Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.     

Co-operative binding of oxytocin to bovine neurophysin II.

The interaction of oxytocin with bovine neurophysin II in 0.1 M-sodium phosphate, pH 5.8, was investigated by equilibrium-dialysis and sedimentation studies. Sigmoidality of the binding curve is attributed to isomerization, either hormone-induced or pre-existing, with preferential binding of oxytocin to one isomeric state. Results are consistent with a binding equation of the form r = (2P[S]+2PQ[S]2)/(1+2P[S]+PQ[S]2) and values of 0.7 X 10(5)M-1 and 1.3 X 10(5)M-1 for P and Q respectively. The significance of these two parameters in relation to current theories of allostery is also discussed.     

Sequence redesign and the assembly mechanism of the oxytocin/bovine neurophysin I biosynthetic precursor

The structural organization of neurohypophysial hormone biosynthetic precursors and the interdependence between intramolecular folding and precursor self-association were examined using sequence-engineered mutants of the semisynthetic oxytocin/bovine neurophysin precursor (pros-OT/BNPI). In [N alpha 1-Ac,N epsilon 30,71-diacetimidyl, Ala2,des-His106] Pro-Ot/BNPI or [N alpha 1-Ac,Ala2]pros-OT/BNPI), two structural elements (Tyr2 and free alpha-amino group) were eliminated which were predicted to be critical for intramolecular conformation by stabilizing contact between hormone and neurophysin domains. This mutant was used to test the dependence of precursor self-association on intramolecular conformation. In the second mutant precursor, [N alpha 30,71-diacetimidyl,D-Pro7,D-Leu8,des-His106]p ro-OT/BNPI (or [D-Pro7,D-Leu8]pros-OT/BNPI), the stereochemistry at L-Pro7-L-Leu8 was changed to test the extent to which precursor conformation depends on ordered structure in the processing/spacer sequence which connects the interacting hormone and neurophysin I domains. Intramolecular conformation was characterized for the precursor and mutants by analytical affinity chromatography on immobilized hormone analog Met-Tyr-Phe and by circular dichroism. Data obtained by both methods showed that, while pros-OT/BNPI is folded, with hormone domain occupying the hormone-binding site of the neurophysin domain, the alpha-acetyl-Ala2 mutant is not so organized intramolecularly. When pros-OT/BNPI and the alpha-acetyl-Ala2 mutant were eluted on immobilized BNPII to measure self-association propensity, the native-like precursor was found to bind with 12-15-fold higher affinity than the assembly mutant. Thus, while pros-OT/BNPI assumes a molecular structure containing a high-affinity self-association surface induced by intramolecular hormone domain-neurophysin domain interaction, [N alpha 1-Ac,Ala2]pros-OT/BNPI does not. The results with the alpha-acetyl-Ala2 mutant show that intramolecular domain-domain interaction is the obligatory "trigger" which induces the high-affinity precursor self-association that likely drives precursor to aggregated forms in the concentrated intragranular environment that exists in peptide hormone-synthesizing cells. In contrast, affinity chromatographic and circular dichroism properties of the D-Pro7,D-Leu8 mutant show that this intramolecular trigger is dependent, but only weakly, on the conformation of the peptide sequence between domains, as judged by native-like interaction properties below 40 degrees C but lowered stability to elevated temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of neurophysin on enzymatic maturation of oxytocin from its precursor

We examined the extent to which rates of enzymatic conversion of the oxytocin biosynthetic precursor to mature peptide are modulated by intramolecular and intermolecular assembly of precursor and polypeptide intermediates. The biosynthesized precursor contains hormone and neurophysin sequences linked by a Gly-Lys-Arg sequence and undergoes enzymatic processing reactions which include endoproteolytic cleavage at the Lys-Arg dibasic sequence, carboxypeptidase B-like exoproteolytic cleavage, and enzymatic amidation. We evaluated the effect of neurophysin on such processing reactions using semisynthetic precursors of oxytocin/bovine neurophysin I and synthetic oxytocinyl precursor intermediates as substrates. Neurophysin I at high concentration (0.7 mM) reduced the rates of carboxy-peptidase B-like conversion of oxytocinyl-Gly-Lys-Arg to oxytocinyl-Gly and the enzymatic amidation of oxytocinyl-Gly to mature (C-terminal amidated) oxytocin. The dependence of rate suppression on the concentrations of peptide substrate and neurophysin I suggested that suppression is due to intermolecular formation of hormone-neurophysin complexes which are aggregated at least to dimers. An analogous intramolecular neurophysin effect was found for endoproteolytic processing of semisynthetic precursors. Endoproteinase Lys-C cleaved the Lys11-Arg12 peptide bond in a native-like semisynthetic precursor at a significantly slower rate than it did an assembly-deficient precursor analogue. The difference in semisynthetic precursor endoproteolysis rates is most substantial at the high concentrations at which the native-like precursor would form dimers but the assembly-deficient analogue would not. The native-like semisynthetic precursor was more stable than the assembly-deficient precursor analogue to tryptic digestion. The concentration-dependent effects of neurophysin, both intramolecularly as a precursor domain and intermolecularly as an interacting protein, are likely to occur in the secretory granules in which the biosynthetic precursors are packaged. The molecular organization of both hormone/neurophysin precursors and the noncovalently complexed hormone-neurophysin intermediates can be expected to play a role in modulating enzymatic processing reactions that lead to mature neurohypophysial hormones.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localisation of oxytocin and neurophysin in the ovine corpus luteum

