Mutations in genes involved in oestrous cycle associated expression of oestrus.

Detection of oestrus is a key determinant of profitability of dairy herds, but oestrus is increasingly difficult to observe in the modern dairy cow, with shorter duration and less intense oestrus. Concurrent with the unfavourable correlation between milk yield and fertility, oestrous detection rates have decreased to less than 50%. A number of mutations have been identified in genes associated with fertility and production traits, but, to date, no single nucleotide polymorphism (SNP) has been associated with oestrous expression. Therefore, the objective of this study was to investigate SNPs, linked to fertility, for the association with oestrous expression. Blood was collected from 205 Holstein Friesian dairy cows and genotyped at 41 loci of 18 genes chosen for their roles in the oestrous cycle and milk production. SNPs were then examined for correlations with increase in activity at oestrus, recorded via activity monitors, using generalised linear models. Physical activity increased at oestrus between two and four fold. Larger increases were associated with mutant alleles in oestrogen receptor-α and gonadotrophin releasing hormone receptor genes (P<0.05) and in the STAT5A gene (P<0.05). Smaller increases were associated with mutant alleles of the activin receptor type IIB and prolactin receptor genes (P<0.10). In conclusion, alleles in these five genes provide the opportunity for selection of animals displaying greater oestrous activity which could aid reversal of the decrease in oestrous detection and thereby contribute to sustainability of the dairy industry worldwide.

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.

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.

Polymorphisms within the coding region of the bovine luteinizing hormone receptor gene and their association with fertility traits.

Mutations within a number of genes have been associated with variations in fertility in various mammals. However, to date there have been no such associations reported for cattle. Herein, we describe three single nucleotide polymorphisms (SNPs) in the luteinizing hormone/choriogonadotropin receptor gene of cattle (Bos taurus). These polymorphisms include two missense mutations and one sense mutation, and all are located in areas of conserved synteny. When assessed in terms of haplotypes, these SNPs were significantly associated with variations in cattle fertility and production traits, most notably on calving interval, days to first service and production index (the UK economic index of milk yield measured in poundGB).

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.

Nongonadal LH/hCG receptors in pig: functional importance and parallels to human.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) share a common receptor in gonadal cells; however, the presence of these receptors has also been detected in several nongonadal but reproduction-associated tissues of pig, human, and other species. There are no data about the ontogeny of the human LH/hCG receptor. The expression of the porcine LH receptor gene in the uterus starts about 10 days after the appearance of this gene in gonads. LH/hCG receptors were found in uterus (myometrium, endometrium), oviduct, cervix, fetal membranes, and umbilical cord in humans and pigs. The main role of LH/hCG receptors in myometrium is stimulation of growth and hyperplasia and relaxation of uterine motility. hCG also increases blood flow in the uterine artery. LH and hCG can increase production of prostaglandins in endometrium, oviduct, and blood vessels. It is suggested that the preovulatory surge of LH plays an important role in controlling oviductal contractions. Human and pig mammary glands also possess LH/hCG receptors through which gonadotropins can affect the metabolism of steroid hormones in this tissue and may play an inhibitory role in mammary carcinogenesis and in the growth of breast tumors.

The effects of lipopolysaccharide and interleukins-1alpha, -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium.

