A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.     

Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.     

Oxytocin and estradiol concentrations in follicular fluid as a means for the classification of large bovine follicles

Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F(2)alpha 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol>/=100 ng/ml and oxytocin<65 pg/ml (preovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol<100 ng/ml and oxytocin>/=65 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol<100 ng/ml and oxytocin<65 pg/ml (atretic follicles). Treatment with prostaglandin F(2)alpha significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol: progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF(2)alpha was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles.     

A comparison of hormone levels in follicle-lutein-cysts and in normal bovine ovarian follicles

Insulin-like growth factors 1 and 2 (IGF-1 and 2), oxytocin, progesterone, estradiol and ubiquitin were measured in bovine follicle-lutein-cysts and in follicular fluid after the classification of ovarian follicles by size (Class I = <4 mm; Class II = 5-8 mm; Class III = 9-12 mm; Class IV = preovulatory; Class V = cystic). It was found that IGF-1 concentrations increased during growth from 280 ng/ml in small follicles to 489 ng/ml in preovulatory follicles; IGF-2 appeared to remain constant in follicular fluid and in cysts (275 ng/ml). Oxytocin values were low in Class I, II and III follicles (30 pg/ml) but increased in preovulatory and cystic follicles (75 pg/ml). Estradiol increased significantly only in preovulatory follicles. Ubiquitin, a protein reflecting cellular replicative activity, could be found in bovine follicular fluid in high concentrations: 1.6 mug/ml in Class I,II and III follicles with the highest amounts in preovulatory follicles (2.3 mug/ml). In contrast with normal follicles, cysts were found to have a minimal concentration of ubiquitin (0.3 mug/ml). Progesterone levels were 5 times higher in cysts (325 ng/ml) and IGF-1 concentrations were markedly higher in cystic follicles (881 ng/ml) than in the other follicles. Simultaneously, maximum gene expression for IGF-1 was found in granulosa/lutein cells of cystic follicles (Class V), suggesting de novo synthesis of IGF-1. Between the different follicle classes progesterone, oxytocin and IGF-1 concentrations correlated positively (r=0.82). Hormonal levels in follicle-lutein-cysts indicated an arrested stage of insufficient luteinization as a possible result from the premature release of LH or from the release of amounts of LH inadequate to cause ovulation.     

Gonadotropin surge induces two separate increases in messenger RNA for progesterone receptor in bovine preovulatory follicles

In mice deficient in progesterone receptor (PR), follicles of ovulatory size develop but fail to ovulate, providing evidence for an essential role for progesterone and PR in ovulation in mice. However, little is known about the expression and regulation of PR mRNA in preovulatory follicles of ruminant species. One objective of this study was to determine whether and when PR mRNA is expressed in bovine follicular cells during the periovulatory period. Luteolysis and the LH/FSH surge were induced with prostaglandin F(2alpha) and a GnRH analogue, respectively, and the preovulatory follicle was obtained at 0, 3.5, 6, 12, 18, or 24 h after GnRH treatment. RNase protection assays revealed a transient increase in levels of PR mRNA, which peaked at 6 h after GnRH and declined to the time 0 value by 12 h and a second increase at 24 h. The second objective was to investigate the mechanisms that regulate PR mRNA expression through in vitro studies on follicular cells of preovulatory follicles obtained before the LH/FSH surge. Theca and granulosa cells were isolated and cultured with or without a luteinizing dose of LH or FSH, progesterone, LH + progesterone, or LH + antiprogestin (RU486). Levels of PR mRNA increased in a time-dependent manner in granulosa cells cultured with LH or FSH and in theca cells cultured with LH, peaking at 10 h of culture. In contrast, progesterone (200 ng/ml) did not upregulate mRNA for its own receptor, and neither progesterone nor RU486 affected LH-stimulated PR mRNA accumulation. Furthermore, RU486 completely blocked LH-stimulated expression of oxytocin mRNA, indicating that PR induced by LH in vitro is functional. These results show that the gonadotropin surge induces a rapid and transient increase in expression of PR mRNA in both theca and granulosa cells of bovine periovulatory follicles followed by a second rise close to the time of ovulation and that the first increase in PR mRNA can be mimicked in vitro by gonadotropins but not by progesterone. These results suggest multiple and time-dependent roles for progesterone and PR in the regulation of periovulatory events in cattle.     

