Effects of nonglycosylated and glycosylated prolactin on basal and gonadotropin-stimulated steroidogenesis in chicken ovarian follicles.

In galliformes, the circulating isoform of prolactin (PRL) significantly changes during different reproductive states. However, the role of the major isoform (glycosylated PRL [G-PRL]) in ovarian steroidogenesis is unknown. The present study aimed to compare the effects of nonglycosylated (NG-) and G-PRL on basal and gonadotropin-stimulated estradiol (E2) and progesterone (P4) production in granulosa cells or follicular walls of chicken of different size class follicles. In the initial experiment, granulosa cells of preovulatory F3-F1 and prehierarchical 6- to 8-mm follicles were incubated for 24 h with different concentrations of NG- or G-PRL (0, 1, 10, 100, or 1,000 ng/mL). In the subsequent experiments, these categorized granulosa cells and follicular walls of prehierarchical 4-6, 2-4, and <2-mm follicles were incubated for 24 h in the absence and presence of 10-ng/mL FSH or LH, or in combination with different concentrations of NG- or G-PRL (10, 100, or 1,000 ng/mL). We observed that lower levels of NG-PRL induced (P < 0.05) E2 and P4 secretion in granulosa cells of either preovulatory or prehierarchical follicles, but at higher levels, this effect was reduced. In contrast, G-PRL promoted (P < 0.05) basal E2 and P4 secretion in preovulatory granulosa cells but was inhibitory (P < 0.05) in prehierarchical granulosa cells. Results obtained by real-time quantitative PCR (qPCR) demonstrated that these effects were mediated through modulation of the expression of StAR, CYP11A1, CYP19A1, and 3ß-HSD. Furthermore, G-PRL was less potent than NG-PRL in inhibiting FSH- or LH-stimulated E2 and P4 production in granulosa cells of preovulatory follicles, whereas NG-PRL enhanced (P < 0.05) but G-PRL reduced (P < 0.05) FSH-induced P4 production in those of prehierarchical follicles. In follicular walls from each group of prehierarchical 4-6, 2-4, and <2-mm follicles, NG- and G-PRL had both stimulatory and inhibitory influences on the actions of FSH on E2 and P4 secretion, but both suppressed (P < 0.05) LH-induced E2 and P4 secretion except for the synergistic effects of LH and G-PRL on P4 secretion by follicular walls of the follicles of 4-6 mm. Taken together, these results suggest that both NG- and G-PRL are biologically active in regulating basal and gonadotropin-stimulated E2 and P4 production in chicken ovarian follicles. However, their effects are different depending on the concentration, the type of gonadotropin (FSH or LH), and the stage of follicle development.

Development of a real-time (Q) PCR assay to measure variation in expression of prolactin receptor mRNA in the hypothalamus and pituitary gland during late embryogenesis in turkeys and chickens.

Changes in levels of PRLR mRNA in the pituitary gland and hypothalamus of chickens and turkeys from embryonic day (ED) 15 and ED21 to 1 day post-hatch, respectively, were measured by real-time PCR. In both species, PRLR mRNA increased from low levels during the last week of ED to reach maxima at the peri-hatch period. Similarly, circulating levels of PRL also increased during this interval and were highly correlated with levels of the PRLR mRNA in both the pituitary gland and hypothalamus. This suggests that PRL was up-regulating its receptor. In support of this, stimulation of the turkey pituitary gland with VIP on ED24 resulted in a 4- and 3-fold increase in PRL and PRLR, respectively. Since VIP had no direct effect on the levels of PRLR transcript in the hypothalamus, it is likely that VIP is acting indirectly through increased PRL to up-regulate the number of receptors.

