Effects of feeding diets containing bacitracin methylene disalicylate to heat-stressed finishing pigs.

The objective of this study was to evaluate the effects of heat stress and dietary bacitracin methylene disalicylate (BMD) on growth performance, carcass characteristics, and immunological responses in finishing pigs. Four groups of 32 finishing pigs (n = 128) with initial BW between 80 to 90 kg were used. Pigs were fed a corn-soybean meal-distillers grains-based control or BMD (31.5 mg/kg) diet for a 14-d adaptation period at the thermal neutral temperature (23°C), and continued to be fed their respective diets when exposed to a constant temperature (23°C) or a cyclical heat stress environment (37°C from 1000 to 1900 h and 27°C from 1900 to 1000 h) for a 28-d experimental period. Each group of pigs was housed in 4 rooms, with 2 pens/room and 4 pigs/pen. Saliva samples from each pig were collected on d -1 (initial baseline), 1, 13, and 27 for cortisol analysis. Concentrations of haptoglobin, IL-1ß, and tumor necrosis factor-α were determined in serum samples on d -1, 1, 13, and 27. Pigs exposed to heat stress had 31% less ADG (P < 0.001), 23% less ADFI (P < 0.001), 9% less G:F (P < 0.001), and 34% greater average daily water intake (P = 0.03) than those in the non-heat-stress conditions. Dietary BMD tended to reduce ADG (P < 0.07) compared with the control (0.66 vs. 0.73 kg/d, respectively). Heat stress increased (P < 0.05) saliva cortisol on d 1, but no effects were observed on subsequent days. Serum haptoglobin concentrations were greater (P < 0.05) in heat-stressed pigs on d 1, and concentrations tended to remain greater (P < 0.1) on d 13. Pigs fed the BMD diet tended to have a longer villus height (P = 0.07) in the duodenum and greater crypt depths in the duodenum (P = 0.09) and jejunum (P = 0.07). Heat-stressed pigs tended to have a decreased proportion of propionate (P = 0.08), greater acetate:propionate (P = 0.08), and a reduced proportion of valerate (P = 0.02) in the cecum. These results indicate that BMD did not counteract the negative effects of heat stress on growth performance, but BMD appears to increase villus height and crypt depth in the duodenum. Furthermore, heat stress appears to alter VFA production in finishing pigs.

Chicken embryo extract mitigates growth and morphological changes in a spontaneously immortalized chicken embryo fibroblast cell line.

The SC-1 spontaneously immortalized chicken embryo fibroblast (CEF) cell line has been established recently. Although this cell line had been in culture for over 3 yr, its growth rate has remained lower than that of primary CEF cells, and the morphology has not been as uniform as observed in primary cells. In the present study, the SC-1 cell line was treated with chicken embryo extract (CEE) to determine whether growth rates could be increased and cell morphology enhanced. The CEE also was tested on primary CEF cells, another spontaneously immortalized CEF cell line (DF-1), and on 2 other nonvirally and nonchemically immortalized CEF cell lines (BCEFi and HCEFi). Results indicated that concentrations of CEE > or = 100 microg/mL inhibited growth of all cells tested. However, addition of 50 microg of CEE/mL enhanced the growth rate and improved the morphology of the SC-1 cells. Addition of CEE to the other immortal or primary CEF cells did not increase the growth rate or change their morphology. Analysis of mRNA expression revealed that SC-1 cells treated with 50 microg of CEE/mL had lower levels of the p16(INK4a) alternate reading frame sequence (ARF) and E2F-1 than untreated SC-1 cells. The increased growth rate and improved morphology of the SC-1 cells achieved with CEE treatment were retained following removal of CEE, and these improvements should aid in increasing the utility of the SC-1 cell line as a cellular/molecular reagent.

The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells.

The steady-state levels of p53 mRNA were dramatically lower in immortal chicken embryo fibroblast (CEF) cell lines compared to primary CEF cells. In the presence of cycloheximide (CHX), the steady-state levels of p53 mRNA markedly increased in immortal CEF cell lines, similar to levels found in primary cells. The de novo synthetic rates of p53 mRNA were relatively similar in primary and immortal cells grown in the presence or absence of CHX. Destabilization of p53 mRNA was observed in the nuclei of immortal, but not primary, CEF cells. The half-life of p53 mRNA in primary cells was found to be a relatively long 23 h compared to only 3 h in immortal cells. The expression of transfected p53 cDNA was inhibited in immortal cells, but restored upon CHX treatment. The 5'-region of the p53 mRNA was shown to be involved in the rapid p53 mRNA destabilization in immortal cells by expression analysis of 5'- and 3'-deleted p53 cDNAs as well as fusion mRNA constructs of N-terminal p53 and N-terminal deleted LacZ genes. Together, it is suggestive that the downregulation of p53 mRNA in immortal CEF cells occurs through a post-transcriptional destabilizing mechanism.

