Characterization of the translated products of the alternatively spliced luteinizing hormone receptor in the ovine ovary throughout the oestrous cycle.

The luteinizing hormone receptor (LHR) is alternatively spliced. It is not known if the alternatively spliced mRNAs are translated in vivo, or indeed if they have any vital role to play. The B splice form has been detected in every species examined, and it encodes a putative protein with a high affinity LH/CG binding domain but no trans-membrane or intra-cellular domains. We raised antisera that recognize the putative protein of the B form, and the closely related G form, and showed that the B form mRNA is translated in the ovine ovary, but not kidney or liver. It localized to the luteal cytosolic and microsomal fractions and the levels declined during regression induced by treatment with prostaglandin F2alpha. We examined alternative splicing by RNase protection analyses and RT-PCR analyses of healthy pre-ovulatory follicles, atretic or steroidogenically-inactive follicles, and of newly formed, mid-luteal and regressing corpora lutea. There was approximately 5-fold more B form mRNA than A form. Thus we have evidence that the LHR B form is translated in vivo, but no evidence that alternative splicing of the LHR mRNA is differentially regulated, throughout the oestrous cycle.

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist.

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.

Influence of the luteinizing hormone-releasing hormone agonist, deslorelin, on patterns of estradiol-17 beta and luteinizing hormone secretion, ovarian follicular responses to superstimulation with follicle-stimulating hormone, and recovery and in vitro development of oocytes in heifer calves.

The objective of this study was to determine the effects of inducing pituitary desensitization by treatment with an LHRH agonist (deslorelin) on reproductive hormone secretion and ovarian follicular status in heifer calves, before and during stimulation with FSH. The recovery and in vitro development of oocytes was also investigated. Brahman (Bos indicus) calves, 6 mo old, received either no treatment from Day 0 to Day 8 and injections of FSH on Days 9, 10, and 11 (controls, n = 10), or bioimplants of deslorelin on Day 0 and injections of FSH on Days 9, 10, and 11 (deslorelin calves, n = 10). Ovarian follicular characteristics were determined on Days -2, 0, and 8 by ultrasonography; follicle sizes (2-4 mm, 5-7 mm, 8-9 mm, or > or = 10 mm) were recorded. Ovaries were removed surgically on Day 12, surface follicle numbers and sizes were recorded, and oocytes were aspirated, graded (A-grade, B-grade, denuded, atretic), and prepared for in vitro fertilization and culture. Blood samples were taken throughout the experiment to monitor plasma concentrations of LH, estradiol-17 beta (estradiol) and progesterone. Treatment with deslorelin desensitized the pituitary in heifer calves and altered patterns of LH and estradiol secretion. There were no apparent consistent effects of deslorelin treatment on follicle numbers and growth. A higher number of combined A-grade and B-grade oocytes were obtained from heifers treated with deslorelin, which, in turn, resulted in twice the number of blastocysts. Treatment with an LHRH agonist provides a model for studying the hormonal requirements for follicle growth and in vivo oocyte maturation in heifer calves.

Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb.

The use of juvenile donors in embryo-transfer (ET) programmes offers considerable potential for accelerated genetic gain in domestic livestock through reduced generation interval. The present paper reviews recent research aimed at optimizing embryo production from oocytes collected from young calves and lambs using in vitro methods of embryo production. Emphasis is placed on criteria for donor selection, oocyte-collection methods, and hormone-stimulation methods designed to produce maximum yields of viable oocytes. In vitro fertilization (IVF) rates of calf and lamb oocytes did not differ significantly whether matured in vivo or in vitro, and rates of development of blastocyst stages in culture were similar to those observed for embryos derived from adult donors. Blastocysts produced by IVF of lamb and calf oocytes established ET pregnancies at rates of 30-45%. Pregnant recipients have reached full term and delivered normal offspring at rates similar to those expected following ET of embryos produced in vivo from superovulated donors. On the basis of current follicle-stimulation protocols, on rates of blastocyst production in vitro under optimal conditions, and on observed pregnancy rates from fresh transfer of IVF embryos, 8-10 pregnancies may be expected per oocyte collection from 10-12-week-old calves and from 6-8-week-old lambs.

Inhibition of cytokine-stimulated thymic lymphocyte proliferation by fatty acids: the role of eicosanoids.