Second messenger systems in the action of LH and oxytocin on porcine endometrial cells in vitro

LH/hCG as well as oxytocin receptors are present in the porcine endometrium. Oxytocin increases phosphatidylinositol hydrolysis in this tissue, but its action on adenylate cyclase activity is disputed. The second messenger system responding to LH/hCG in endometrial cells has not been established. In this study, we investigated the involvement of protein kinase A and C signaling mechanisms in the action of LH on porcine endometrial cells in vitro. The possibility of cAMP accumulation after treatment of endometrial cells with oxytocin was also investigated. Endometrial tissue was obtained from gilts during Days 12-15 of the estrous cycle. To study the adenylate cyclase system, endometrial cells were cultured for 48 h and then incubated with different doses of LH or oxytocin for 15, 30, 60, and 180 min. To study the phospholipase C system, dispersed cells were first labeled with myo-[3H]inositol and then treated with increasing doses of LH or 100 nM of oxytocin for 30 min. Time- and dose-dependent effect of LH and oxytocin on cAMP concentration was observed. After 30 min of incubation only the highest dose of LH (100 ng/ml) was able to increase cAMP concentration in medium (P < 0.05). Longer periods (1 and 3 h) caused increased cAMP accumulation after treatment with 10 and 100 ng/ml of LH (P < 0.001). Oxytocin-stimulated cAMP concentration was observed after 1 h when only the highest dose (1000 nM) of hormone was used (P < 0.01) and after 3 h of incubation with doses of 10-1000 nM (P < 0.01). LH (10 and 100 ng/ml) increased inositol phosphates (IPs) accumulation in endometrial cells after 30 min of incubation (P < 0.01). Oxytocin involvement in IPs synthesis was more apparent than was LH (P < 0.001 versus P < 0.01). This is the first demonstration that LH receptor signaling leads to increased cAMP generation as well as IPs turnover in porcine endometrium. Oxytocin-dependent cAMP production in endometrial cells of swine was found after longer periods (3 h) of incubation. Our observations lead to the conclusion that both protein kinase A and C second messenger systems are involved in LH action and that oxytocin is able to stimulate adenylate cyclase activity in porcine endometrial cells.     

Insulin-like growth factor I enhances gonadotropin-releasing hormone-stimulated luteinizing hormone release from bovine anterior pituitary cells

The role of insulin-like growth factor I (IGF-I) in the release of luteinizing hormone (LH) is unclear in ruminants. In the present study, the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) were examined in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17beta (E(2)) and IGF-I was characterized. GnRH(100nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36h after addition of IGF-I (250ng/ml), with a maximum at 12h (48.4ng/ml media versus 35.4ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500ng/ml increased the release by 18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2ng/ml media). E(2) (10nM), IGF-I (250ng/ml) and combined treatment of E(2) plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E(2) alone was 37.3% greater than with IGF-I alone (39.0ng/ml media versus 28.4ng/ml media) (P<0.05). When E(2) and IGF-I were added together (45.6ng/ml media), the release of LH was significantly greater than with either E(2) alone or IGF-I alone (P<0.05). E(2) (10nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250ng/ml) failed to increase GnRH binding. These results show that IGF-I enhances GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interacts with E(2) to increase the response to GnRH.     

Naloxone evokes large-amplitude GnRH pulses in luteal-phase ewes

Ewes were sampled during the mid-late luteal phase of the oestrous cycle. Hypophysial portal and jugular venous blood samples were collected at 5-10 min intervals for a minimum of 3 h, before i.v. infusions of saline (12 ml/h; N = 6) or naloxone (40 mg/h; N = 6) for 2 h. During the 2-h saline infusion 2/6 sheep exhibited a GnRH/LH pulse; 3/6 saline infused ewes did not show a pulse during the 6-8-h portal blood sampling period. In contrast, large amplitude GnRH/LH pulses were observed during naloxone treatment in 5/6 ewes. The mean (+/- s.e.m.) amplitude of the LH secretory episodes during the naloxone infusion (1.07 +/- 0.11 ng/ml) was significantly (P less than 0.05) greater than that before the infusion in the same sheep (0.54 +/- 0.15 ng/ml). Naloxone significantly (P less than 0.005) increased the mean GnRH pulse amplitude in the 5/6 responding ewes from a pre-infusion value of 0.99 +/- 0.22 pg/min to 4.39 +/- 1.10 pg/min during infusion. This episodic GnRH secretory rate during naloxone treatment was also significantly (P less than 0.05) greater than in the saline-infused sheep (1.53 +/- 0.28 pg/min). Plasma FSH and prolactin concentrations did not change in response to the opiate antagonist. Perturbation of the endogenous opioid peptide system in the ewe by naloxone therefore increases the secretion of hypothalamic GnRH into the hypophysial portal vasculature. The response is characterized by a large-amplitude GnRH pulse which, in turn, causes a large-amplitude pulse of LH to be released by the pituitary gland.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Restoration of endocrine and ovarian function after stopping GnRH antagonist treatment in goats

