Prepubertal Sheep Research Articles

Regional Differences in the Distribution of Gonadotropin-Releasing Hormone Cells between Rapidly Growing and Growth-Restricted Prepubertal Female Sheep

Regional Differences in the Distribution of Gonadotropin-Releasing Hormone Cells between Rapidly Growing and Growth-Restricted Prepubertal Female Sheep

Regional differences in the distribution of gonadotropin-releasing hormone cells between rapidly growing and growth-restricted prepubertal female sheep.

Growth retardation induced by dietary restriction in the lamb results in a low GnRH pulse frequency, and thus, puberty is delayed. In our experimental model, in which ovariectomized lambs are maintained at weaning weight (approximately 20 kg BW), hypothalamic GnRH is present and releasable, suggesting that central mechanisms limit the release of GnRH during chronic growth restriction. Our study compared the number and distribution of GnRH-containing neurons in growth-restricted (n = 5) and rapidly growing (n = 5) ovariectomized prepubertal female lambs at 24 weeks of age (normal age of puberty is about 30 weeks). Immunoreactive cells were labeled using LR-1 antiserum (R. Benoit) and an avidin-biotin-immunoperoxidase procedure. GnRH neurons were localized in 60-micron coronal sections from the level of the diagonal band of Broca to the mammillary bodies. The estimated total number of GnRH neurons in the growth-restricted and rapidly growing lambs was similar (3364.8 +/- 513.8 vs. 3151.2 +/- 279.8, respectively). In addition, the percent distributions of GnRH neurons in the diagonal band of Broca, the anterior hypothalamus, the lateral hypothalamus, and the posterior hypothalamus were not different. A trend (P = 0.07) toward a smaller percent distribution in the preoptic area was noted in growth-restricted lambs (30.6 +/- 3.6) compared to rapidly growing lambs (44.0 +/- 5.2). By contrast, the percent distribution of GnRH neurons in the medial basal hypothalamus was significantly greater in the growth-restricted lambs compared with the rapidly growing lambs (17.7 +/- 2.2 vs. 6.7 +/- 1.4, respectively; P < 0.005). It is of interest that the percent distribution of GnRH-containing neurons in the medial basal hypothalamus of the hypogonadotropic growth-restricted lamb is similar to that observed in the fetal lamb, whereas the eugonadotropic rapidly growing lamb is more similar to the adult female. In this context, decreased GnRH secretion and delayed puberty during diet-induced growth restriction may arise from alterations in the GnRH neurosecretory system.

Analysis of proteins and their synthesis in bovine oocytes and associated cumulus cells during maturation in vitro

The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P < 0.05) for oocytes obtained from ewes (G1, G4, and C1) compared with those from ewe lambs (G2, G3, and C2). The greatest increment in MPF activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM = 3.62) followed by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89, G1: 1.87, and G4: 1.86). In summary, coincubation with AAE during the 24-hour IVM had a positive impact on ovine oocyte competence and ensuing in vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes.

Postpubertal Maturation of Endogenous Opioid Regulation of Luteinizing Hormone Secretion in the Female Sheep1

This study investigated whether the role of endogenous opioid peptides in the suppression of LH secretion during seasonal anestrus in the sheep changes with age. The experimental approach was to determine the effect of blockade of opioid receptors with naloxone on LH secretion at different times of year within the anestrous season, and to compare responses between seasonally anestrous sheep of different ages. Sheep, all past the normal age of puberty, were ovariectomized before the study and treated s.c. with estradiol implants to provide a fixed estradiol feedback signal. One-year-old females responded to naloxone with a rapid increase in LH pulse frequency in the early (April) and late (August) phases of their first anestrous season. This response was similar to that previously found in prepubertal female sheep. Only 5 of the 8 females responded to the same naloxone challenge in mid anestrus (June), suggesting that the contribution of opioid pathways to the inhibition of LH secretion at this time of year is not necessarily the same as that in early and late anestrus. None of the older anestrous sheep (greater than or equal to 2 yr) responded to naloxone in June, indicating age-related changes in the role of endogenous opioid mechanisms in the inhibition of LH secretion. Ovary-intact mature sheep did not respond to naloxone, in contrast to our previous observations in intact prepubertal females. We infer that the neural mechanisms underlying the superficially similar hypogonadotropic states that occur during the prepubertal period, first anestrous season, and later anestrous seasons are not identical.(ABSTRACT TRUNCATED AT 250 WORDS)

