Sequence variants in the bovine gonadotrophin releasing hormone receptor gene and their associations with fertility.

Seven sequence variants (SVs) have been identified in exon 1 and in the promoter region upstream of the bovine gonadotrophin releasing hormone (GnRH) receptor gene, at nucleotides g.-331A>G, g.-108T>C, g.+206G>A, g.+260C>T, g.+341C>T, g.+383C>T and g.+410C>T relative to the translation start site. The SVs at nucleotides g.-108, g.260, g.341 and g.410 and those at g.206 and g.383 formed two groups with complete linkage disequilibrium within groups, but incomplete linkage disequilibrium between groups, and none of the SVs altered receptor amino acid sequence. The g.-108T>C allelic variants were associated with an approximately 0.4 day reduction in predicted transmitting ability for days to first service. None of the allelic variants affected the pattern of circulating LH following administration of GnRH. The g.260C>T alteration introduced a new transcription factor binding site in a region of DNA with relatively low nucleosome formation potential. The data suggest that selection for animals carrying the g.-108T>C group of alterations will improve fertility in the dairy cow.

Genetic variation of metabolite and hormone concentration in UK Holstein-Friesian calves and the genetic relationship with economically important traits.

The decline of dairy cattle fertility worldwide remains a major concern, with conception rates to first service commonly below 40%. The length and severity of negative energy balance postpartum are unfavorably correlated with fertility, suggesting that the length and severity of negative energy balance and fertility are linked via several hormones or metabolites. These compounds therefore have the potential to predict fertility at a genetic level. The addition of a predictor trait for fertility into present fertility indices would accelerate genetic gain, particularly if it was expressed before adulthood. The objective of this work was to estimate the genetic variation in several metabolites and hormones in calves, and to determine their genetic relationships with fertility and production through sire predicted transmitting abilities (PTA; sires of calves sampled). Circulating concentrations of free fatty acids (FFA), glucose, growth hormone (GH), insulin, and insulin-like growth factor 1 (IGF-1) in male and female UK Holstein-Friesian dairy calves (average age +/- SD; 126 +/- 12.7 d) were analyzed during 2 studies: data set 1 (n = 496 females; 1996-2001; 7 commercial dairy herds) and data set 2 (n = 326 females, n = 256 males; 2002-2006; multiple ovulation and embryo transfer breeding scheme). Univariate mixed models were fitted to the data using ASREML. Basal concentrations of FFA, glucose, GH, insulin and total IGF-1 were all moderately heritable in both sexes (heritability range +/- SE; 0.09 +/- 0.05 to 0.66 +/- 0.14). The sire PTA for protein percentage had significant regression coefficients and approximate genetic correlations with FFA and insulin, and the sire PTA for calving interval had significant regression coefficients and approximate genetic correlations with GH. Additive genetic variance seems responsible for a moderate proportion of the phenotypic variation in important metabolites and regulatory hormones in male and female UK Holstein-Friesian dairy calves, therefore supporting further investigation into their use as juvenile predictors for fertility in the mature female.

Incidence and treatment of inadequate postovulatory progesterone concentrations in repeat breeder cows.

The incidence of low day 5 milk progesterone in dairy cows has been investigated and the efficacy of treating the problem assessed. The incidence of inadequate milk progesterone (empirically defined as <3ng/mL) in repeat breeder cows was 34% compared with 11.4% in first insemination cows. Treatment with an intravaginal progesterone device for 7 days starting from day 5 or 6 did not improve pregnancy rate. Treatment with 1500 iu human chorionic gonadotrophin (hCG) on day 5 gave an increase in pregnancy rate that was dependent on initial progesterone concentration and significant (P<0.05) in multiparous but not primiparous cows. While the incidence of inadequate day 5 progesterone was high in repeat breeder cows, it was responsive to hCG treatment, although only in multiparous and not primiparous animals.

A PPAR-independent pathway to PUFA-induced COX-2 expression.