Genetic variability of the cytosolic phosphoenolpyruvate carboxykinase gene in white leghorn chickens.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. Genetic variations in this gene may therefore affect a wide variety of traits, including tumor growth that is heavily dependent on glucose metabolism. We have previously shown the gene coding for the mitochondrial form of PEPCK (PEPCK-M) segregates for markers that are collected with resistance to Marek's disease. In this communication we analyze the genetic variability of PEPCK-C, the gene which codes for the cytosolic form of PEPCK. A 3,792-bp segment of 5'-region of the PEPCK-C gene (position -1723 to 2069) was sequenced in four individuals from eight different strains of White Leghorn chickens (a total of 64 genomes). A total of 19 single nucleotide polymorphisms (SNP) were identified. Neither deletions nor insertions were present. The most frequent SNP were transitions (79%), and in most cases the ancestral allele coincided with CpG dinucleotides (10-fold excess after correcting for dinucleotide frequencies). A gene tree was constructed assuming maximal parsimony. It led to the delineation of 6 haplotypes (combination of alleles). Two of the SNP coincided with RFLP detectable by the restriction enzymes AciI and BstEII, respectively. Based on this analysis we can now identify individuals with the evolutionary most distant PEPCK-C haplotypes, establish strains of these haplotypes, and analyze trait associations and epistasis with other genes.

Molecular cloning of chicken vasoactive intestinal polypeptide receptor complementary DNA, tissue distribution and chromosomal localization.

Chicken vasoactive intestinal polypeptide receptor (VIPR) cDNA was cloned by the reverse transcription-polymerase chain reaction method using primers designed on the basis of other species of VIPR cDNA. The cDNA obtained was sequenced by the dideoxy-mediated chain-termination method. Of the 2227 nucleotides that were sequenced, 84, 855, and 1338 bases represent the 5'-untranslated region (UTR), the 3'-UTR, and the open reading frame that predicts a peptide of 446 amino acids. The cDNA of the chicken VIPR shows 65% and 60% homologies to human cDNA of VIP1 and VIP2 receptors, respectively. The clone had the expected similarity to highly conserved features of the other G protein-coupled receptors (GPCRs) such as six cysteine residues that are functionally important in the VIPR subfamily. In addition, the seven potential membrane-spanning domains characteristic of the family B group III GPCR superfamily and highly conserved motif within the third cellular loop between transmembrane regions 5 and 6. Northern blot hybridization analysis in this study indicated mRNA expression of VIPRs in the various tissues of the chicken. Strong signal was detected in the brain and anterior pituitary gland. High levels of VIPR mRNA in the brain was consistent with VIP-binding experiments and with the function of VIP in the brain as a neuroendocrine factor or neurotransmitter. Expression of VIPR was detected in the anterior pituitary gland of chick embryos. The expression of VIPR mRNA in the chick anterior pituitary gland may indicate a regulatory function of VIP on prolactin (PRL) production or PRL cell proliferation during embryogenesis. Chicken VIPR shows high homology with mammalian type I VIPR but, in some part, possesses similarity of amino acid sequence. Expression of VIPR in various tissues supports diverse functions for VIP in the chicken.

In vitro release of isoforms of prolactin from pituitary glands of turkey hens at different physiological stages.

To study the in vitro release of PRL isoforms, anterior pituitary glands from medium white turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Pituitaries were cut into hemi-pituitaries after collection and placed into separate perifusion chambers. Medium (M199) was continuously perifused through the system and pituitaries were stimulated with cVIP (10(-7) M). Total PRL content was monitored by RIA and, the ratio of immunoreactive PRL isoforms in the perifusate was estimated by Western blotting. After exposure to X-ray film for autoradiography, the relative intensity of the bands was analyzed by densitometry. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands with relative molecular weights of 24 and 27 kDa were detected by Western blotting. The immunoreactive band corresponding to the glycosylated isoforms of PRL (27 kDa) was predominant in samples from egg-laying and incubating hens and the band corresponding to the nonglycosylated isoform (24 kDa) was predominant in samples from out-of-lay and molting stages. No changes in the ratio of isoform released were detected during cVIP stimulation. Our data clearly show that glycosylated and nonglycosylated PRL isoforms are released by the pituitary gland in vitro in the same relative proportion that was previously observed in pituitary extracts and thus are likely to reflect the secreted forms of PRL in the blood during various physiological stages. In addition, the PRL-releasing activity of VIP does not affect the ratio of isoforms secreted by the pituitary gland in vitro.

Mitochondrial PEPCK: a highly polymorphic gene with alleles co-selected with Marek's disease resistance in chickens.

