We have studied the action of GH on the production of hormones, growth factors, growth factor-binding protein and the occurrence of apoptosis in bovine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these effects. For this purpose we investigated the effects of exogenous bovine GH (0.001-10 microgram/ml), PKA blockers KT5720 (100 ng/ml) and adenosine-3',5'-monophosphothiodate (Rp-cAMPS) (1 micromol), alone and in combination, on IGF-I, IGF-binding protein (IGFBP)-3, oxytocin, progesterone and estradiol secretion, cAMP and PKA content and the occurrence of apoptosis.The secretion of hormones, IGF-I and IGFBP-3 into the culture medium was measured using RIA/IRMA. The presence of PKA was detected using immunocytochemistry and Western immunoblotting. The presence of cAMP in cells was demonstrated using immunocytochemistry, whilst the proportion of apoptotic cells was determined by the TUNEL method.It was found that the addition of GH to the culture medium strongly (P<0.05) stimulated IGF-I (at a concentration of 0.001-10 microgram GH/ml medium), IGFBP-3 (0.001-1 microgram GH/ml) and oxytocin (0.01-10 microgram GH/ml) secretion. Low concentrations (1-100 ng/ml) of GH stimulated, whilst a higher concentration (10 microgram/ml) inhibited estradiol output. GH slightly (P<0.05) inhibited progesterone (1-100 ng GH/ml) secretion and significantly (P<0.05) decreased the incidence of apoptosis (0.01-1 microgram GH/ml) in cultured cells. The addition of GH (100 ng/ml) caused a dramatic (P<0.05) increase in the proportion of cells possessing the immunoreactive catalytic subunit of PKA and a slight decrease in the proportion of cells containing the regulatory PKA subunit.PKA blockers KT5720 and Rp-cAMPS significantly (P<0.05) reduced the proportion of granulosa cells containing cAMP, and the catalytic and (in the case of KT5720) regulatory subunits of PKA. KT5720 given alone significantly (P<0.05) inhibited the secretion of IGFBP-3, but not that of IGF-I or progesterone. Rp-cAMPS decreased (P<0.05) the secretion of oxytocin but not that of estradiol output or the occurrence of apoptosis. KT5720 and Rp-cAMPS fully or partially prevented the GH effect on IGF-I, IGFBP-3, oxytocin, progesterone, estradiol and apoptosis.These observations suggest the involvement of GH and a cAMP/PKA-dependent intracellular cascade in the control of IGF-I, IGFBP-3, oxytocin, progesterone, estradiol, cAMP and apoptosis in bovine ovarian granulosa cells. The stimulation of PKA by GH and the prevention of GH-induced effects by PKA blockers suggest that the observed GH effects on bovine ovarian cells are probably mediated by the cAMP/PKA system.
E Hydbring, A Madej, E MacDonald, G Drugge-Boholm, B Berglund, and K Olsson
Parturition is a natural event that involves stress and pain for the mother. We thus hypothesized that levels of stress hormones measured during parturition could reflect levels reached in response to severe discomfort and pain of other kinds as well. The aim of this study was therefore to determine whether plasma concentrations of cortisol, adrenaline, noradrenaline, beta-endorphin, met-enkephalin, vasopressin and oxytocin vary depending on the phase and severity of labour in dairy heifers (ten) and dairy goats (six), and how these hormones interact with each other. Blood samples were taken once a day for 3 days before labour and for 3 days afterwards and at predetermined phases during labour. All heifers delivered one calf and five of them needed obstetrical assistance. Two of the goats delivered one kid, and four had twins; all kidded without help. The cortisol concentration peaked when the calf and the first kid were born. In the heifers, plasma adrenaline increased after delivery, while the noradrenaline concentration did not change significantly in heifers that needed assistance, but increased during expulsion in heifers calving without help. In the goats, adrenaline and noradrenaline concentrations increased in association with expulsion of the first kid. The beta-endorphin concentration increased during labour in goats. In heifers that needed assistance, beta-endorphin concentration increased 1 h after labour but there was no change in heifers that did not need assistance. The met-enkephalin concentration was elevated during expulsion in heifers and fluctuated in the goats. Both oxytocin and vasopressin increased during expulsion in both groups of heifers, but vasopressin increased four times more in heifers needing assistance. In the goats, oxytocin reached its highest levels just as the feet of the first kid became visible, and vasopressin peaked as the head emerged. Parturition took longer in heifers that needed assistance than in those that did not. It is concluded that, even though the pattern of change differed between hormones during labour, the changes were related to the phases of labour. A longer labour therefore meant that the hormone concentrations stayed elevated for longer. Vasopressin reached high levels in goats and was the only hormone for which plasma concentrations were higher in heifers that needed assistance than in those that did not, indicating that this hormone is released in order to deal with the pain-related stress associated with labour.