Polyunsaturated fatty acids (PUFAs) induce COX-2 in bovine endometrial stromal cells through activation of peroxisome-proliferator-activated receptor alpha (PPARalpha). We have investigated alternative (PPAR-independent) pathways to COX-2 induction using a reporter construct driven by a COX-2 gene promoter sequence lacking a PPAR response element. This construct was induced by PUFAs, but not by PPAR agonists. PPAR-independent reporter gene expression occurred 6h after PPAR-dependent induction of the endogenous COX-2 gene. In contrast to PPAR-dependent COX-2 induction, which is not affected by NF-kappaB inhibitors, the PPAR-independent pathway was blocked by the NF-kappaB inhibitor MG132 or following deletion of NF-kappaB sites in the COX-2 promoter. The PPAR-independent effect of PUFA was mimicked by the PKC activators 4beta-PMA and prostaglandin F(2alpha), but was not blocked by the PKC inhibitor RO318425. The results demonstrate a pathway to the induction of COX-2 by PUFAs requiring NF-kappaB but not PPAR or PKC.

Genetic analysis of postpartum measures of luteal activity in dairy cows.

The objective of this study was to estimate genetic parameters for measures of luteal activity during the first 60 d postpartum. Analyses were made with different sampling intervals to investigate the possibility of combining progesterone measurement with routinely performed milk recording. Progesterone level in milk as an indicator of female fertility when selecting sires in a progeny-testing scheme was also examined. Data were collected from 1996 to 1999, and comprised 1,212 lactations from 1,080 British Holstein-Friesian cows at 8 commercial dairy farms in the United Kingdom. Milk samples for progesterone analysis were collected thrice weekly. Mixed linear animal models were used to analyze the data. Heritability for the percentage of samples with luteal activity during the first 60 d postpartum (PLA) was 0.30 and decreased with more infrequent sampling to 0.25, 0.20, and 0.14 for weekly, twice-monthly, and monthly sampling, respectively. Measures of PLA had a high negative genetic correlation with prolonged anovulation (-0.53 for monthly sampling, < -0.87 otherwise) and a moderate positive genetic correlation with persistent corpus luteum in the first estrus cycle (>0.65 if at least twice-monthly sampling). Genetic correlations with interval from calving to commencement of luteal activity were close to -1 for all PLA measurements and the selection index calculations showed that monthly progesterone sampling could be used with high accuracy (0.80 with 50 daughters per bull) to predict breeding values for commencement of luteal activity. Progesterone analysis at the time of regular milk recording could thereby be used to select for an early interval from calving to commencement of luteal activity and, at the same time, a decreased frequency of prolonged anovulation during the postpartum period.

Interactions between the cytomegalovirus promoter and the estrogen response element: implications for design of estrogen-responsive reporter plasmids.

We aimed to produce an estrogen-responsive reporter plasmid that would permit monitoring of estrogen receptor function in the uterus in vivo. The plasmid pBL-tk-CAT(+)ERE was induced by estrogen in bovine endometrial stromal cells. When the CAT gene was replaced by the secreted alkaline phosphatase SeAP, the resulting construct pBL-tk-SeAP(+)ERE remained estrogen responsive. However when the tk promoter was replaced by the cytomegalovirus (cmv) promoter, the resulting plasmid (pBL-cmv-SeAP(+)ERE) was not estrogen responsive. Inhibition of ERE function was not due to an effect in trans or due to lack of estrogen receptor. It was not due to an interaction between the cmv promoter and the SeAP gene. cmv promoter function was dependent on NF-kappaB, and mutagenesis in the NF-kappaB sites reduced basal reporter expression without imparting responsiveness to estrogen. A mutation in the TATA box also failed to impart estrogen responsiveness. Modeling of DNA accessibility indicated the ERE was inserted at a site accessible to transcription factors. We conclude that the cmv promoter inhibits ERE function in cis when the two sequences are located in the same construct, and that this effect does not involve an interaction between cmv and reporter gene, NF-kappaB sites or the TATA box, or DNA inaccessibility.

Large luteal cells the source of luteal oxytocin in the sheep.