The effect of individual fatty acids on the proliferation of thymic lymphocytes in response to interleukin-1 (IL-1) was investigated. Proliferation was estimated by measuring [3H]thymidine incorporation into the acid insoluble fraction of the thymocytes. A concentration-dependent inhibition (in the range 1-100 microM) in the IL-1-stimulated proliferation was observed with the C20 fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid and eicosapentaenoic acid (EPA). A less pronounced concentration-dependent inhibitory response was observed with the C18 fatty acids linoleic acid, alpha-linolenic acid and gamma-linolenic acid. Palmitic acid and oleic did not have any effect on either basal or IL-1-stimulated proliferation at concentrations up to 100 microM. The potencies of each fatty acid for this effect at a concentration of 100 microM were: arachidonic acid > EPA > or = DGLA > linoleic acid. DGLA, arachidonic acid and EPA also attenuated IL-2-stimulated proliferation. The inhibitory action of these fatty acids was not mediated by conversion to prostaglandins or other eicosanoids as the cyclooxygenase inhibitor, ketoprofen and NDGA did not alter their action. Incubation of thymocytes with radiolabelled DGLA and EPA followed by reverse-phase HPLC analysis revealed that DGLA is predominantly converted to a more polar metabolite which is not PGE1 whereas EPA does not appear to be converted to any other detectable metabolite. The data indicate that the inhibitory actions of fatty acids on cell proliferation do not occur as a result of conversion to other metabolites but may be direct effects. The inhibition of cytokine-stimulated lymphocyte proliferation by unsaturated fatty acids would imply that they may attenuate cell-mediated immune reactions.

Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes.

Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; P<0.05). Of 33 unselected immature oocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that attainable by conventional breeding or by embryo transfer in adult cattle.

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin.

The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E2 release was studied in monocytes (M phi). Both IL-1 alpha and IL-1 beta increased the release of PGE2 in a concentration-dependent manner, with EC50s of 0.48 nM and 0.12 nM, respectively. Intact M phi were prelabelled with [3H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [3H]phosphatidylinositol-4,5-bisphosphate to aqueous soluble radioactivity by M phi homogenates. IL-1 alpha (5.8 nM) increased the accumulation of IPs within 1-4 minutes and increases in IP3 and IP4 occurred before the increase in IP1+2 whereas LPS only increased the IPs level after at least 30 min. IL-1 alpha increased PIC activity in M phi homogenates within 15 min with an EC50 of 0.58 nM and IL-1 beta (0.1 nM) also increased activity. Neither IL-1 alpha nor IL-1 beta affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in M phi.

Mechanisms controlling the offset of melatonin secretion in the ewe.

Melatonin secretion was investigated in ewes maintained in continuous darkness for 6 days and following an acute delay of lights off as part of a study of factors controlling the melatonin rhythm. In continuous darkness, the interval between successive offsets in circulating melatonin was always greater than 24 hr, indicating that the decline in melatonin secretion in sheep is controlled by endogenous mechanisms having a period longer than 24 hr. In ewes placed under extended darkness with no delay in the time of lights off, the decline in circulating melatonin was delayed by 2.5 +/- 0.2 hr. Animals that had dusk delayed by 4 hr continued to secrete melatonin for at least 4 hr after subjective lights on. Under these conditions, it is proposed that the offset of melatonin secretion is influenced by the timing of lights off and the onset of melatonin secretion. The brain centers controlling melatonin secretion in sheep appear to operate in a manner similar to several species, and thus the sheep may prove to be a useful experimental animal for further studies of circadian mechanisms.

Temporal changes in the pattern of melatonin secretion in sheep held in constant darkness.

The pattern of melatonin secretion was investigated in six 2 yr-old Suffolk ewes transferred from a long-day lighting regime (14L:10D) to continuous darkness. During the 9 d in continuous darkness, five of the ewes maintained a characteristic diurnal rhythm in melatonin secretion. However, there was a systematic change in the duration of each episode of melatonin secretion with the result that it extended from 10 h under the entraining lighting regime to about 19 h by the fourth cycle and then contracted to about 14 h by the eighth cycle under continuous darkness. The pattern of individual ewes varied, but overall the time of offset showed a consistent delay each successive day of about 1 h, indicative of control by a pacemaker with a period of greater than 24 h. By contrast, the time of onset tended to occur earlier each day during the first 4 d of continuous darkness, then changed so that after 8 d, it was delayed in all animals compared to the time of onset on d 4. If, as previously postulated, onset is controlled by a separate pacemaker, then this change is accounted for by interaction with the dominant offset control pacemaker; however, control of both onset and offset through a single pacemaker cannot be excluded. The study confirms that the patterns of melatonin secretion developed in sheep held in continuous darkness may be used to gain insight into the activity of the light-sensitive pacemaker centre(s) controlling the offset and onset of pineal function. Further experimentation is required to differentiate whether control is being exerted by one or two or more independent but interacting pacemaker centres.

Pituitary and ovarian responses of post-partum acyclic beef cows to continuous long-term GnRH and GnRH agonist treatment.