We have tested if the high number of unfertilized ova and degenerated embryos found in superovulated goats previously treated with GnRH antagonist can be related to a prolongation of gonadotrophin down-regulation and/or alterations in follicular function during the period of administration of the superovulatory treatment, around 4 days after the end of the antagonist treatment. A total of 15 does were treated with intravaginal progestagen sponges and daily injections of 0.5mg of the GnRH antagonist Antarelix for 6 days, while 5 does acted as controls receiving saline. During the antagonist treatment, the mean plasma LH concentration was lower in treated than control goats (0.5 +/- 0.2 versus 0.7 +/- 0.5 ng/ml, P < 0.0005 ); however, the FSH levels remained unaffected (0.8 +/- 0.4 versus 0.8 +/- 0.5 ng/ml). In this period, treated does also showed an increase in the number of small follicles with 2-3 mm in size ( 10.7 +/- 0.7 versus 8.4 +/- 0.6, P < 0.05), and a decrease in both the number of follicles > or =4 mm in size ( 5.0 +/- 0.3 versus 6.8 +/- 0.5, P < 0.005) and the secretion of inhibin A (120.9 +/- 10.7 versus 151.6 +/- 12.6 pg/ml, P < 0.05). After cessation of the antagonist treatment, there was an increase in LH levels in treated goats from the day after the last Antarelix injection (Day 1), so that LH levels were the same as controls on Day 3 (0.6 +/- 0.1 versus 0.6 +/- 0.2 ng/ml). However, there were even greater numbers of small follicles than during the period of antagonist injections (15.4 +/- 0.6 in treated versus 8.9 +/- 0.7 in control, P < 0.0005 ). Moreover, the number of > or =4 mm follicles and the secretion of inhibin A remained lower in treated goats (3.9 +/- 0.3 follicles and 84.4 +/- 7.0 pg/ml versus 5.4 +/- 0.5 follicles, P < 0.05 and 128.9 +/- 14.2 pg/ml, P < 0.05 ). These results indicate that pituitary secretion of gonadotrophins is restored shortly after the end of antagonist treatment, but activity of ovarian follicles is affected.     

Exogenous GnRH induces ovulation in seasonally anoestrous lactating goats (Capra hircus)

A specific sheep LH radioimmunoassay was validated for the measurement of goat LH, and used to monitor luteal-phase LH episodes and the preavulatory LH surge in progestagen sponge-synchronized cycling goats. No luteal-phase LH episodes were detected during 12 h of frequent (15-min) blood sampling in 2 goats. A preovulatory LH surge was recorded in 5/5 goats, with a mean amplitude of 45.4 +/- 7.2 ng/ml and a mean time of onset of 38.4 +/- 1.2 h after removal of a progestagen-impregnated sponge. In anoestrous goats, single i.v. injections of 1000 and 2000 ng GnRH induced LH episodes with a mean amplitude of 2.04 +/- 0.11 and 3.67 +/- 0.06 ng/ml respectively, but injections of 250 or 500 ng did not consistently elevate LH concentrations. Progestagen-primed, seasonally anoestrous lactating goats were treated with repeated injections of 1500 ng GnRH (every 2 h for 52 or 78 h) in May 1985 or 1986. All 10 had kidded in March of the same year, and were consequently at peak lactation at the time of GnRH treatment. A preovulatory LH surge was detected in 9 goats with a mean time of onset of 59.5 +/- 2.9 h (1985) or 39.6 +/- 3.3 h (1986) after vaginal sponge removal. All animals displayed oestrus and ovulated, and 9 of the goats were mated: in 5 of these animals pregnancies were successfully carried to term. The results show episodic LH release in response to GnRH and indicate that ovulation can be induced in seasonally anoestrous goats, even at peak lactation, and normal pregnancies may result.     