Up-regulation of endometrial oxytocin receptor (OTR) expression followed by an increase in pulsatile endometrial prostaglandin (PG) F(2alpha) secretion causes luteolysis in cattle. Inhibition of luteolysis is essential for the maternal recognition of pregnancy but also occurs in association with endometritis. The factors regulating OTR expression at this time are unclear. The OTR gene promoter region contains binding elements for acute phase proteins but their function has not been established. This study investigated the effects of various cytokines on OTR expression and on PGF(2alpha) and PGE(2) production in explant cultures of bovine endometrium. Endometrium was collected in the late luteal phase (mean day of cycle 15.4+/-0.50) or early luteolysis (mean day of cycle 16.4+/-0.24) as determined by the initial concentration of endometrial OTR. Explants were treated for 48 h with: (i) lipopolysaccharide (LPS) and/or dexamethasone (DEX), (ii) ovine interferon-tau (oIFN-tau), or (iii) human recombinant interleukin (IL)-1alpha, -2 or -6. OTR mRNA was then measured in the explants by in situ hybridisation and the medium was collected for measurement of PGF(2alpha) and PGE(2) by RIA. LPS treatment stimulated production of PGF(2alpha), whereas DEX either alone or in combination with LPS was inhibitory to both PGF(2alpha) and PGE(2). Neither of these treatments altered OTR mRNA expression. oIFN-tau reduced OTR mRNA expression but stimulated production of both PGF(2alpha) and PGE(2). In endometrial samples collected in the late luteal phase, IL-1alpha, -2 and -6 all inhibited OTR mRNA expression, but IL-1alpha and -2 both stimulated PGF(2alpha) production. In contrast, when endometrium was collected in early luteolysis, none of the interleukins altered OTR expression or caused a significant stimulation of PGF(2alpha) production but IL-2 increased PGE(2). Neither IL-1alpha nor -2 altered OTR promoter activity in Chinese hamster ovary cells transfected with a bovine OTR promoter/chloramphenicol acetyl transferase reporter gene construct. In conclusion, the action of interleukins on both OTR mRNA expression and endometrial prostaglandin production alters around luteolysis. Pro-inflammatory interleukins suppress OTR expression in the late luteal phase, while LPS stimulates PGF(2alpha) without altering OTR mRNA expression. IL-I and -2 and LPS are therefore unlikely to initiate luteolysis but may cause raised production of PGF(2alpha) during uterine infection.

Trophoblast interferon and pregnancy.

The maternal recognition of pregnancy in ruminants requires the production of interferons by the preimplantation blastocyst. These proteins, the trophoblast interferons (IFN-tau), are the products of a number of similar genes, the expression of which is controlled by characteristic promoter regions. They are expressed for a short period in high concentrations, and have antiluteolytic, antiviral, antiproliferative and immunomodulatory effects, through receptors on the endometrial epithelium. The antiluteolytic effects of IFN-tau result from inhibition of endometrial expression of the oxytocin receptor, through which circulating oxytocin stimulates episodic prostaglandin F2a production. Some of the properties of IFN-tau differ from those of other type I interferons, and they may have novel therapeutic effects. Because of their central role in early gestation, these proteins have excited the interest of reproductive physiologists. However, their other properties, and the fact that their expression is controlled so precisely, have made them of interest to a wide range of biologists.

Gene expression and peptide localization for LH/hCG receptor in porcine small and large luteal cells: possible regulation by opioid peptides.

The aim of the present studies was to investigate (1) the presence of LH receptor (LHR) in porcine separated small (SLCs) and large (LLCs) luteal cells taken from mid-luteal corpora lutea and (2) the influence of opioid agonist, FK 33-824 (FK) on LHR gene expression in these cells. Immunocytochemistry revealed intense staining for LHR in both SLCs and LLCs. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization were used to check the effect of FK and hCG on LHR gene expression. The LHR gene expression was observed in non-stimulated LLCs and in both types of cells after treatment with FK or hCG. FK stimulated LHR gene expression in SLCs and inhibited the gene expression in LLCs. Moreover, FK inhibited and potentiated stimulatory influence of hCG on the gene expression in LLCs and SLCs, respectively. These results suggest that LHR gene expression in porcine luteal cells can be regulated by opioid peptides.

Uterine lymphocyte distribution and interleukin expression during early pregnancy in cows.

Both the production of cytokines and the distribution of immune cells within the uterus change during early pregnancy. Evidence obtained mainly from mice indicates that these changes are important for implantation and in preventing a maternal immune response to the conceptus. The ruminant embryo also produces interferon tau at this time, the signal for the maternal recognition of pregnancy. The relationship between these events in cows was studied using uteri from three groups of animals on day 16 after observed oestrus: (i) cyclic controls, (ii) pregnant and (iii) inseminated but with no embryo present. Embryo size and the antiviral activity in uterine flushings (indicative of the interferon tau concentration) were measured. Sections of intact uterus were frozen for the localization and quantitation of CD4(+) (T lymphocytes), CD14(+) (macrophages) and CD21(+) (B lymphocytes) uterine cells by immunohistochemistry. The expression of interleukin (IL)-1alpha, IL-2, IL-6 and IL-10 mRNAs in uterine extracts was measured by RT-PCR. Neither embryo size, interferon tau concentration nor pregnancy status influenced the distribution of CD4(+), CD14(+) or CD21(+) cells in the day 16 uterus. Endometrial IL-1alpha mRNA was detected in most cows across the groups, whereas IL-2 mRNA was only present in the non-pregnant uterus. IL-6 and IL-10 mRNAs were not detectable in any uteri. In conclusion, IL-2 mRNA expression is detectable in the non-pregnant but not the pregnant uterus on day 16 and interferon t is unlikely to play a role in the redistribution of immune cells in the uterus during early bovine pregnancy.