The gene coding for the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), a pivotal component in gluconeogenesis from lactate via the Cori cycle, was highly polymorphic in strains of egg-type chickens (White Leghorn) of different origins. Based on MspI restriction fragment polymorphisms a total of seven alleles could be distinguished. The allele frequencies were determined in six pairs of strains derived from different genetic base populations. Each pair consisted of two strains which differed in their susceptibility to Marek's disease (MD), a virus-induced neoplastic disease. The frequency of the most common haplotype (M2) was consistently higher in the susceptible strains than in the corresponding resistant strains (P < 0.05, Wilcoxon signed-ranks test), indicating that the observed differences were not due to random genetic drift. This result suggests that PEPCK-M may be a candidate gene which contains genetic variants affecting MD susceptibility. Variations in gluconeogenesis may affect the interplay between proliferation of neoplasia and host metabolism.

Prevention of incubation behavior expression in turkey hens by active immunization against prolactin.

The consequences of active immunization against prolactin on expression of incubation, reproductive performance and hormonal profiles were evaluated in turkey hens. Hens were injected weekly for 4 wk starting 8 wk before being submitted to a stimulatory photoperiod and 3 times thereafter at intervals of 4 to 5 wk. The hens were injected i.d. with 0.5 mL of a mixture diluted half in Freund's adjuvant. The mixture was prediluted in .9% saline and contained 100 micrograms of a fusion protein (GST-tPRL), GST, oPRL or vehicle. The results indicate that active immunizations with GST-tPRL or oPRL both induce production of specific prolactin antibodies. The onset of egg production was unaffected but higher egg production was observed for the GST-tPRL immunized hens. No GST-tPRL immunized hens expressed incubation behavior, whereas 20 to 30% of hens in the other experimental groups did so. Apparent hyperprolactinemia was detected by RIA for the GST-tPRL immunized groups starting before photostimulation and lasting until Week 10 of egg production but not in other groups. No significant differences were observed in either plasma LH or estradiol concentrations of immunized and nonimmunized turkey hens. In conclusion, both GST-tPRL or oPRL induced the production of antibodies against prolactin in turkey hens. However, only active immunization using GST-tPRL induced higher antibody titers as well as full prevention of incubation behavior expression. Such a pharmacological approach is of great practical interest, although its uses need to be carefully evaluated under commercial conditions.

Prevention of the expression of incubation behaviour using passive immunisation against prolactin in turkey hens (Meleagris gallopavo).

The efficacy of injecting antibodies raised against turkey prolactin to prevent the expression of incubation behaviour has been investigated in turkey hens. Medium white turkey hens (n = 15 x 2) were injected three times weekly for 4 consecutive weeks starting on week 5 of egg production. The hens were injected im with a volume of 1 mL per injection for the 1st week and 0.5 mL thereafter, of normal rabbit serum or serum containing antibodies raised against turkey prolactin (Guémené et al, 1994a). None of the 15 passively immunised hens expressed incubation behaviour, whereas, more than half (53%) of the control hens did express it. Plasma prolactin concentrations observed in the two groups presented comparable profiles until week 9 and from week 19 of egg production onward. Differences were, therefore, observed from week 10 until week 17 with the non immunised hens showing higher plasma prolactin concentrations than the immunised ones. This difference was related to the presence of incubating hens in the control group. A higher percentage of non immunised hens disrupted egg production during the course of the study and consequently immunised hens laid more eggs than the control ones. No change in plasma LH and oestradiol concentrations can be related to the immunisation procedure. We conclude that prevention of incubation behaviour can be achieved using passive immunisation against prolactin, prevention which resulted in more egg production under our experimental protocol.

Molecular cloning, tissue distribution, and expression of the prolactin receptor during various reproductive states in Meleagris gallopavo.