Effect of suckling on basal and GnRH-induced LH release in post-partum dairy buffaloes

Suckling, a common practice in smallholder dairy-farming systems in the developing world, delays the onset of post-partum ovarian activity in dairy buffalo. The present study was designed to assess the effect of suckling on pituitary function in lactating buffaloes 25-35 days post-partum. Six suckled and nine non-suckled buffaloes were challenged intravenously with a bolus injection of GnRH (20microg buserelin acetate; Receptal). Heparinized venous blood samples were collected at 15min intervals for 2h before and up to 4h after GnRH for luteinizing hormone (LH) estimation. Pretreatment basal LH concentrations were similar in the suckled (0.6+/-0.2ng/ml) and the non-suckled (0.5+/-0.1ng/ml) buffaloes. All but one suckled buffaloes released a LH surge, starting 15-60min post-GnRH treatment, which lasted for 180-225min. While one suckled buffalo did not respond to GnRH, the LH response in the remaining suckled buffaloes was significantly less than in the non-suckled buffaloes in terms of peak LH concentrations (14.3+/-2.7ng/ml versus 26.2+/-4.3ng/ml) and area under the LH curve (1575.6+/-197.4mm(2) versus 2108.9+/-323.9mm(2)). The LH response was least in suckled buffaloes challenged with GnRH while in the luteal phase of an oestrus cycle and with plasma progesterone concentration >1ng/ml. In conclusion, suckling suppressed pituitary responsiveness to exogenous GnRH challenge in post-partum buffaloes.     

Effect of oxytocin infusion on secretion of progesterone and luteinizing hormone and the concentration of uterine oxytocin receptors during the periovulatory period in cloprostenol-treated ewes.

Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH.

The pattern of GnRH-like stimuli capable of inducing follicular growth, ovulation, and luteal function was evaluated in ewes passively immunized against GnRH. The estrous cycles of 30 regularly cyclic sheep were synchronized using vaginal pessaries impregnated with a synthetic progestogen. Animals were passively immunized against GnRH (groups 2-5, n = 6) or the carrier protein, keyhole limpet hemocyanin (KLH; group 1, n = 6), at the time of pessary removal (PR). Circhoral delivery of saline (groups 1, 2, and 5) or low amplitude GnRH agonist (des-Gly10 GnRH ethylamide [100 ng/hourly pulse]; groups 3 and 4) was initiated at PR and continued for 3 (groups 4 and 5) or 12 days (groups 1-3). In groups 4 and 5, the amplitude of the GnRH-like stimulus was increased to 800 ng/hourly pulse (stimulus-shift) during the 24-h period beginning 72 h after PR. The amplitude of the hourly stimulus was adjusted to 100 ng/pulse 96 h after PR and continued at that level to Day 12. The endocrine changes associated with follicle growth and maturation (serum concentrations of estradiol [E2] above 10 pg/ml), ovulation (surge-like secretion of LH and FSH), and normal luteal function (serum concentrations of progesterone [P] above 2 ng/ml) were evident in ewes passively immunized against KLH (group 1). In this group, the preovulatory surge of gonadotropins was noted 48.7 +/- 1.2 h after PR. These endocrine events were blocked by passive immunization against GnRH (group 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influences of GnRH, oxytocin and vasoactive intestinal peptide on LH and PRL secretion by porcine pituitary cells in vitro.

The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n = 8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n = 8) and Group 3 (n = 8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h before slaughter; and in Group 4 (n = 9) with progesterone (P4) at the dose 120 mg/ 100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (10(6)/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37 degrees C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37 degrees C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p < 0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p < 0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p < 0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals.     

Failure of naloxone to stimulate luteinizing hormone secretion during pregnancy and steroid treatment of ovariectomized beef cows

The response of serum luteinizing hormone (LH) to naloxone, an opiate antagonist, and gonadotropin-releasing hormone (GnRH) was measured in cows in late pregnancy to assess opioid inhibition of LH. Blood samples were collected at 15-min intervals for 7 h. In a Latin Square arrangement, each cow (n = 6) received naloxone (0, 0.5, and 1.0 mg/kg BW, i.v.; 2 cows each) at Hour 2 on 3 consecutive days (9 +/- 2 days prepartum). GnRH (7 ng/kg body weight, i.v.) was administered at Hour 5 to all cows on each day. Mean serum LH concentrations (x +/- SE) before naloxone injection were similar (0.4 +/- 0.1 ng/ml), with no serum LH pulses observed during the experiment. Mean serum LH concentrations post-naloxone were similar (0.4 +/- 0.1 ng/ml) to concentrations pre-naloxone. Mean serum LH concentrations increased (p less than 0.05) following GnRH administration (7 ng/kg) and did not differ among cows receiving different dosages of naloxone (0 mg/kg, 1.44 +/- 0.20; 0.5 mg/kg, 1.0 +/- 0.1; 1.0 mg/kg, 0.9 +/- 0.1 ng/ml). In Experiment 2, LH response to naloxone and GnRH was measured in 12 ovariectomized cows on Day 19 of estrogen and progesterone treatment (5 micrograms/kg BW estrogen: 0.2 mg/kg BW progesterone) and on Days 7 and 14 after steroid treatment. On Day 19, naloxone failed to increase serum LH concentrations (Pre: 0.4 +/- 0.1; Post: 0.4 +/- 0.1 ng/ml) after 0, 0.5, or 1.0 mg/kg BW.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient uterine refractoriness after oxytocin administration in ewes