LH/hCG receptors in the porcine uterus--a new evidence of their presence in the cervix.

High-affinity LH/hCG binding sites have been characterized in bovine, lepine, murine, human uteri and porcine myometrium and endometrium. In the present studies we analyzed these receptors in the porcine cervix. Radioreceptor ligand assays were performed with cell membrane preparations of the cervix which were analyzed for binding sites specificity, capacity and affinity. Corpus luteum and myometrium were used as positive control tissues. In the cervix there was little competition for receptor occupancy between hCG and porcine FSH (1.2%) or bovineTSH, porcine GH and porcine PRL (0.1%, 0.1% and < 0.001%; respectively) but porcine LH could completely inhibit the binding of [125I] hCG. There was not binding for LH/hCG in crude membrane preparations of kidney or skeletal muscle. The concentration (fmol/mg protein) of cervical LH/hCG receptor did not vary significantly during particular phases of the estrous cycle, except the early luteal phase (Days 6-7) when the level of LH receptors was very low (p < 0.05). The affinity of uterine LH/hCG binding sites in the cervix and the myometrium was not different from the affinity of LH/hCG binding sites in luteal cells. The porcine cervix as well as the myometrium contains a 75- and 48-kDa immunoreactive LH/cCG receptor proteins similar to corpus luteum. Southern blot of RT-PCR products performed to enhance the specificity and sensitivity of LH receptor transcripts determination in uterine tissues revealed that expected fragments of 740 and 470 bp were present in myometrium and corpus luteum. The cervix showed only 740 bp fragment. In situ hydridization showed the expression of mRNA for LH receptor in the epithelium of the cervix. Immunoreactive staining for LH/hCG receptors was also observed only in epithelial cells of the cervical tissue. Our studies are probably the first evidence demonstrating the specific LH/hCG binding sites in female cervix.

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.

Ontogeny of LH receptor gene expression in the pig reproductive tract.

In view of the recently documented expression of the LH receptor gene in several nongonadal reproductive tissues, the aim of this study was to analyse further the ontogeny of expression of this gene in the pig reproductive tract in fetal and neonatal life. RT-PCR was used to investigate the expression of the extracellular and transmembrane receptor domains, and to identify the time of onset of transcription of the full-length LH receptor mRNA and its shorter splice variants. Expression of the LH receptor gene was first detected around day 30 of fetal life in both ovary and testis, coinciding with morphological differentiation. The pattern of expression of LH receptor splice variants did not change during postnatal gonadal maturation. Expression of the LH receptor gene in the pig non-gonadal reproductive tract started during fetal life and continued during sexual maturation. A novel pig LH receptor spliced variant, lacking exon 10, was detected for the first time. The transmembrane receptor domain was expressed in fetal tissues, but not in neonatal control tissues. On the basis of the transmembrane domain of the LH receptor mRNA, it is concluded that the ovary and extragonadal tissues of the pig reproductive tract, like the pig testis, synthesize functional LH receptors during fetal life. The presence of LH receptor mRNA in extragonadal reproductive tissues indicates that LH is involved in the control and regulation of reproductive tract maturation.

Evidence for the presence of luteinizing hormone-chorionic gonadotrophin receptors in the pig umbilical cord.

