RESUMEN
The aims of this study were to investigate the role of inhibin in the distribution of healthy and atretic antral follicles and the secretion patterns of gonadotrophins. Ewes were actively immunized against either alphaN or alphaC of the inhibin alpha subunit with a primary injection and three booster injections. The control ewes received adjuvant only. The ovaries were removed either before or at 24 h after hCG administration in a synchronized follicular phase 48 h after removal of intravaginal progesterone pessaries. Morphological observations were made on every fifth section of the complete ovary (one per ewe) stained with haematoxylin and eosin. The mean number of corpora lutea observed per ewe with corpora lutea was not significantly different in ewes immunized against alphaN (2.4; alphaN-immunized ewes) or alphaC (2.6; alphaC-immunized ewes), and control (2.4) ewes, although some corpora lutea appeared cystic in the immunized ovaries. Compared with luteal phase concentrations, mean basal FSH concentrations in the early follicular phase were significantly increased in the alphaC-immunized ewes, similar in alphaN-immunized ewes and reduced in control ewes. No differences were observed in any of the LH parameters. Before hCG treatment, healthy antral follicles > 1 mm in diameter were not observed in any of the 52 follicles in the aC-immunized ewes and were observed in one of 37 follicles from alphaN-immunized ewes compared with 19 of 28 follicles in control ewes (P < 0.0001). For healthy antral follicles < 1 mm in diameter, there were 72 of 85 follicles in the alphaC-immunized ewes, 79 of 81 follicles in the alphaN-immunized ewes and 81 of 82 follicles in the control ewes. Similar results were obtained in healthy antral follicles < 1 mm in diameter at 24 h after hCG administration. In contrast to the control ewes, no healthy preovulatory follicles (> 6 mm in diameter) were observed in alphaN- and alphaC-immunized ewes either before or 24 h after hCG administration. Two newly formed corpora lutea from alphaC-immunized ovaries contained retained oocytes compared with none in control and alphaN-immunized ovaries. In conclusion, immunization against alphaN and alphaC may result in disruption of the normal processes of antral follicular growth and maturation independent of the concentrations of FSH and LH.
Asunto(s)
Atresia Folicular/fisiología , Gonadotropinas Hipofisarias/metabolismo , Inmunización , Inhibinas/inmunología , Folículo Ovárico/fisiología , Fragmentos de Péptidos/inmunología , Ovinos/fisiología , Animales , Anticuerpos/sangre , Apoptosis , Gonadotropina Coriónica/administración & dosificación , Cuerpo Lúteo/anatomía & histología , Femenino , Hormona Folículo Estimulante/metabolismo , Fase Folicular , Células de la Granulosa/citología , Inmunización Secundaria , Etiquetado Corte-Fin in Situ , Fase Luteínica , Hormona Luteinizante/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Células Tecales/citologíaRESUMEN
Whilst there have been many studies in various species examining the effects of leptin on food intake, there is a paucity of data comparing responsiveness in the two sexes. We have, therefore, addressed this issue in sheep. Because this species shows seasonal variation in voluntary food intake (VFI), we also considered the possibility that there might be seasonal variation in the responsivity to leptin. Centrally administered leptin was relatively ineffective as a satiety factor in either sex during AUTUMN: In Spring, leptin had a profound inhibitory effect on VFI in the females, but only a slight effect in males. These data indicate that responsiveness to leptin depends on sex and also on season in animals that are substantially affected by photoperiod.