SJ Yankey, BA Hicks, KG Carnahan, AM Assiri, SJ Sinor, K Kodali, JN Stellflug, JN Stellflug, and TL Ott
Interferon-tau (IFN tau) acts locally on the endometrium to suppress estrogen and oxytocin receptor expression and block luteolysis in ruminants. Systemic administration of conceptus homogenates or recombinant ovine IFN tau does not block luteolysis or enhance pregnancy rates in sheep or cattle, respectively. However, IFN tau up-regulates expression of the antiviral protein Mx throughout the entire uterine wall during early pregnancy. These studies determined if conceptus-derived IFN tau also up-regulates Mx expression in components of the circulating immune system that migrate through the endometrial wall. In experiment one, peripheral blood mononuclear cells (PBMC) were isolated from ewes at D26 post-artificial insemination (AI) and Mx mRNA levels examined by Northern and slot-blot hybridization. Pregnancy resulted in a two-fold increase in Mx mRNA levels compared to bred, non-pregnant ewes at D26. In experiment two, PBMC were isolated from ewes at AI, and every three days from D9 to D30. Results showed a four-fold increase in Mx mRNA levels in PBMC from pregnant versus bred, non-pregnant ewes at D15. Increased Mx mRNA, which remained elevated through D30, was accompanied by increased levels of Mx protein. These results show that pregnancy recognition signaling rapidly induces Mx gene expression in PBMC, and are the first to suggest that IFN tau activates gene expression in components of the circulating immune system.
CB Brenninkmeijer, SA Price, A Lopez Bernal, and S Phaneuf
There is evidence for hormonal receptor desensitisation in human myometrium, but little is known about the mechanisms involved in the loss of myometrial response to agonists such as beta(2)-adrenergic agonists, prostaglandin gamma and oxytocin. It is well known that the receptors for these hormones are coupled to G-proteins. The first step of receptor desensitisation is the phosphorylation of activated receptors by a G-protein-coupled receptor kinase (GRK). GRKs are members of a multigene family and the various subtypes differ in their localisation, regulation and mode of action. We have used Western blotting and reverse transcription PCR to identify the GRKs present in human myometrium from pregnant and non-pregnant women as well as in cultured human myometrial cells. We have found that human myometrium expresses the GRK subtypes 2, 4gamma, 5 and 6. On the other hand, GRK3 and the isoforms GRK4alpha, beta and delta were not found in myometrial tissue. Our data indicate that GRK2 is only expressed in pregnant term myometrium and is not found in non-pregnant tissue. Moreover, GRK6 appears to be expressed at a much higher level in pregnant term tissue than in non-pregnant myometrium. Our observations suggest that GRK2 and GRK6 may contribute to the regulation of uterine contractility at term. Further work is necessary to determine whether GRKs and receptor desensitisation play a role in disorders of uterine contractility.
S Asai, R Ohta, M Shirota, A Tohei, G Watanabe, and K Taya
Hatano high-avoidance (HAA) and low-avoidance (LAA) animals were originally selected from Sprague-Dawley rats for good and poor active avoidance learning in a shuttle box. We studied the endocrinological profile in lactating rats to determine the effect of suckling during mid-lactation in HAA and LAA rats. The pups were separated from their mother rats 6 h before the onset of suckling and blood samples were drawn from unanaesthetized mother rats via a jugular cannula at 0, 5 and 15 min after the suckling stimulus and then 15, 45 and 105 min after pups were removed. Plasma concentrations of oxytocin in HAA rats were significantly higher than in LAA rats during the suckling period. Plasma concentrations of prolactin and ACTH in HAA rats were significantly higher than in LAA rats during the suckling period, and at 15 min and 45 min after the pups were removed. However, there were no strain differences in circulating corticosterone between the two lines, indicating that the response of the hypothalamo-pituitary axis to the suckling stimulus was greater in HAA rats than in LAA rats, whereas the ACTH-induced adrenal response of corticosterone release was higher in LAA rats than in HAA rats. Since dopamine from the median eminence inhibits prolactin secretion from the lactotrophs of the anterior pituitary, and tuberoinfundibular dopaminergic neurones are partially regulated by the level of circulating prolactin, we evaluated the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. TH, measured by the accumulation of 3,4-dihydroxyphenylalanine, was significantly higher in HAA rats than in LAA rats before the suckling stimulus. After the suckling stimulus, TH activity in HAA rats was significantly lower than before suckling, whereas TH activity in LAA rats was not changed. These findings clearly demonstrated that apparent differences between the two Hatano lines exist in endocrinological profiles during suckling. These strain differences probably originate from neurotransmitter changes, such as dopamine.