Summary Polyclonal rabbit antisera raised against oxytocin, bovine neurophysin I and vasopressin were used, together with an immunogold complex, to localise the peptides in ultrathin sections of ovine corpus luteum. The only organelle which consistently showed gold labelling was the secretory granule of the large luteal cell. In non-consecutive sections of the same large luteal cell all the granules showed a similar level of labelling after oxytocin or neurophysin I antisera: however no immunolabelling was detected for vasopressin. Oxytocin and neurophysin seem to be rapidly lost after secretion since exocytosed granule cores showed no labelling above background levels.     

Immunocytochemical localization of oxytocin and neurophysin in human corpora lutea

Corpora lutea, corpora albicantia, and ovarian stroma from normal human premenopausal ovaries were examined for the presence of oxytocin and neurophysin by using highly specific antisera and peroxidase-antiperoxidase light-microscopic immunohistochemistry. Oxytocin and neurophysin immunoreactivity was found in some but not all cells of the corpora lutea obtained on days 19 to 24 of the menstrual cycle. Stromal tissue and corpora albicantia did not give a positive reaction for either of these peptides, and negative results were also obtained with corpora lutea of mid- and term-pregnancy and preovulatory follicles. Specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive oxytocin and neurophysin in corpora lutea of the human menstrual cycle directly demonstrates the presence of oxytocin- and neurophysin-positive cells within the human corpus luteum.     

Immunocytochemical localization of neurophysin and oxytocin in ovine corpora lutea

The cellular distribution of neurophysin and oxytocin within ovine corpora lutea obtained on Days 4, 10 and 16 of the estrous cycle was examined immunocytochemically. Serial sections (8-10 micron-thick) prepared from corpora lutea that had been fixed in Bouin's solution and embedded in paraffin were immunostained for neurophysin or oxytocin using the peroxidase-antiperoxidase (PAP) procedure. Irrespective of the day of the cycle examined, immunoreactivity was restricted to large luteal cells. However, on Days 4 and 10 of the cycle, the intensity of staining in large luteal cells was highly variable; and, within the same section some cells were heavily stained, others were only lightly stained, and still others were not stained at all. In contrast, on Day 16 of the cycle, the intensity of staining was uniform and essentially all of the large luteal cells were immunoreactive. Based on the results obtained, it is evident that immunoreactive neurophysin and oxytocin can be detected as early as Day 4 of the cycle, persists through Day 15, and is restricted to large luteal cells.     

Oxytocin,oxytocin-associated neurophysin and the oxytocin receptor in the human prostate

Oxytocin has been implicated in the regulation of prostate growth. However, the cellular localisation of oxytocin in the normal and diseased human prostate is not known. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were detected by immunohistochemistry in tissues from patients undergoing routine prostatectomy and in normal human prostate epithelial and stromal cell lines. Western blot analysis detected a single band at 14 kDa with neurophysin antiserum and a 66-kDa band with oxytocin receptor antiserum in epithelial and stromal cell lines. Similar sized bands were also detected in extracts of hyperplastic and adenocarcinomic prostate tissues. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were present in stromal and epithelial cell lines and in tissue from patients with benign prostatic hyperplasia. The peptides were localised predominantly to the epithelial cells, although discrete areas of stromal staining were also observed. There was a significant difference in the intensity of oxytocin-staining between tissue displaying benign prostatic hyperplasia and invasive carcinoma, with less immunoreactivity being present in the malignant epithelial cells. Thus, oxytocin and its neurophysin and receptor are present in epithelial and stromal cells of the human prostate. Oxytocin expression is reduced with tumour progression and may provide a marker for invasive disease.This work was supported by a Project Grant (007756) from the Wellcome Trust and from Lottery Health Research     