Asunto(s)
Ingestión de Alimentos/efectos de los fármacos , Leptina/farmacología , Estaciones del Año , Caracteres Sexuales , Animales , Peso Corporal , Femenino , Leptina/administración & dosificación , Masculino , Orquiectomía , Ovariectomía , Fotoperiodo , Saciedad/efectos de los fármacos , OvinosRESUMEN
Ewes actively immunized against alpha N, the N-terminal peptide of inhibin alpha 43 precursor, have lowered fertility associated with ovulation failure, restricted tissue remodelling and reduced matrix metalloproteinase-2 activity in the follicular fluid at the time of expected ovulation. This could be due to altered ratios of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase (TIMP-1), or to the onset of atresia in antral follicles destined to ovulate. The objectives of the present study were to investigate the effects of immunization against alpha N on the localization of TIMP-1 in ovine follicles, and on follicular growth and atresia in the follicular phase. Ewes were either immunized against alpha N or remained as controls and the ovaries were removed before (0, n = 4) and at 12 h (n = 4) and 24 h (n = 4) after hCG administration in a synchronized follicular phase, 48 h after removal of intravaginal pessaries. Observations were made on a single section taken through the largest follicle present in the ovaries of each ewe. There were no healthy antral follicles > 1 mm in immunized ovaries (0/29) compared with controls (16/31) (P < 0.001), whereas the proportion of healthy antral follicles < 1 mm was the same in each group (9/19 versus 5/12). TIMP-1 immunoactivity was localized in large luteal cells, smooth muscle and endothelial cells, and in all antral follicles, including oocytes. At the time of hCG administration, no TIMP-1 immunoreactivity was detected in the apical region of the follicular wall of large follicles (> 6 mm) compared with the rest of the follicle wall, but staining appeared in the apical granulosa layer 24 h later. In newly formed corpora lutea, TIMP-1 expression was found along the invaginating vascular layer. There was no effect of immunization on the patterns of TIMP-1 immunoreactivity, suggesting that changes in TIMP-1 are not involved in the effects of alpha N. These data are consistent with a paracrine role for alpha N in the selection and atresia of antral follicles, and for TIMP-1 in tissue reorganization and steroidogenesis at the time of ovulation.
Asunto(s)
Atresia Folicular , Inhibinas/fisiología , Folículo Ovárico/metabolismo , Precursores de Proteínas/fisiología , Ovinos/fisiología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Análisis de Varianza , Animales , Gonadotropina Coriónica/farmacología , Femenino , Fase Folicular , Inmunización , Inmunohistoquímica , Inhibinas/inmunología , Folículo Ovárico/efectos de los fármacos , Precursores de Proteínas/inmunología , Inhibidor Tisular de Metaloproteinasa-1/análisisRESUMEN
Endothelin, which has potent vasoconstrictor and mitogenic actions, was measured by radioimmunoassay in tissue extracts of sheep endometrium and myometrium and was found to be present in similar amounts in both tissues during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy (250-630 pg g-1 wet weight). Immunoreactive endothelin extracted from endometrium eluted at the same position as standard endothelin-1 on reverse-phase HPLC. Immunohistochemical techniques demonstrated that during the oestrous cycle endothelin immunoreactivity was very low in caruncular and intercaruncular stroma, luminal epithelium, outer and inner glandular epithelium, myometrium and blood vessels until after day 12 (oestrus: day 0). Staining increased in all but the inner glands to day 16 and the most intense staining was found in intercaruncular luminal epithelium and outer glands and in myometrium, although endothelin in tissue extracts did not change over this period. During early pregnancy (days 4-20), staining in intercaruncular areas and in myometrium increased slightly from day 4 to day 12 to a maximum which was maintained from day 15 to day 20. Intensity of staining in caruncles increased only from day 15, particularly in the epithelium. Immunoreactive endothelin was also present in the trophoblast cells of the embryo on day 20 of pregnancy. Strong endothelin immunostaining was observed in uteri from ovariectomized ewes, particularly in epithelial cells and in blood vessels. The intensity of immunostaining in epithelium and epithelial cells and in blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Endotelinas/metabolismo , Estro/metabolismo , Preñez/metabolismo , Ovinos/metabolismo , Útero/metabolismo , Animales , Femenino , Miometrio/metabolismo , Ovariectomía , Embarazo , RadioinmunoensayoRESUMEN