To determine the cellular origin of oxytocin produced by the cyclical corpus luteum (CL) of the sheep, enriched fractions of enzymatically dispersed small and large luteal cells from 12 CL were prepared on a Ficoll 400 gradient. Oxytocin was measured by RIA. Large luteal cells contained 1.08 +/- (SD) 0.70 fg/cell oxytocin, which was congruent to 30 X the content of small luteal cells. Endothelial cells contained little if any oxytocin. During a 12-h incubation, large luteal cells produced 0.28 fg/cell.h oxytocin: small luteal cells did not produce measurable amounts of oxytocin. It is concluded that the large luteal cells are the source of the oxytocin produced by the CL of the sheep.

Plasma concentrations of prostaglandins during late human pregnancy: influence of normal and preterm labor.

Highly sensitive and specific RIA procedures have been used to measure prostaglandin concentrations in the peripheral circulation of late pregnant and parturient women. The concentrations of prostaglandin E (PGE) and prostaglandin F (PGF) in plasma samples assayed within 4 weeks of collection were not significantly different among the groups studied, the levels (mean +/- SEM, picograms per ml) were: late pregnancy (n = 13): PGE, 4.8 +/- 1.0; PGF, 6.2 +/- 0.5; early term labor (n = 5): PGE, 6.8 +/- 1.5; PGF, 7.9 +/- 0.7; late term labor (n = 5): PGE, 5.4 +/- 2.2; PGF, 12.4 +/- 3.5; and preterm labor (n = 7): PGE, 4.4 +/- 0.4; PGF, 6.9 +/- 1.4. The concentration of 13,14-dihydro-15-keto-prostaglandin F (PGFM) in late pregnancy was 59.0 +/- 7.8 pg/ml. During spontaneous term labor, the concentration of PGFM was significantly elevated (P less than 0.01) to 142.8 +/- 32.3 pg/ml in early labor and 282.7 +/- 55.3 pg/ml in late labor. The concentration of PGFM in plasma from patients in preterm labor (62.7 +/- 17.4 pg/ml) was not significantly different from that found during late pregnancy, but was significantly lower than levels found at term during early labor (P less than 0.05). The concentration of PGE increased significantly in frozen plasma samples stored for more than 4 weeks in all groups studied; the concentration of PGF was significantly elevated after storage only in the late pregnancy group (P less than 0.01). The plasma concentration of PGFM in all groups studied was unaffected by storage. It is concluded that measurement of PGFM concentrations is the most reliable method available of monitoring prostaglandins in the peripheral circulation and that great care must be exercised in the assay and interpretation of prostaglandin levels in human plasma.

Heterogeneity in the third intracytoplasmic region of the oxytocin receptor-encoding gene.

The oxytocin receptor (OTR), a member of the seven-transmembrane domain guanine-nucleotide-binding protein (G protein) coupled receptor family plays a central role in lactation, ovarian cyclicity and reproductive behaviour. Recent cloning and sequencing unexpectedly revealed that the third intracytoplasmic region (3ICR) of the sheep receptor has 3 and 2 additional amino acids (aa) relative to the rat and human receptors, respectively. We have now confirmed, by sequencing polymerase chain reaction (PCR)-derived genomic fragments coding for the OTR 3ICR from a variety of ruminant and non-ruminant species, that additional aa are a general phenomenon in ruminants.

Sequencing analysis of prion genes from red deer and camel.

An abnormal isoform of the prion protein (PrP) appears to be the agent responsible for transmissible spongiform encephalopathies (TSE). The normal isoform of PrP is host-encoded and expressed in the central nervous system. The recent bovine spongiform encephalopathy (BSE) epidemic in the UK and the incidence of prion-related diseases in other animals could indicate that ruminants are highly susceptible to infection via ingestion of prion-contaminated food. Sequence analysis of PrP gene open reading frames from red deer and camel was carried out to investigate sequence variability of these genes among ruminants.

Acute effects of interferon on estrogen receptor function do not involve the extracellular signal-regulated kinases p42mapk and p44mapk.