Hypothalamic modulation of immunoreactive oxytocin in the rat thymus

Immunoreactive oxytocin was determined in a peptidic extract of rat thymus by means of a highly specific radioimmunoassay combined with high pressure liquid chromatography fractionation. Rat thymus was found to contain 80 +/- 7.5 pg/g wet tissue (congruent to 0.56 pg/mg protein) of oxytocin-like immunoreactivity, which behaved like synthetic oxytocin in the radioimmunoassay and in two different high pressure liquid chromatography columns. Oxytocin concentration was increased by bilateral electrolytic lesion of the paraventricular nucleus of the hypothalamus (PVN), and by high doses of corticosterone (10 mg/kg IM for 7 days) but was not modified by low doses of corticosterone (1 mg/kg IM for 7 days) or by hypophysectomy. The results suggest that rat thymus synthesizes oxytocin and that thymic oxytocin concentration is modulated by the hypothalamus.     

Evidence for an oscillator other than luteinizing hormone controlling the secretion of progesterone in cattle

The objective of this study was to quantify and compare the frequencies of pulses in ovarian and systemic concentrations of progesterone, systemic concentrations of luteinizing hormone (LH) and rate of ovarian blood flow. Blood was collected simultaneously from previously implanted catheters in the ovarian venous circulation and jugular vein on Day 12 or 13 of estrous cycles from 4 nonlactating dairy cows. Blood was collected at a rate of 2.5 ml/min for 5 min out of every 10 min over an 8-h period. The mean rate of blood flow in the ovarian artery during the 5-min collection period was estimated by an electromagnetic blood flow transducer. Pulses were observed over time in both ovarian and systemic concentrations of progesterone at frequencies that ranged between 0.625 and 0.875 cycles/h (1.1 to 1.5 h/cycle) among the animals. Only one or two episodes of release of LH were observed during the 8-h period, and transient increases in blood flow to the ovaries were associated temporally with each episode of LH release. The estimated frequencies for release of LH and increased blood flow were the same for each animal and ranged between 0.250 and 0.375 cycles/h. A second cycle with a frequency similar to that for LH was evident in the spectral density functions for ovarian and systemic concentrations of progesterone. This cycle was eliminated when the cycle for LH was removed from the data for progesterone, but the magnitude and frequency of the pulses in progesterone were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

Source of immunoreactive inhibin in the chicken ovary.

High concentrations of immunoreactive inhibin were detected in the plasma of the laying domestic hen using a heterologous RIA validated for use in the chicken. Cessation of egg production induced by restricting the intake of nutrients decreased circulating inhibin to approximately 20% of its original concentration within 8 days, indicating that the ovary is the major source of the measured material. Dissection of ovarian follicles revealed that inhibin is nearly exclusively produced in the granulosa cell layer. When expressed per milligram cell protein the concentration of inhibin decreased significantly in granulosa layers of follicles of succeeding order in the hierarchy (F4 to F1). The concentration of progesterone increased in the granulosa layers of the same follicles whereas oestradiol in the surrounding theca layers decreased. In vitro culture of granulosa cells derived from follicles at different stages of development confirmed the decrease in inhibin secretion as a function of follicular growth observed in vivo. The granulosa cell inhibin secretion is stimulated by LH as well as by FSH, the former being the most effective one. The physiological significance of these changes in inhibin concentration during follicular maturation requires further investigation. It may be concluded, however, that the chicken presents a useful model for the study of the endocrine as well as the paracrine function of ovarian inhibin.     

Extension of oestrous cycles and prolonged secretion of progesterone in non-pregnant cattle infused continuously with oxytocin