Disappearance of Opioidergic Tone on LH Secretion in Underfed Prepubertal Sheep

A study was conducted to determine whether an opioid tonus inhibitory of LH secretion is present in underfed prepubertal sheep. Ten Suffolk ewe lambs were subjected to food restriction during 60 days. During this period they were allowed to pasture only 2 hours per day while control ewe lambs were allowed for 10 hours. Body weight and plasma blood levels of glucose, urea and total proteins were measured weekly. At the end of this period, an intravenous injection of Naloxone (NAL, 1.5 mg/kg BW) was given to control and underfed animals followed 60 min later by an intravenous injection of LHRH to test the pituitary responsiveness. Underfed animals did not show an increase in plasma LH while control animals presented a rise from 0.28 +/- 0.08 to 2.02 +/- 0.6 ng/ml after the NAL stimulus (P less than 0.05). The response to LHRH was similar in both group of animals. Basal plasma levels of insulin were lower in underfed ewe lambs than in control animals (P less than 0.05). Underfed animals were placed on plain feeding with a schedule similar to control lambs for 30 days and the same experiment was repeated. During this occasion, NAL increased plasma LH concentration in both group of lambs. Levels of plasma insulin were not different in both groups. The lack of effect of NAL on LH secretion in food restricted ewe lambs suggests that the opioid modulation of LH secretion is absent by underfeeding in female prepubertal sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous opioid regulation of pulsatile luteinizing hormone secretion during sexual maturation in the female sheep.

The developing female sheep, which attains puberty after 25 weeks of age, was used as an experimental model to investigate the role of endogenous opioid peptides in the control of pulsatile LH secretion during sexual maturation. Treatment of ovary-intact prepubertal sheep at 12 weeks of age with the opiate antagonist naloxone resulted in a dose-dependent increase in LH secretion. Subsequent studies used ovariectomized (OVX) lambs implanted with capsules containing 17 beta-estradiol to provide a constant, ovarian steroid feedback signal throughout development. Naloxone treatment (hourly iv injections of 1 mg/kg BW for 4 h) produced an increase in the frequency of episodic LH secretion at all prepubertal ages, when lambs were highly sensitive to the estradiol negative feedback. However, increases in LH pulse frequency were also induced by naloxone treatment at a postpubertal age in estradiol-treated OVX sheep, indicating that opioid inhibition is still present at a time when sensitivity to the feedback effects of ovarian steroids is markedly reduced and endogenous LH secretion is increased. These observations in ovary-intact and estradiol-treated OVX lambs suggest that opioid mechanisms inhibit pulsatile tonic LH secretion during both the prepubertal and postpubertal periods. Endogenous opioid inhibition of LH secretion is not dependent on the presence of ovarian steroids, as evidenced by the response to naloxone 3 weeks after removal of an estradiol implant from OVX lambs, when LH pulse frequency was already high. Naloxone treatment increased LH pulse frequency further, at both a prepubertal age (18 weeks) and a postpubertal age (38 weeks). Naloxone also increased LH pulse frequency in OVX lambs in which LH secretion was inhibited chronically by progesterone rather than by estradiol. The response to naloxone was similar in postpubertal P-treated OVX lambs and age-matched prepubertal P-treated OVX controls in which puberty had been delayed by means of an inhibitory seasonal photoperiod. In addition, after removal of steroid implants to allow LH secretion to increase, the degree of inhibition of LH secretion by the opiate agonist morphine was similar between age-matched postpubertal sheep and those with photoperiodically delayed puberty. We conclude that endogenous opioid mechanisms are an important inhibitory mechanism controlling pulsatile LH secretion in the developing sheep. However, changes in opioid inhibition are unlikely to underlie the decrease in sensitivity to steroid negative feedback and increase in pulsatile LH secretion that occur at puberty.

Melatonin and the pituitary response to GnRH in prepubertal and adult ewes.

GnRH-induced LH-release was studied in female lambs from 5 weeks of age until puberty and in adult anoestrous ewes. In pre-pubertal animals LH was released rapidly after GnRH treatment but after puberty the responses became slower and more sustained although the peak concentration did not change. In neither pre-pubertal nor adult sheep did prior treatment with melatonin influence LH release after GnRH treatment.