The primary sequence of the prolactin receptor (PRL-R) in turkeys was deduced from a cDNA clone isolated from a kidney cDNA library and from a polymerase chain reaction (PCR) product. The open reading frame of the turkey PRL-R (tPRL-R) predicted an 831-amino acid protein composed of a leader peptide, an extracellular domain, a single transmembrane domain, and an intracellular domain. The extracellular domain contained two homologous repeat units with 63% amino acid sequence identity to each other. Each repeat unit contained all of the conserved cysteine pairs and a WSXWS motif found in mammalian PRL-Rs. A tPRL-R transcript with a molecular size of about 3000 nucleotides was identified by Northern blot analysis. The tPRL-R transcripts were detected in all 26 tissues examined using reverse transcriptase PCR (RT-PCR). The pituitary gland, hypothalamus, crop sac, duodenum, and gizzard were found to express the highest levels of tPRL-R among the 26 tissues. The expression levels of tPRL-R in 17 tissues were compared using semi-quantitative RT-PCR in nonphotostimulated, laying, out-of-lay, incubating, and maternal hens, and male birds. In most tissues examined there was no obvious relationship between blood levels of PRL, reproductive states, and estimated concentrations of the receptor mRNA. In the pituitary gland and hypothalamus, plasma levels of PRL and levels of tPRL-R transcript were inversely correlated. In the hypothalamus, increasing blood levels of PRL were associated with decreasing levels of the receptor transcript (p < or = 0.05), whereas the opposite was observed in the pituitary gland (p < or = 0.05). These findings support the hypothesis that PRL itself may participate in the neuroendocrine control of incubation behavior through actions on both the hypothalamus via a short-loop feedback mechanism and the pituitary gland via autocrine and/or paracrine effects.

Production and characterization of recombinant turkey prolactin.

1. Recombinant turkey prolactin (rctPRL) was produced as a fusion protein in E. coli, purified by affinity chromatography followed by cleavage with thrombin. The final yield of the released rctPRL (> 90% purity) was 1-2 mg/l of bacterial culture. 2. Recombinant tPRL co-migrated with the main immunoreactive band (25 kDa) in turkey pituitary extracts and was identical to natural tPRL except for the addition of three amino acids (Gly-Ser-Ser) resulting from the cloning strategy at the amino terminal end. 3. The bioactivity of the rctPRL was equipotent to ovine PRL in a rabbit mammary explant system and in the Nb2 lymphoma mitogenic assay.

Synergism between the endogenous viral loci ev6 and ev9 in inducing immunological tolerance to avian leukosis virus.

1. The course of infection by exogenous avian leukosis virus was followed in a commercial strain of White Leghorn domestic fowls by measuring viral antigen in feather pulp and egg albumin. Ten days after hatching, 2 out of 360 birds tested positive and at 286 days of age about 60% of the birds had been antigen positive at least once. 2. Among the antigen positive birds, two groups could be distinguished: those which permanently and those which transiently expressed viral antigen. Permanent antigen expression was associated with low antibody titres, while transient antigen expression was associated with high antibody titres. 3. The strain segregated for the two endogenous viral genes ev6 and ev9, both of which express endogenous viral envelope protein, and have been implicated in affecting immune-responsiveness. The antibody titre in individuals positive for both ev6 and ev9, was significantly lower than in those which had none or only one of the two ev-genes. In addition, individuals positive for both ev-genes occurred more frequently in the group permanently positive for viral antigen than in the group transiently antigen positive. 4. The results indicate that there was a strong synergism between ev6 and ev9 in reducing the antibody response to exogenous avian leukosis virus infection, perhaps by inducing immune tolerance or interfering with antibody formation.

Gonadotropin-stimulated estradiol production in small ovarian follicles of the hen is suppressed by physiological concentrations of prolactin in vitro.

The in vitro effects of physiological levels of prolactin (PRL) on luteinizing hormone (LH)-stimulated estradiol production by four classes of small follicles (super small (SS) less than 1 mm; small white (SW) 1-2 mm; large white (LW) 3-5 mm; and small yellow (SY) 5-10 mm) were compared in laying and out-of-lay (OL) Gifujidori hens. All classes of follicles responded to increasing doses of LH in a dose-dependent manner and the estradiol content of the media increased two to five-fold when 8 ng/ml of ovine LH was included in the incubation media. Ovine PRL (up to 1 micrograms/ml) had no effect on the LH-stimulated rise in media content of estradiol from SW or LW follicles from either the laying or OL hens, whereas the rise in the estradiol content of the media from SS follicles was blocked or reduced by 500 ng/ml PRL in the laying and OL hens, respectively. This suggests that the SS follicles are a target organ for PRL and that PRL may act, in part, at the level of the ovary to reduce estradiol production at the onset of incubation behavior and thus cause ovarian regression.