WX Wu, XH Ma, Q Zhang, and PW Nathanielsz
Our objective was to examine the topology-, gestation- and labor-related changes of estrogen receptor (ER)alpha, progesterone receptor (PR), oxytocin receptor (OTR) and thrombospondin-1 (TSP1) mRNA in pregnant baboon myometrium. ER alpha, PR, OTR and TSP1 mRNAs extracted from the lower uterine segment and fundal myometrium of pregnant baboons not in labor between 121 and 180 days of gestational age (n=9) and in established spontaneous labor between 164 and 193 days of gestational age (n=5) were analyzed by Northern blot. There were no topology-, gestation- or labor-related changes of ER alpha and PR mRNA in or between the lower uterine segment or/and the fundus. OTR mRNA was the same in the lower uterine segment and the fundus from baboons not in labor and non-labor fundal, but not lower uterine segment, myometrial OTR mRNA increased with gestation (R(2)=0.81, P<0.05). Fundal OTR mRNA rose significantly compared with the lower uterine segment during spontaneous labor. TSP1 mRNA increased significantly in both the fundus and lower uterine segment during labor. TSP1 mRNA in the lower uterine segment during spontaneous labor was higher than in the fundus during spontaneous labor. In conclusion, fundal and lower uterine segment ER alpha and PR mRNA remained unchanged in late gestation and spontaneous labor. The increased OTR mRNA may serve as a mechanism to increase uterine sensitivity to OT during late gestation. The higher fundal OTR mRNA compared with the lower uterine segment provides polarity which assists fetal expulsion by uterine contractions during labor. The significance of increased TSP1 mRNA during labor may relate to homeostasis and merits further study.
ME Guibbolini and M Avella
Neurohypophysial hormone receptors were studied in primary cultures of sea bass (Dicentrarchus labrax) gill respiratory-like cells grown on permeable supports. This preparation was previously shown to provide a functional model for investigating the hormonal regulation of Cl- secretion. Under control conditions, the cultured monolayered epithelium had a short-circuit current (ISC) of 3.5+/-1.1 micro A x cm(-2). This current had previously been identified as an active Cl- secretion. The addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the ISC. Maximal increases of 60.9+/-12.1% and 117.7+/-28.0% above the basal ISC value were obtained for 10(-7) M AVT and IT respectively. Half-maximal effects were obtained for 3.1 x 10(-9) M AVT and for 1.4 x 10(-9) M IT. Mucosal application of 1.0 mM diphenylalamine-2-carboxylic acid (a specific blocker of Cl- channels) after serosal addition of 5 x 10(-8) M AVT or IT inhibited not only the basal but also the stimulated current, revealing a correlation with a hormone-dependent Cl- transport. Specific V1 or V2 receptor analogues of vasopressin (mammalian hormone) were used to characterize the type of neurohypophysial hormone receptors pharmacologically. While the V1 agonist [Phe2,Orn8]-oxytocin stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist [Deamino1,Val4,d -Arg8]-vasopressin had no effect. The V1 antagonist [d(CH2)5 1,O-Me-Tyr2,Arg8]-vasopressin used at a concentration of 5 x 10(-7) M totally reversed the 10-8 M AVT-stimulated Cl- secretion, whereas the V2 antagonist [d(CH2)5 1,d -Ile2,Ile4,Arg8,Ala9]-vasopressin used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10(-8) M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with the efficiency of the V1 receptor antagonist being significantly greater than that of the V2. This study provides evidence for neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests that on a physiological basis the hormonal effect is shared by the two peptides present in fish neurohypophysis (AVT and IT), acting by means of two distinct, although pharmacologically similar, V1-type receptors (according to the mammalian classification). These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish.