The experimental objective was to evaluate how continuous infusion of oxytocin during the anticipated period of luteolysis in cattle would influence secretion of progesterone, oestradiol and 13,14-dihydro-15-keto-prostaglandin F-2 alpha (PGFM). In Exp. I, 6 non-lactating Holstein cows were infused with saline or oxytocin (20 IU/h, i.v.) from Day 13 to Day 20 of an oestrous cycle in a cross-over experimental design (Day 0 = oestrus). During saline cycles, concentrations of progesterone decreased from 11.0 +/- 2.0 ng/ml on Day 14 to 2.0 +/- 1.3 ng/ml on Day 23; however, during oxytocin cycles, luteolysis was delayed and progesterone secretion remained near 11 ng/ml until after Day 22 (P less than 0.05). Interoestrous interval was 1.6 days longer in oxytocin than in saline cycles (P = 0.07). Baseline PGFM and amplitude and frequency of PGFM peaks in blood samples collected hourly on Day 18 did not differ between saline and oxytocin cycles. In Exp. II, 7 non-lactating Holstein cows were infused with saline or oxytocin from Day 13 to Day 25 after oestrus in a cross-over experimental design. Secretion of progesterone decreased from 6.8 +/- 0.7 ng/ml on Day 16 to less than 2 ng/ml on Day 22 of saline cycles; however, during oxytocin cycles, luteolysis did not occur until after Day 25 (P less than 0.05). Interoestrous interval was 5.9 days longer for oxytocin than for saline cycles (P less than 0.05). In blood samples taken every 2 h from Day 17 to Day 23, PGFM peak amplitude was higher (P less than 0.05) in saline (142.1 +/- 25.1 pg/ml) than in oxytocin cycles (109.8 +/- 15.2 pg/ml). Nevertheless, pulsatile secretion of PGFM was detected during 6 of 7 oxytocin cycles. In both experiments, the anticipated rise in serum oestradiol concentrations before oestrus, around Days 18-20, was observed during saline cycles, but during oxytocin cycles, concentrations of oestradiol remained at basal levels until after oxytocin infusion was discontinued. We concluded that continuous infusion of oxytocin caused extended oestrous cycles, prolonged the secretion of progesterone, and reduced the amplitude of PGFM pulses. Moreover, when oxytocin was infused, pulsatile secretion of PGFM was not abolished, but oestrogen secretion did not increase until oxytocin infusion stopped.     

Evidence for a receptor-mediated endocytosis of Alu I in Chinese hamster ovary cells

Pretreatment of Chinese hamster ovary cells with proteases or with NaN3 leads to less chromosomal aberrations when the cells are posttreated with Alu I compared to the treatment of cells with Alu I alone. The same result is obtained when the cells are treated with Alu I at 0 degree C instead of 37 degrees C. The cells recover from the protease treatment when they are kept in medium before treatment with Alu I. These results are interpreted to mean that Alu I is bound by surface receptors and that the Alu I-receptor complexes are internalized by an energy-dependent endocytotic process.     

Evidence for the presence of oxytocin in the ovine epididymis

The testes of several species contain oxytocin and/or neurophysin, but the content or localization of oxytocin in epididymal tissue has not been studied. The present study was undertaken to localize oxytocin and neurophysin in epididymal tissue of the ram, and to quantify oxytocin in the ductus epididymidis and fluids entering and leaving the ductus epididymidis. Neurophysin was not detected in the epididymis; thus, synthesis of oxytocin by the epididymis is unlikely. Immunohistochemical localization of oxytocin was confined to the epithelium and capillaries. Oxytocin immunostaining was most intense for epithelium of the caput and declined in corpus and cauda regions. However, based on radioimmunoassay, no difference in oxytocin concentration was detected among regions of the epididymis. Since rete testis fluid entering and cauda epididymal fluid leaving the epididymis contained at least fourfold more oxytocin than testicular venous plasma, it was concluded that regional differences in epithelial concentration of oxytocin may have been masked by oxytocin contained in the luminal fluid. It was concluded further that the epididymis of the ram does not synthesize oxytocin, but about 22 ng/day enters the epididymis in rete testis fluid. Most of this luminal oxytocin apparently is absorbed by the epithelium of the caput epididymidis, with additional adsorption in the corpus and cauda. Although a role for oxytocin in ductal contractility cannot be excluded, it is more likely that the luminal oxytocin influences epithelial or sperm function.     

Impact of ether anesthesia on the hypophyseal content of oxytocin neurophysin I and II: a comparative study with ketamine in the rat

The effect of anesthetic stress on the major hormones of the posterior pituitary (PP), such as oxytocin (OT), oxytocin-neurophysin (OTNP-I) and its metabolic product, OTNP-II, was studied. Rats were treated with either a combination of atropine (0.87 mg/kg) and diphenylhydantoin (85 mg/kg) and then anesthetized with ketamine (42 mg/kg) or were directly anesthetized with diethyl-ether, and then killed. Controls were killed with a laboratory guillotine. Our study revealed that 1.) animals killed with a guillotine or being medicated with our drug combination prior to sacrifice had similar concentrations of OT, OTNP-I and OTNP-II per PP and ml of blood; 2.) animals anesthetized with either prior to sacrifice had a decreased concentration of neuropeptides per PP; the blood concentration of OT was 1.6 times higher than in animals treated with the drug combination or killed directly with a guillotine. In addition plasma concentrations of OTNP-I and OTNP-II were above the baseline. We conclude that ether is not an adequate anesthetic for studying the neurophysins from the PP in vivo. Treatment of animals with atropine and diphenylhydantoin in combination with ketamine does not alter the profile of the major hormones from the PP during anesthetic stress.