Exposure to type I interferons (IFN) increased estrogen receptor (ER) ligand binding and induced protein kinase C (PKC) translocation within 30 min but had no effect on net incorporation of [32P] into ER in Madin Darby bovine kidney (MDBK) cells. Ligand binding was also increased within 30 min by phorbol ester and the protein phosphatase inhibitor okadaic acid. Mitogen-activated protein (MAP) kinase phosphorylation was initially inhibited between 2 and 30 min and subsequently activated between 30 and 60 min after treatment with IFN. The activatory response was blocked by the PKC inhibitor Ro 31-8220. Following transient transfection with an ERE-CAT reporter construct, IFN increased CAT expression after 6 h but decreased ER ligand binding, transcriptional activity and phosphorylation after 48 h, probably as a result of decreased ER concentrations. The results rule out rapid activation of ER ligand binding through phosphorylation at Ser118 by MAP kinase because (1) the increase in ligand binding preceded activation of MAP kinase, and (2) IFN had no short-term effect on [32P]incorporation or ER transcriptional activity. The rapid effect of IFN on ER ligand binding is postulated to reflect phosphorylation of the receptor at Tyr537 by p56lck, a member of the Src family of PKC-activated tyrosine kinases.

Use of pertussis toxin to investigate the mechanism of action of prostaglandin F2 alpha on the corpus luteum in sheep.

Pertussis toxin catalysed the ADP-ribosylation of a protein of M(r) 40,000 in ovine luteal tissue. Ribosylation of 45% of this protein in whole cell incubations (as judged by subsequent ribosylation of cell-free preparations in the presence of [32P]NAD) attenuated the prostaglandin (PG)F2 alpha-stimulated hydrolysis of [3H]inositol-labelled phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate by 60%, but did not affect the inhibition by PGF2 alpha of LH-stimulated accumulation of cyclic AMP. It is concluded that activation of phospholipase C by PGF2 alpha involves a pertussis toxin-sensitive protein, probably a G protein, and that the inhibitory effect of PGF2 alpha on LH-stimulated adenylate cyclase is unlikely to be directly mediated by such a protein.

Negative regulatory domains in a trophoblast interferon promoter.

A bovine trophoblast interferon (IFN-tau) gene promoter sequence (-450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between - 338 and - 247 bp, and between - 150 and - 71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-tau genes.

Structure of an interferon-alpha 2 gene expressed in the bovine conceptus early in gestation.

Genomic and cDNA clones for bovine trophoblast interferon (IFN) have been isolated by probing a bovine genomic library and a bovine embryonic (day-18 post coitus) cDNA library respectively with the ovine trophoblast IFN cDNA. The two DNA sequences were identical; sequence analysis demonstrated 80% identity between the amino acid sequence of bovine trophoblast IFN and ovine trophoblast IFN, and 70% identity with a previously identified bovine IFN-alpha 2. Southern blotting of bovine genomic DNA indicated the presence of a minimum of three trophoblast IFN genes. Primer extension analysis identified the transcription start site in the 5' flanking region of the bovine IFN gene. Computer-aided analysis of the 5' flanking sequence demonstrated a similarity with that of bovine IFN-alpha 2 and the existence of a possible viral induction sequence.

Isolation and structure of a partial sheep oxytocin receptor cDNA and its use as a probe for northern analysis of endometrial RNA.

The polymerase chain reaction (PCR) was used to generate a 131 bp cDNA encoding part of the sheep endometrial oxytocin receptor. The nucleotide sequence of this cDNA was 93.8% identical to the human oxytocin receptor sequence in this region. When used to probe Northern blots of sheep endometrial RNA the PCR product identified a 6.7 kb mRNA which appeared and disappeared during the oestrous cycle in parallel with the oxytocin receptor molecule as measured by ligand binding. The sheep endometrial receptor mRNA was significantly larger than the human myometrial mRNA (4.7 kb). It is suggested that the cloned cDNA described here is an appropriate probe for use where it is required to measure sheep oxytocin receptor mRNA.

Structure and expression of an ovine endometrial oxytocin receptor cDNA.

A sheep endometrial oxytocin receptor (OTR) cDNA (1.5 kb) was isolated from a lambda-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.

Localization of oxytocin receptor mRNA in the ovine uterus during the oestrous cycle and early pregnancy.