Immunization of ewes against the amino-terminal peptide (alpha N) of the pro-alpha-subunit of inhibin has been shown to reduce fertility, thought to be due to disruption of ovulation. The aims of this study were to examine the effects of active immunization of ewes against alpha N on circulating concentrations of FSH, LH and on ovarian inhibin and progesterone, and to relate these observations to number of corpora lutea and oocyte recovery rates. Ewes were immunized against one or both of two recombinant full length bovine-alpha N immunogens (FP1 and FP2). Three experiments were performed in which jugular venous plasma was sampled from control and immunized ewes: (1) hourly across the oestrous surge of gonadotrophins (Expt 1); (2) daily for one entire oestrous cycle, and in the subsequent cycle, oviducts were flushed to recover ovulated eggs (Expt 2); and (3) samples were taken at 10 min intervals during the follicular and luteal phases (Expt 3). Binding of 125I-labelled alpha N1-26 to serum was greater (P < 0.05) in immunized groups than in controls for all experiments. The number of eggs per corpus luteum recovered from the oviducts was lower (P < 0.05) in the alpha N-immunized groups (39%) than in controls (88%). There were more (P < 0.05) corpora lutea per ewe in FP2 immunized groups 4 (1.8 +/- 0.45) and 5 (1.75 +/- 0.5) than in the control group (1.13 +/- 0.13), but no increase in group 3 (FP1; 1.4 +/- 0.24).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Inhibinas/fisiología , Ovario/fisiología , Hipófisis/fisiología , Animales , Cuerpo Lúteo/efectos de los fármacos , Femenino , Hormona Folículo Estimulante/sangre , Inmunización Pasiva , Inhibinas/sangre , Inhibinas/inmunología , Hormona Luteinizante/sangre , Ovulación/fisiología , OvinosRESUMEN
The aim of this study was to measure the peripheral concentrations of immunoreactive inhibin (ir-inhibin) and progesterone (P) during pregnancy and parturition in the ewe and to relate the concentrations of ir-inhibin to P and to the number and sex of the fetuses. P increased across pregnancy with higher levels in ewes with 2 fetuses (n = 5) than in those with 1 fetus (n = 6), and concentrations falling before birth. ir-Inhibin concentrations were relatively stable during the first 40 days of pregnancy for example (Day 20, 34.7 +/- 2.9 pmol L-1; mean +/- s.e.m., n = 11). After Day 40, inhibin fell in all ewes to reach less than or equal to 2.5 pmol L-1 after Day 80 (mean on Day 103, 6.3 +/- 1.2 pmol L-1), and remained low until 2 days before parturition when concentrations rose sharply, peaking at or around the day of birth in all ewes (21.5 +/- 2.1 pmol L-1). Thereafter, ir-inhibin fell and remained low or undetectable for up to 10 days in the six ewes still being sampled. ir-Inhibin concentrations in ewes carrying one (n = 6), two (n = 5) or three fetuses (n = 1) did not differ at any stage of pregnancy examined. The sex of the fetus did not appear to influence the peripheral concentrations of ir-inhibin in the ewe.
Asunto(s)
Trabajo de Parto/fisiología , Preñez/metabolismo , Análisis de Varianza , Animales , Femenino , Inhibinas/análisis , Tamaño de la Camada , Embarazo , Progesterona/análisis , Radioinmunoensayo , Factores Sexuales , OvinosRESUMEN
Ovariectomized ewes were treated with 100 IU insulin, iv, which caused reductions in blood sugar and plasma LH concentrations. The effect was prevented by the infusion (iv) of glucose, suggesting that neuroglycopenia and not a direct action of insulin was the cause of reduced LH secretion. An iv infusion of naloxone (40 mg/h for 2 h), which commenced 25 min before the insulin injection, blocked the inhibitory effect of insulin on LH secretion, but it did not prevent the decrease in plasma glucose concentrations. In this treatment group and in a group treated only with naloxone, the opioid antagonist significantly stimulated LH secretion. To determine whether CRF might be involved in the insulin-induced decrease in LH secretion, 50 micrograms CRF were injected into ovariectomized sheep. Despite producing very high circulating concentrations of CRF within 2 min of injection and the stimulation of cortisol secretion during most of the 4-h posttreatment period, plasma LH levels were not affected. In addition, the intracerebroventricular administration of 10 micrograms CRF or 10 micrograms CRF plus 10 micrograms arginine vasopressin (AVP) did not affect LH secretion. These observations suggest that insulin-induced hypoglycemia decreased LH secretion by neuroglycopenia. This may involve an opioidergic mechanism, but does not involve activation of the hypothalamo-pituitary-adrenocortical axis.
