effects of close intra-arterial infusion of acetylcholine and adrenalin on ovarian secretion of progesterone and oxytocin were examined on Day 10 of the estrous cycle in goats (estrus = Day 0). Acetylcholine (15 micrograms/min) was without effect, but adrenalin (10 micrograms/min) significantly (P < 0.001) raised both progesterone and oxytocin concentrations in ovarian vein plasma. These results show that luteal hormone secretion is enhanced in the goat by beta-adrenergic stimulation and suggest that, as in the sheep and cow, there may be neuroendocrine involvement in the regulation of caprine luteal function.     

Regression of the corpora lutea in sheep in response to cloprostenol is not affected by loss of luteal oxytocin after hysterectomy

Concentrations of oxytocin in corpora lutea were reduced from 1706 to less than 15 ng/g wet wt after hysterectomy in sheep during the oestrous cycle. Hysterectomy also blocked the appearance of raised levels of oxytocin in ovarian and jugular venous plasma caused by cloprostenol. Administration of cloprostenol to hysterectomized ewes resulted in luteal regression, which occurred as rapidly as in intact animals. Therefore oxytocin in the corpus luteum during the oestrous cycle is unlikely to be involved in intraluteal events mediating prostaglandin-induced luteolysis.     

Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. Effects of these hormones on progesterone secretion in vitro

The relative levels of mRNAs for relaxin, prolactin, inhibin and oxytocin have been measured in porcine granulosa as well as luteal cells by hybridisation to single-stranded synthetic DNA. The likelihood of a paracrine function of oxytocin and prolactin in the porcine ovary was inferred from the in vitro effects of both hormones on progesterone secretion of ovarian cells. Both hormones were found to inhibit progesterone secretion of luteal cells. In contrast, only prolactin but not oxytocin stimulated progesterone secretion in granulosa cells.     

Variation in the oxytocin content of caprine corpora lutea across the breeding season

Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season.     

Role of oxytocin in activation of spontaneous electrical activity of uterine body and uterine tubes in non-pregnant rats

The work studies effects of various doses of oxytocin (0.01, 0.1, 1 and 10 microg/kg) on duration of discharges of spontaneous electrical activity and frequency of spikes in various parts of uterine tubes and of uterine body of non-pregnant rats. Under these conditions, changes in these parameters for ovarian parts of the uterine tubes had similar character unlike those in cervical parts of the tubes and in the middle part of the uterine body, so the latter parts can be grouped together owing to peculiarities of their changes. The longest duration of genesis of electric discharges has been shown for the ovarian part of uterine tubes at a concentration of 10 microg/kg of oxytocin. Morphological experiments revealed that among all studies areas the ovarian parts of uterine tubes were characterized by the highest amount of atypical cells that have the maximally pronounced functional activity.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

Oxytocin in the semen and gonads of the stallion

It has been suggested that oxytocin is involved in sperm transport and motility in domestic animals. Immunoreactive oxytocin was measured in seminal fractions (pre-ejaculatory fluid, seminal plasma, gel and sperm) and in extracts of testis and epididymis from stallions. In addition, sections of gonadal tissue from stallions were immunostained for the presence of oxytocin and its neurophysin. Oxytocin was detected in all of the seminal fractions, being highest in the gel. It was also present in washed, lysed sperm and in extracts from the testis and epididymis. Immunostaining for oxytocin was present in occasional interstitial cells in the testis and in the epididymal epithelium and smooth muscle. However, immunostaining for neurophysin was detected in a few interstitial cells in the testis of only 1 of 8 stallions and was absent from all areas of the epididymis. These data demonstrate for the first time the presence of oxytocin in stallion semen and gonadal tissue; however, lack of immunostaining for neurophysin indicated that it was unlikely that there was local synthesis within the gonads.     

Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture

Bovine granulosa cells were cultured in collagen (Vitrogen 100)-pretreated wells using defined medium to study the secretion of oxytocin and progesterone under serum-free conditions. Secretion of oxytocin began spontaneously after the first day and was maintained at a high level during a 1 week culture period. Addition of serum to the medium reduced oxytocin concentrations by up to 90%. There were positive exponential relationships between oxytocin and progesterone concentrations and the inoculated cell density (range, 1.67 to 23.4 X 10(5) cells/ml/well). The results indicate that neither serum nor gonadotrophins are required for in vitro differentiation of bovine granulosa cells and that addition of serum may attenuate subsequent hormone secretion. This culture system should provide a better in vitro model for the study of ovarian oxytocin secretion than those previously described.     