Post-partum acyclic beef cows received continuous long-term treatment with GnRH (200 or 400 ng/kg body wt/h) or the GnRH agonist buserelin (5.5 or 11 ng/kg body wt/h) using s.c. osmotic minipumps which were designed to remain active for 28 days. All treatments increased circulating LH concentrations whereas FSH remained unchanged. Ovulation and corpus luteum (CL) formation as judged by progesterone concentrations greater than or equal to 1 ng/ml occurred in 0/5 control, 4/5 200 ng GnRH, 4/4 400 ng GnRH, 4/5 5.5 ng buserelin and 3/5 11 ng buserelin cows. The outstanding features of the progesterone profiles were the synchrony, both within and across groups, in values greater than or equal to 1 ng/ml around Day 6, and the fact that most CL were short-lived (4-6 days). Only 3 cows, one each from the 400 ng GnRH, 5.5 ng buserelin and 11 ng buserelin groups, showed evidence of extended CL function. Cows failed to show a second ovulation which was anticipated around Day 10 and this could have been due to insufficient FSH to stimulate early follicular development, or the absence of an endogenously driven LH surge. The highest LH concentrations for the respective groups were observed on Days 2 and 6 and by Day 10 LH was declining, although concentrations did remain higher than in controls up to Day 20.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase delay of the rhythm of 6-sulphatoxy melatonin excretion by artificial light.

The purpose of this study was to investigate the effect of bright artificial light exposure on the rhythms of 6-sulphatoxy melatonin and cortisol excretion in urine. Six healthy males were exposed to light (greater than 3,000 lux) from 1900 to 0200 h (sunset 1928 h) on one occasion. The artificial light delayed the onset of 6-sulphatoxy melatonin excretion. On the next evening the onset of 6-sulphatoxy melatonin excretion in normal light/darkness was delayed by 1 h. The timing of the peak excretion of cortisol was not affected by the light treatment; however, cortisol excretion rate was maintained at a significantly higher rate in the morning and afternoon after the treatment. These results demonstrate the inhibitory action of high intensity light in humans and suggest that one 6-h period of extra light in the evening can phase delay the melatonin onset.

Effects of lipid fatty acyl chain structure on the activity of the (Ca2+ + Mg2+)-ATPase.

The (Ca2+ + Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum has been reconstituted into a series of phosphatidylcholines in the liquid crystalline phase. For phosphatidylcholines containing monounsaturated fatty acyl chains, optimal activity is observed for a chain length of C18, with longer or shorter chains supporting lower activities. Phospholipids with methyl-branched chain saturated fatty acids support somewhat lower activities than the corresponding phospholipids with mono-unsaturated fatty acids. Mixed chain phospholipids support ATPase activities comparable to those shown by an unmixed chain phospholipid with the same average chain length. However, the response of the ATPase reconstituted with mixed chain phospholipids to the addition of oleyl alcohol is dominated by the longest fatty acyl chain. Based on their ability to displace brominated phospholipids, relative binding constants to the ATPase of a series of phosphatidylcholines have been determined. Binding to the ATPase is virtually unaffected by fatty acyl chain length or the presence of methyl branches.

Serum melatonin profiles and endocrine responses of ewes exposed to a pulse of light late in the dark phase.

To determine whether effects of light pulses on the photoperiodic time measuring system involve changes in pineal gland function, melatonin profiles were determined in groups of ewes maintained under 10-h light, 14-h dark (10L:14D) or 10L:10D:1L:3D. Ewes exposed to 10L:14D had a significantly (P less than 0.01) longer duration of melatonin secretion (15.0 +/- 0.4 h, mean +/- SE) than ewes under 10L:10D:1L:3D (9.0 +/- 0.4 h). The 1-h pulse of light therefore acted as a dawn signal in the latter group. During a period of extended darkness imposed to study endogenous control of melatonin release, there was no change in the duration of elevated melatonin in control ewes (16.1 +/- 0.5 h), but a significant (P less than 0.05) lengthening occurred in pulsed ewes (13.2 +/- 1.4 h). PRL responses to a bolus iv injection of TRH (50 ng/kg BW) were significantly (P less than 0.01) smaller in control ewes (478 +/- 134 ng/ml) compared with pulsed ewes (1578 +/- 175 ng/ml), with responses in the latter group resembling those observed in ewes on long days. A 1-h pulse of light late in the dark phase, therefore, resulted in a melatonin pattern normally observed under long days in ewes, and this was associated with other endocrine functions also characteristic of sheep on long days. It is concluded that pulses of light modify activity of the pineal gland which in turn interacts with the photoperiodic time-measuring system via melatonin. The increase in duration of melatonin secretion observed in pulsed ewes under extended darkness suggests that the melatonin rhythm is under the control of two oscillators coupled to dusk and dawn, and that these oscillators interact more strongly when compressed by an interrupted dark phase.