Asunto(s)
Hipoglucemia/fisiopatología , Insulina , Hormona Luteinizante/metabolismo , Ovariectomía , Animales , Arginina Vasopresina/farmacología , Hormona Liberadora de Corticotropina/administración & dosificación , Hormona Liberadora de Corticotropina/farmacología , Femenino , Glucosa/farmacología , Hidrocortisona/metabolismo , Hipoglucemia/inducido químicamente , Cinética , Hormona Luteinizante/sangre , Naloxona/farmacología , OvinosRESUMEN
The effects of dopamine, noradrenaline and 3,4-dihydroxyphenylacetic acid (DOPAC) on the release of prolactin were examined in ovariectomized ewes. Infusion of dopamine (0.5 or 1 microgram/kg per min for 2 h i.v.) reduced plasma prolactin concentrations in a dose-dependent manner, whereas DOPAC (5 or 10 micrograms/kg per min for 2 h i.v.) had no effect. In a further series of experiments, ovariectomized hypothalamopituitary disconnected ewes were given dopamine or noradrenaline (each at 0.5 or 1 microgram/kg per min for 2 h i.v.), and both amines reduced mean plasma concentrations of prolactin with similar potency in a dose-dependent manner. These effects were blocked by treatment with pimozide and prazosin respectively. During the infusion of dopamine, the peripheral plasma concentrations of DOPAC and 3,4-dihydroxyphenylethyleneglycol (DHPG) were increased (DOPAC, 22 +/- 7 (S.E.M.) to 131 +/- 11 nmol/l; DHPG, 2.9 +/- 0.3 to 6.4 +/- 0.2 nmol/l), but plasma concentrations of dopamine and noradrenaline did not change. Finally, administration of domperidone, a specific dopamine receptor antagonist that does not cross the blood-brain barrier, resulted in a sustained increase in plasma prolactin concentrations in ovariectomized ewes. We conclude that the secretion of prolactin from the pituitary gland is under dual inhibitory regulation by both dopamine and noradrenaline in the sheep.
Asunto(s)
Dopamina/farmacología , Norepinefrina/farmacología , Adenohipófisis/efectos de los fármacos , Prolactina/metabolismo , Ácido 3,4-Dihidroxifenilacético/farmacología , Animales , Femenino , Adenohipófisis/metabolismo , OvinosRESUMEN
A series of experiments was performed to monitor plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin responses to human gonadotropin-releasing hormone (GnRH) associated peptide (GAP) and related peptides. Ovariectomized hypothalamo-pituitary disconnected (HPD) ewes were challenged with injections (1-10 micrograms i.v.) of GAP, or given, with and without estradiol, hourly 500- or 1,000-ng pulses of GAP for 5-7 days. In all cases GAP failed to cause the release of LH or FSH from the pituitary gland or to alter mean plasma prolactin concentrations. When the same HPD ewes were given hourly or 2-hourly pulses of 250 ng GnRH, LH responded in a a pulsatile manner, and FSH secretion was maintained, thus confirming the functional integrity of the pituitary gland after HPD. Fragments of the GAP molecule (pro-GnRH 14-36, 28-36, 38-49, and 51-66) and GAP dimer did not stimulate LH or FSH or inhibit prolactin release in HPD ewes. GAP and GAP dimer did not affect pituitary responsiveness to GnRH administration. GAP also failed to inhibit the thyrotropin-releasing hormone-induced rise in prolactin. Finally, GAP injections (100 micrograms i.v.) given to lactating ewes did not cause any change in plasma prolactin concentrations. These data show that human GAP, GAP dimer, or putative processed GAP peptides do not act on the sheep pituitary gland in a variety of physiological states to regulate gonadotropin or prolactin secretion.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/metabolismo , Prolactina/metabolismo , Precursores de Proteínas/farmacología , Ovinos/metabolismo , Animales , FemeninoRESUMEN