Pig umbilical cord, like that of humans, contains two arteries and a vein surrounded by Wharton's jelly with amnion covering the exterior surface. The aim of the present study was to investigate whether LH-hCG receptors are present in the pig umbilical cord, using light microscope immunohistochemistry, semiquantitative autoradiography, western blotting and reverse transcription-polymerase chain reaction. Umbilical cords were collected on days 48, 71 and 103 of fetal life (n = 6). Monoclonal and polyclonal anti-LH receptor antibodies were used to study receptor distribution. Immunoreactivity was observed in the umbilical blood vessels, the epithelium of umbilical amnion and cells in the Wharton's jelly. No differences in LH-hCG receptor distribution related to the sex of the fetus, period of fetal life or section of the umbilical cord were observed. Strong immunostaining was observed in umbilical vein and in umbilical arteries. However, in the arteries, the tunica media expressed weaker receptor immunostaining than did the tunica intima and tunica adventitia. No immunoactivity was detected in non-target tissue (skeletal muscle) but LH receptors were immunostained in the pig ovary. Topical autoradiography showed that vein and arteries in the umbilical cord bind 125I-labelled hCG, which was highly diminished after co-incubation with an excess of unlabelled hCG. The binding of 125I-labelled hCG to the Wharton's jelly and epithelial amnion was less intense than it was to vessels. Gonadotrophin binding sites were not present in the skeletal muscle. The pig umbilical arteries, vein and Wharton's jelly contained a 75 kDa immunoreactive LH-hCG receptor protein similar to that found in corpora lutea. Southern blot analysis of reverse transcription-polymerase chain reaction products, performed to enhance the sensitivity and specificity of LH receptor transcripts determination in umbilical cord tissues, revealed that the expected fragments of 740 and 470 bp were present in the arteries, vein, Wharton's jelly and corpora lutea (positive control). An additional product of 670 bp was found in the corpora lutea and arteries of umbilical cord, but not in the vein and Wharton's jelly. This is probably the first reported evidence of the presence of LH-hCG receptors in the umbilical cord of a non-human female mammal.

Ontogeny of LH/hCG receptor gene expression in male and female pig reproductive tracts.

The receptors for LH, located in the gonadal tissues of the male and female, mediate stimulation of ovarian and testicular steroidogenesis by the pituitary. However, it is known that several animal and human nongonadal reproductive tissues also contain functional LH receptors. This study was undertaken to compare expression of LH/hCG receptor gene expression in gonadal and nongonadal reproductive tissue of the male and female fetal and neonatal pigs. We used reverse transcription-polymerase chain reaction (RT-PCR) to examine the onset of LH/hCG receptor gene expression in both gonadal and nongonadal tissues during ontogeny of the pig. According to our results the expression of LH/hCG receptor gene as measured by the presence in its extracellular domain starts between 30 and 40 d post coitum in the testis and between 40 and 48 d post coitum in the ovary. The expression of extragonadal LH/hCG receptor gene in the uterus occurred between Days 48 and 103 post coitum. When detected, the extracellular domain of LH/hCG receptor gene was expressed permanently both in gonadal and nongonadal porcine reproductive tissues. We also showed that LH/hCG receptors are expressed in the pig epididymis on Day 1 of neonatal life.

Cycle dependent LH/hCG receptor gene expression in porcine nongonadal reproductive tissues.

Uterine and other nongonadal reproductive tissues are a new target for LH and CG action in pig and many other mammalian species, including humans. Since rat and pig LH/CG receptor cDNAs have been cloned we have been prompted to look for the expression of this gene in porcine uteri and oviducts. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out on total RNA isolated from endometrium, myometrium and oviduct. Ovarian and testicular RNA served as positive and skeletal muscle RNA as negative control. RT-PCR was designed to amplify part of the extracellular domain (exons II-IX) of the LH/CG receptor. Southern blot with cDNA probe was used to verify the authenticity of the cDNA species generated. A PCR product of an expected size of 619 bp corresponding to exons II-IX was detected in, besides gonads, both the endometrial and myometrial uterine layers as well as in oviducts. Southern blot revealed presence of additional, shorter fragment (about 400 bp) in corpus luteum, myometrium and oviduct. This additional fragment was found in myometrium during the follicular and late luteal phases as well in oviducts during early luteal, early and late follicular phases. Also, testes showed two RT-PCR products. There was no DNA amplification after reverse transcription in the reaction mixture containing skeletal muscle total RNA. The results of our paper have demonstrated that the LH/CG receptor gene is expressed in the porcine nongonadal reproductive tissues and the expression is dependent on the estrous cycle phase and probably is also tissue specific.