S Kobayashi, B Berisha, WM Amselgruber, D Schams, and A Miyamoto
The newly formed corpus luteum (CL) rapidly develops after ovulation and has the features of active vascularisation and mitosis of steroidogenic cells. These stage-specific mechanisms also may contribute to gain the function of prostaglandin F2 alpha (PGF2 alpha)-resistant CL at this stage. Recent studies suggest that the vasoactive peptide angiotensin II (Ang II) regulates luteal function. Thus, this study aimed to investigate (i) the expression of angiotensin-converting enzyme (ACE) mRNA by RT-PCR and the ACE protein expression by immunohistochemistry, (ii) the effects of angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), on the secretion of Ang II, PGF2 alpha, progesterone and oxytocin (OT), and (iii) the effects of luteal vasoactive peptides (Ang II and endothelin-1 (ET-1)) or OT on the secretion of PGF2 alpha, progesterone and OT from bovine early CL (days 3--4 of the oestrous cycle), and evaluate a possible interaction of these substances with PGF2 alpha. The expression of mRNA for ACE was found in theca interna of mature follicle, early CL and endothelial cells from developing CL as well as pituitary and kidney, but granulosa cells of mature follicle were negative. The immunohistochemical analysis revealed that blood capillaries (endothelial cells) were stained for ACE, but luteal cells were negative in early CL. To examine the effects of substances on the secretory function of the CL, an in vitro microdialysis system was used as a model. The infusion of bFGF and VEGF stimulated Ang II and PGF2 alpha secretion as well as progesterone, but not OT secretion in early CL. The infusion of Ang II after PGF2 alpha infusion continued the stimulatory effect on progesterone and OT release within early CL until 3 h thereafter. However, the infusion of ET-1 alone had no effect on progesterone or OT release. The infusion of luteal peptides such as Ang II and OT stimulated PGF2 alpha secretion, whereas the infusion of ET-1 did not. In conclusion, the overall results of this study indicate that a functional angiotensin system exists on the endothelial cells of early CL, and that angiogenic factors bFGF and VEGF upregulate luteal Ang II and PGF2 alpha secretion, which fundamentally supports the mechanism of progesterone secretion in bovine early CL. This idea supports the concept that the local regulatory mechanism involved in active angiogenesis ensures the progesterone secretion in the developing CL in vivo.
AV Sirotkin, AV Makarevich, J Kotwica, PG Marnet, HB Kwon, and L Hetenyi
The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.
Z Cheng, RS Robinson, PG Pushpakumara, RJ Mansbridge, and DC Wathes
Dietary polyunsaturated fatty acid (PUFA) intake in humans can affect the incidence of a variety of diseases including coronary heart disease. Feeding high PUFA diets to cows can alter the PUFA content of milk for human consumption. PUFAs supply the precursors for prostaglandin (PG) synthesis and PGs in turn influence many aspects of reproduction. This study examined the effects of a control (CONT), a high n-6 PUFA diet (derived from protected soya) and a high n-3 diet (derived from protected linseed) on uterine PG synthesis in the lactating dairy cow. Endometrial explants obtained on days 15-17 of the oestrous cycle were cultured for an initial 42 h in vitro in fully defined medium (basal production) and then challenged with control medium, oxytocin (OT; 20 or 200 nM) or calcium ionophore A23187 (CaI; 10 microM). PGF(2 alpha), PGE(2) and 6-keto-PGF(1 alpha) were measured in the spent medium. The experiments were repeated using tissue from two groups of cows, nine in Experiment 1 (three cows per diet) and seven in Experiment 2 (four CONT and three n-6). Results of the two experiments were consistent. The basal concentrations of all three PGs were significantly lower (>50% reduction) in the n-6-fed group in comparison with CONT and n-3 groups. The n-3 diet did not alter basal PGF(2 alpha) and PGE(2) but increased 6-keto-PGF(1 alpha). The n-6 diet also inhibited the ability of the tissue to respond to both OT and CaI, with significant reductions in the stimulated levels of all three PGs. In contrast, the n-3 diet only had minor effects; it did not alter the response to OT but did reduce the long-term response to CaI at 24 h post treatment. In conclusion, dietary PUFA intake can inhibit PG production in bovine endometrial explants, with a more pronounced effect following n-6 rather than n-3 supplementation. These data suggest that a high n-6 diet reduces the endometrial capacity to produce PGs and may therefore have implications for the control of luteolysis and other PG-mediated events such as ovulation.