Experiments were conducted in ovariectomized ewes after hypothalamo-pituitary disconnection (HPD) to examine LH and FSH secretion during constant infusion of gonadotrophin-releasing hormone (GnRH) or physiological saline and to determine whether or not a constant GnRH background enhances or diminishes pituitary responsiveness to GnRH pulses. Whereas pulsatile GnRH infusions maintained LH and FSH secretion, constant infusions (125 or 250 ng/h) led to the complete cessation of LH secretion and reduced FSH secretion. The rate of decline of plasma FSH concentrations was significantly (P less than 0.01) greater in animals receiving 250 ng GnRH/h than in saline-treated animals, whereas that in animals receiving 125 ng/h was not significantly different. When GnRH pulses were administered during constant GnRH infusion, the plasma LH pulse amplitudes were similar to those seen without the GnRH background. These data show that, in ovariectomized-HPD ewes FSH secretion does not require GnRH pulses and may merely reflect ongoing FSH synthesis and a constant low background of GnRH does not affect pituitary responsiveness to GnRH pulses.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Sistema Hipotálamo-Hipofisario/fisiología , Hormona Luteinizante/metabolismo , Hormonas Liberadoras de Hormona Hipofisaria/farmacología , Animales , Femenino , Ovariectomía , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , OvinosRESUMEN
Epithelial endometrial cells were isolated on day 13 from nonpregnant (NPr) and pregnant (Pr) ewes and cultured in the presence or absence of conditioned media from 15-day blastocysts (BM) under optimized conditions in the presence of [35S]methionine (S-met). The incorporation of S-met into secreted protein was analyzed, and individual proteins were identified by autoradiography of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Incorporation of S-met into secreted protein was higher (P less than 0.05) for cells from Pr than NPr animals. Secretion by cells from NPr ewes was increased in three of four cases by addition of BM to the culture medium. Autoradiography of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed five secreted proteins [mol wt range 74,000-120,000; isoelectric point (pI) less than 6.5] which were either absent from or present in only small amounts in secretions from cells from NPr animals. The proportion of these proteins was greatly increased in secretions from cells from Pr animals. The presence of BM in cultures from NPr ewes enhanced the secretion of these same proteins, this effect being maintained even after heat treatment of the BM. One of these proteins had previously been shown to be maximally induced by the presence of estrogen and progesterone. Three other similarly controlled proteins were also enhanced, though to a lesser extent, by the presence of a blastocyst in vivo but were not stimulated by BM in vitro. It is concluded that endometrial epithelial cells from Pr ewes are metabolically more active than those from ewes on day 13, and the blastocyst and its secretions induce the secretion of several specific proteins by epithelial cells, some of these proteins being the same as those controlled by the combination of estrogen and progesterone.
Asunto(s)
Blastocisto/fisiología , Desarrollo Embrionario , Endometrio/metabolismo , Biosíntesis de Proteínas , Animales , Células Cultivadas , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Epitelio/metabolismo , Femenino , Cinética , Metionina/metabolismo , Embarazo , Proteínas/metabolismo , OvinosRESUMEN
The inhibitory effects of follicular fluid on FSH secretion were similar in gonadectomized male and female sheep, and in the anoestrous and breeding seasons. Significant suppression of LH was variable and was observed only at the highest dose of follicular fluid when suppression rarely exceeded 50% of pretreatment values. Basal plasma FSH and LH concentrations were higher in castrated males than in ovariectomized females in both seasons. Plasma FSH concentrations in gonadectomized males and females and LH concentrations in the males were lower in the anoestrous than the breeding season. Therefore, in the absence of the gonads, sex and photoperiod can influence hypothalamic control of basal pituitary gonadotrophin secretion in males and females, whereas the feedback effect of non-steroidal factors in follicular fluid (inhibin) on FSH secretion is not influenced by photoperiod or sex.