Heterogeneity in the third intracytoplasmic region of the oxytocin receptor-encoding gene

The oxytocin receptor (OTR), a member of the seven-transmembrane domain guanine-nucleotide-binding protein (G protein) coupled receptor family plays a central role in lactation, ovarian cyclicity and reproductive behaviour. Recent cloning and sequencing unexpectedly revealed that the third intracytoplasmic region ('3ICR) of the sheep receptor has 3 and 2 additional amino acids (aa) relative to the rat and human receptors, respectively. We have now confirmed, by sequencing polymerase chain reaction (PCR)-derived genomic fragments coding for the OTR 3ICR from a variety of ruminant and non-ruminant species, that additional aa are a general phenomenon in ruminants.     

Local exchange of oxytocin from the ovarian vein to ovarian arteries in sheep

Local transfer of 125I-labeled oxytocin from the ovarian vein to arteries supplying the ovary, the oviduct, and the tip of the uterine born has been investigated. In five sheep, 10 infusions of 125I-oxytocin over a period of 1 h were performed, and the concentration of labeled polypeptide in the peripheral plasma was compared to ovarian arterial plasma. During 2 consecutive infusions into each animal's ovarian vein, blood was collected simultaneously from the following sites: ovarian branch of the ovarian artery (OBOA), tubal branch of the ovarian artery (TBOA), uterine branch of the ovarian artery (UBOA), and from the jugular vein. In all experiments the concentration of 125I-oxytocin in ovarian arterial plasma was higher than in peripheral plasma. The ratio of ovarian artery/jugular vein for 125I-oxytocin was: OBOA 2.8, TBOA 1.8, UBOA 1.6. Based on a 4 ml/min blood flow through ovarian arteries supplying ovary, oviduct, and the tip of the uterine horn, the local transfer of the total amount of oxytocin infused was estimated to be about 1% (range: 0.1-4.4%). Analysis of variance did not reveal significant differences in the exchange ratios between OBOA, TBOA, and OBOA. However, the variances within these groups are significant, presumably because of anatomical variation in the degree of surface contact area between arteries and veins at the ovarian pedicle. It is concluded that polypeptides are locally recirculated to ovaries, oviduct, and the tip of the uterine horn in a higher concentration than is supplied by peripheral blood. This could provide a mechanism for local distribution and concentration of the ovarian peptides that regulate reproductive function.     

Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep

Jugular venous concentrations of oxytocin and progesterone changed in parallel during the oestrous cycle in the ewe, falling at luteal regression and rising with formation of the new corpus luteum. These fluctuations in the circulating concentration of oxytocin were not caused by changes in its metabolic clearance rate. On Days 6-9 of the cycle circulating oxytocin concentrations exhibited a diurnal rhythm, peaking at 09:00 h; this rhythm was absent on Days 11-14. Although there was no evidence for increased production of oxytocin at or preceding luteal regression in samples taken daily, more frequent sampling revealed that two thirds of detected surges of uterine secretion of prostaglandin (PG) F-2 alpha were accompanied by raised levels of oxytocin. This oxytocin was not of pituitary origin. Luteal regression induced with cloprostenol on Day 8 after oestrus caused a decrease in circulating progesterone level followed after 24 h by a fall in oxytocin. Measurements of oxytocin in the ovary and other organs before and after treatment with cloprostenol identified the corpora lutea as a major potential source of oxytocin, and suggested that 98% of luteal oxytocin was available for secretion in response to prostaglandin stimulation. The data are consistent with a role for ovarian secretion of oxytocin in response to uterine release of PGF-2 alpha in the control of luteal regression.     

Peripheral administration of oxytocin increases social affiliation in the naked mole-rat (Heterocephalus glaber)

The neuropeptide oxytocin regulates a wide variety of social behaviors across diverse species. However, the types of behaviors that are influenced by this hormone are constrained by the species in question and the social organization that a particular species exhibits. Therefore, the present experiments investigated behaviors regulated by oxytocin in a eusocial mammalian species by using the naked mole-rat (Heterocephalus glaber). In Experiment 1, adult non-breeding mole-rats were given intraperitoneal injections of either oxytocin (1 mg/kg or 10 mg/kg) or saline on alternate days. Animals were then returned to their colony and behavior was recorded for minutes 15–30 post-injection. Both doses of oxytocin increased huddling behavior during this time period. In Experiment 2, animals received intraperitoneal injections of either oxytocin (1 mg/kg), an oxytocin-receptor antagonist (0.1 mg/kg), a cocktail of oxytocin and the antagonist, or saline across 4 testing days in a counterbalanced design. Animals were placed in either a 2-chamber arena with a familiar conspecific or in a small chamber with 1 week old pups from their home colony and behaviors were recorded for minutes 15–30 post-injection. Oxytocin increased investigation of, and time spent in close proximity to, a familiar conspecific; these effects were blocked by the oxytocin antagonist. No effects were seen on pup-directed behavior. These data suggest that oxytocin is capable of modulating affiliative-like behavior in this eusocial species.