Changes in plasma levels of prolactin and estradiol, nutrient intake, and time spent nesting during the incubation phase of broodiness in the Chabo hen (Japanese bantam).

The plasma concentrations of prolactin (PRL) and estradiol, time spent nesting, consumption of feed and water, body weight, and hematocrit were measured during egg laying, incubating, and brooding of the chicks in a Japanese strain of dwarf bantam hen, Chabo. Plasma levels of PRL increased before incubation, were maintained at high levels during incubation, and decreased rapidly at the onset of hatching the young. The concentration of estradiol decreased before incubation and was maintained at low levels when circulating levels of PRL were high. During incubation, hens spent greater than 95% of the day on the nest, reduced their daily intake of feed and water by 63 and 78%, respectively, and decreased their body weight by 19% by the end of incubation. Thus, Chabo hens show a mode of incubation behavior similar to that reported in larger chickens. The temporal changes between nutrient intake and plasma levels of PRL at the start and end of incubation behavior suggested that changes in nutrient intake may not cause changes in the concentration of PRL, whereas the association between increased levels of PRL and decreased levels of estradiol suggested that they may be causally associated. Plasma levels of PRL also appeared to be associated with time spent on the nest.

Effect of pregnant mare serum gonadotropin on plasma prolactin, luteinizing hormone, estradiol, and ovarian growth in incubating and out-of-lay turkeys.

The effect of pregnant mare serum gonadotropin (PMSG) on ovarian growth and estradiol production was assessed in out-of-lay (OL) turkey hens that have low plasma concentrations of prolactin (PRL) and in incubating hens that have high plasma levels of PRL. In OL hens after injection with 400 or 2,000 IU PMSG, plasma concentration of PRL did not change, whereas ovarian weight and plasma concentration of estradiol increased by greater than 20-fold to levels comparable to those of laying hens. Follicles were not arranged in a hierarchy following the injection of either 400 or 2,000 IU PMSG into OL hens. There was a linear relationship between the dose of PMSG injected into incubating hens and the subsequent increase in plasma concentration of estradiol. Plasma levels of PRL were not different among hens injected with 0, 16, 80, or 400 IU PMSG, whereas plasma levels of PRL decreased in incubating hens injected with 2,000 IU PMSG. A significant increase in ovarian weight occurred only in hens injected with 2,000 IU PMSG. None of the hens deserted the nest following the injection of PMSG, indicating that the maintenance of incubation does not require a steroidogenically quiescent ovary. Although the regressed ovaries of both OL and incubating hens are responsive to gonadotropin stimulation, it would seem that the higher levels of PRL in incubating hens may act, in part, to suppress PMSG-induced ovarian growth and steroidogenesis.

Effects of ovariectomy or force feeding on the plasma concentrations of prolactin and luteinizing hormone in incubating turkey hens.

The effect of ovariectomy (OVX) on plasma concentrations of prolactin (PRL) and luteinizing hormone (LH) in incubating turkey hens was studied. Neither the sham-operated nor the OVX hens exhibited any change in the pattern of incubation behavior as a result of the surgery. Plasma concentrations of estradiol decreased to less than approximately 3 pg/ml by 2 days after surgery in the OVX hens. There were no significant differences in plasma levels of PRL between the sham-operated and OVX hens throughout the study. The concentration of PRL did not change in either the sham-operated or OVX hens and was maintained at high levels after surgery and during incubation of the eggs. By 2 days after hens were placed into cages, plasma levels of PRL significantly decreased and were maintained at low levels in both groups. The concentration of LH did not change in either group during the two wk after surgery when the hens were incubating eggs. After the hens were placed into cages, the concentration of LH increased in the OVX hens and was maintained at significantly higher levels than in the sham-operated hens. By contrast, the concentration of LH increased within 4 days after OVX of out-of-lay but nonincubating hens. The delay in the postcastration increase in plasma level of LH in the OVX hens was not associated with anorexia of incubating hens, since plasma levels of LH were not affected by force-feeding unless plasma levels of PRI were suppressed by nest deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)