Asunto(s)
Estro , Hormona Folículo Estimulante/metabolismo , Inhibinas/farmacología , Estaciones del Año , Anestro , Animales , Castración , Depresión Química , Retroalimentación , Femenino , Hormona Folículo Estimulante/sangre , Hormona Luteinizante/sangre , Masculino , Embarazo , Factores Sexuales , OvinosRESUMEN
The effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion of various regimens of pulsatile gonadotropin-releasing hormone (GnRH) replacement were examined in ovariectomized (OVX) ewes after hypothalamo-pituitary disconnection (HPD). Hourly pulses of 500 ng GnRH restored gonadotropin secretion in OVX-HPD sheep. Replacement beginning 2 days after HPD gave consistent responses of LH and FSH within a week. Replacement beginning 61-96 days after HPD caused more gradual re-establishment of LH and FSH secretion with LH responses appearing immediately and FSH responses appearing 2 weeks later. When hourly GnRH pulses were increased in amplitudes from 250 to 500 ng the plasma LH baseline, peak values and pulse amplitudes were increased. There was no significant change in plasma FSH levels over 10 pulses at the higher dose. Decreases in GnRH pulse frequency led to increases in LH pulse amplitude and decreases in plasma LH baseline. In contrast, immediately after a change from a 2-hourly to an hourly mode, an increase in LH baseline occurred without an immediate reduction in LH pulse amplitude. Mean plasma FSH concentrations increased when the frequency was reduced from hourly to 2-hourly or 4-hourly. However, a change from 4-hourly to hourly pulses did not reduce FSH values within 7 days. It is concluded that changes in the pattern of LH secretion observed during the ovine estrous cycle could be accounted for, in part, by changes in GnRH pulse frequency.
Asunto(s)
Castración , Hormona Folículo Estimulante/sangre , Hipotálamo/fisiología , Hormona Luteinizante/sangre , Hipófisis/fisiología , Hormonas Liberadoras de Hormona Hipofisaria/sangre , Animales , Relación Dosis-Respuesta a Droga , Femenino , Hormonas Liberadoras de Hormona Hipofisaria/farmacología , Ovinos , Factores de TiempoRESUMEN
Effects of various anaesthetics on plasma LH, FSH and prolactin levels were studied in ovariectomized ewes. In the first experimental series, conducted between June and November (late breeding season, early anoestrous season), the following treatments were given: saline (i.v.) (n = 7); single thiopentone injection (i.v.) (n = 4); induction of anaesthesia for 2 h with thiopentone (n = 5), ketamine/thiopentone mixture (n = 6), Alphathesin (n = 6) or induction with thiopentone and maintenance with halothane (n = 6). The major findings were: (1) halothane anaesthesia reduced mean plasma LH levels by preventing pulsatile secretion of LH; (2) Alphathesin had the least effect on tonic LH concentration; (3) a single thiopentone injection did not affect LH levels; (4) continuous thiopentone anaesthesia increased LH pulse amplitude; (5) plasma FSH concentration was not affected by any of the treatments; (6) ketamine/thiopentone-induced and Alphathesin-induced anaesthesia increased plasma prolactin levels. In a second experimental series four ovariectomized ewes were anaesthetized with thiopentone for 3 h in January. In contrast to the results obtained with thiopentone in August, treatment in January reduced plasma LH pulse amplitude and mean plasma LH levels. These latter results support the hypothesis that there may be seasonal variation in responses to barbiturate anaesthesia.