Steroid-primed, ovariectomized ewes were treated intravenously with 2 doses of 1 microgram oxytocin at intervals of 1, 2, 4 or 6 h. The initial dose resulted in increases in 13,14-dihydro-15-keto-PGF-2 alpha in the peripheral circulation from 173 to 667 pg/ml within 5 min; subsequent doses caused responses of 23 +/- 1, 23 +/- 6, 54 +/- 12 and 62 +/- 10% respectively of the initial dose. Concentrations of oxytocin receptor in myometrium, caruncular endometrium and intercaruncular endometrium were, respectively, 185 +/- 33, 128 +/- 7 and 105 +/- 14 fmol/mg protein at 2 h after saline injection and 147 +/- 27, 195 +/- 52 and 170 +/- 50 fmol/mg protein at 2 h after administration of 1 microgram oxytocin. The dose of oxytocin administered was shown to raise circulating concentrations to levels characteristic of those observed during spontaneous episodes of release of oxytocin at luteolysis. Oxytocin administration therefore results in transitory uterine refractoriness which may be due to failure of a post-receptor response and this may contribute to the episodic nature of uterine prostaglandin secretion.     

Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I.

Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents. Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion. The two hormones had different patterns of secretion during the course of incubation. OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9. The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak. Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts. Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein. However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea. These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.     

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of ³H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10⁻¹¹ M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED₅₀ = 1.58 ± 0.22 × 10⁻¹⁰ M) and native GnRH (ED₅₀ = 1.4 ± 0.3 × 10⁻⁶ M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10⁻⁸ M could prevent the inhibition elicited by 10⁻⁷ M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc.     

Effects of oxytocin alone and in combination with selected hypothalamic hormones on ACTH, beta-endorphin, LH and PRL secretion by anterior pituitary cells of cyclic pigs

Oxytocin (OT) is involved in the stimulation of secretion of anterior pituitary hormones in females during the periovulatory and periparturient periods. In the present study we examined the role of OT in control of ACTH, beta-endorphin, LH and PRL secretion in vitro from dispersed anterior pituitary cells collected from gilts during the luteal (Days 10-12; n=6) and follicular (Days 18-20; n=5) phases of the estrous cycle. Isolated anterior pituitary cells (1 x 10(6)/ml) were transferred into 24-well plates, separately for each animal, and were pre-incubated for three days at 37 degrees C in atmosphere of 5% CO(2) and 95% air. The cells which attached to the dishes were incubated (3.5 h, 37 degrees C) in McCoy's medium in the absence (control) or in the presence of the following factors: CRH alone (10(-10), 10(-9), 10(-8), 10(-7) M), OT alone (10(-8), 10(-7), 10(-6) M), LVP alone (10(-7) M), OT (10(-7) M) plus CRH (10(-9) M) and LVP (10(-7) M) plus CRH (10(-9) M) for studying ACTH and beta-endorphin secretion; OT alone (10(-8), 10(-7), 10(-6) M), GnRH alone (100 ng/ml), CRH alone (10(-9) M), OT (10(-7) M) plus GnRH (100 ng/ml) and OT (10(-7) M) plus CRH (10(-9) M) for studying LH and PRL secretion. Concentrations of the studied hormones in media were analyzed by RIA. Oxytocin alone increased ACTH (at doses 10(-7), 10(-6) M), beta-endorphin (at dose 10(-8) M), LH (at dose 10(-8) M) and PRL (at doses 10(-7), 10(-6) M) secretion by pituitary cells isolated only from luteal-phase gilts. None of the studied hormone concentrations in the medium was increased in response to OT when pituitary cells of follicular-phase gilts were examined. Oxytocin in combination with CRH exerted an additive effect on beta-endorphin secretion during the luteal phase. Summarizing, in the present study the stimulatory effect of oxytocin on ACTH, beta-endorphin, LH and PRL secretion by pituitary cells isolated from gilts during the luteal phase was demonstrated. However, the cells collected from follicular-phase gilts appeared to be unresponsive to OT. Moreover, interaction between OT and CRH in affecting beta-endorphin secretion was shown. These results suggest that OT may be transiently involved in the modulation of anterior pituitary hormone secretion in cyclic pigs.