A synthetic 45-mer oligonucleotide corresponding to part of the ovine endometrial oxytocin receptor cDNA was hybridized to sections of ovine uterus collected from 40 ewes at different stages during the oestrous cycle, the first 3 weeks of pregnancy and seasonal anoestrus. The quantity of oxytocin receptor mRNA was measured as the optical density (OD) value on autoradiographs using image analysis. Message first appeared in the luminal epithelium on days 14-15 of the cycle, increasing to a peak OD of 0.48 at oestrus and decreasing again between days 2 and 5. Oxytocin receptor mRNA in the superficial glands, deep glands and caruncular stroma increased between day 15 and oestrous to peak OD values of 0.17, 0.11 and 0.11 respectively, declining again by day 2 and reaching basal values (OD < 0.015) by day 5. Hybridization to the myometrium tended to rise from a mean OD value of 0.01 on days 2-15 to a peak of 0.03 +/- 0.01 (mean +/- S.E.M.) on days 0-1, but the change was not significant. In pregnant ewes there was no detectable oxytocin receptor mRNA on days 14-15 in any region, but hybridization to the luminal epithelium was present in two of three ewes on day 21. In anoestrous ewes oxytocin receptor mRNA concentrations in all areas of the endometrium were approximately half those measured at oestrus. Optical density readings for oxytocin receptor mRNA in the various uterine compartments were compared with measurements of oxytocin receptors in the same regions as assessed by binding studies using the 125I-labelled oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin (125I-labelled OTA). In the endometrium, receptor mRNA and 125I-labelled OTA binding patterns changed in parallel, and both sets of measurements were significantly correlated (P < 0.01). In the myometrium, a significant increase in 125I-labelled OTA binding occurred at oestrus; this was not accompanied by a similar increase in oxytocin receptor mRNA hybridization. This study helps to confirm that the previously identified cDNA clone is derived from the ovine oxytocin receptor, as patterns of oxytocin receptor mRNA expression in the endometrium closely resembled those of oxytocin binding. Maximum expression and binding both occurred at oestrus, suggesting that regulation of the oxytocin receptor gene in the uterus occurs principally at the transcriptional, rather than at the translational, level. Failure to detect a significant increase in myometrial mRNA expression at oestrus may indicate that the endometrial and myometrial oxytocin receptors are of different isoforms.

Structure of an ovine interferon receptor and its expression in endometrium.

A sheep type I interferon receptor (oIFNAR1) cDNA was isolated from a lambda-ZAP library using a reverse transcription (RT)-PCR product probe generated from oestrous endometrial RNA. The oIFNAR1 cDNA was 79, 66 and 95% homologous to human, murine and bovine IFNAR1 cDNAs respectively. The encoded receptor was a 560-amino acid transmembrane protein 80, 66 and 95% similar to human, murine and bovine IFNAR1 respectively. Northern blot analysis of endometrial mRNA revealed the presence of 6.5, 4.3 and 3.7 kb transcripts. Using semi-quantitative RT-PCR the oIFNAR1 mRNA was not found to be down-regulated after 72 h treatment with bovine recombinant IFN-alpha I in in vitro experiments with endometrial explants.

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels.

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-alpha family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

Pertussis toxin-catalysed ADP-ribosylation of endometrial proteins in sheep.

Membrane fractions prepared from the uterine endometrium of untreated ovariectomized sheep contained a 41 x 10(3) Mr protein that was [32P]ADP-ribosylated by pertussis toxin in the presence of [32P]NAD. Progestin and progestin plus oestrogen treatment in vivo increased the concentration of this protein 2.7- and 3.6-fold respectively. Endometrial extracts from untreated or progestin-treated sheep also contained proteins of Mr 69 x 10(3) and 120 x 10(3) which were ADP-ribosylated in the absence of pertussis toxin; these proteins were not ADP-ribosylated in sheep receiving oestrogen. Incubation of endometrial slices from progestin plus oestrogen-treated sheep with oxytocin in vitro increased phosphoinositide hydrolysis 11-fold. This effect was not altered by prior incubation with pertussis toxin, although toxin treatment reduced by 64% subsequent labelling of the 41 x 10(3) Mr protein when membrane fractions prepared from pretreated slices were incubated with pertussis toxin and [32P]NAD. Thus the endometrium contains a pertussis toxin-sensitive protein which is induced by steroid treatment, but this protein is not involved in the phosphoinositide response to oxytocin.