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase.

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.

Alterations in p53 and E2F-1 function common to immortalized chicken embryo fibroblasts.

A number of non-virally and non-chemically immortalized chicken embryo fibroblast (CEF) cells have been established recently in continuous cell culture. All immortal CEF cells tested showed common genetic alterations in the expression patterns of p53 and E2F-1 mRNA and protein which were down- and up-regulated, respectively. The biological effects of differentially regulated p53 and E2F-1 were determined by reporter gene transcriptional activity assays, DNA binding assays, and Northern blot analysis of the expression patterns of down-stream genes. In addition, expression of most of the cyclin genes was up-regulated in immortal CEF cells, which may be associated with the rapid cell division rates and serum-independent growth patterns seen in immortal CEF cells. The telomeric lengths and chromosome integrity were maintained in all immortal CEF cell lines without detectable telomerase activity. Although the functional inactivations of the p53 and Rb regulatory pathways are known to be common events for cellular immortalization, the genetic changes leading to alteration of p53 and E2F-1 function through transcriptional and post-transcriptional regulation seem to be unique in immortal CEF cells.

Post-transcriptional inactivation of p53 in immortalized murine embryo fibroblast cells.

The steady-state levels of p53 mRNA and protein were barely detectable by Northern and Western blot analysis in spontaneously immortalized (10)3 and (10)7 murine embryo fibroblast (MEF) cells. But when cells were treated with cycloheximide (CHX) or emetine, expression levels were restored to those observed in primary and immortal (10)10 MEF cells. However, levels of p53 mRNA were not changed in primary or (10)10 MEF cells by CHX treatment. De novo p53 mRNA synthetic rates were similar in primary, (10)10, (10)3, and (10)7 MEF cells treated with or without CHX. Treatment with actinomycin D (ActD) showed that p53 mRNA in primary and (10)10 MEF cells had a relatively long half-life of 22 h, compared to less than 2 h for (10)3 and (10)7 MEF cells. Pulse-chase analysis of p53 mRNA turnover using CHX and ActD showed that the rapid destabilization of p53 mRNA in (10)3 and (10)7 MEF cells could be regulated at the transcriptional and translational levels. In addition, the destabilization of p53 mRNA appeared to occur in the nucleus for (10)3 and (10)7 cells, but not for primary and (10)10 MEF cells. Taken together, the present study demonstrates that inactivation of the p53 gene occurs at the post-transcriptional level by rapid destabilization of its mRNA in the nucleus of spontaneously immortalized (10)3 and (10)7 MEF cells.

Increased mitochondrial-encoded gene transcription in immortal DF-1 cells.

We have established, in continuous cell culture, a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (DF-1) as well as several other immortal CEF cell lines. The immortal DF-1 cells divided more rapidly than primary and other immortal CEF cells. To identify the genes involved in rapidly dividing DF-1 cells, we have used differential display RT-PCR. Of the numerous genes analyzed, three mitochondrial-encoded genes (ATPase 8/6, 16S rRNA, and cytochrome b) were shown to express at higher levels in DF-1 cells compared to primary and other immortal CEF cells. The inhibition of mitochondrial translation by treatment with chloramphenicol markedly decreased ATP production and cell proliferation in DF-1 cells, while not affecting growth in either primary or other immortal CEF cells. This result suggests a correlation between rapid cell proliferation and the increased mitochondrial respiratory functions. We also determined that the increased transcription of mitochondrial-encoded genes in DF-1 cells is due to increased de novo transcript synthesis as shown by mitochondrial run-on assays, and not the result of either increased mitochondrial biogenesis or mitochondrial transcript half-lives. Together, the present studies suggest that the transcriptional activation of mitochondrial-encoded genes and the elevated respiratory function should be one of the characteristics of rapidly dividing immortal cells.

Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha.

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHalpha. The steady-state mRNA levels of cDcoHalpha were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHalpha showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHalpha increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHalpha mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHalpha mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHalpha probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHalpha showed similar functional activity as measured by increased expression of a reporter gene, as well as alpha-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHalpha might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states.

Molecular cloning and expression of turkey inhibin-alpha and -betaA subunits.

We isolated cDNA encoding turkey inhibin-alpha (tINH-alpha) and -betaA (tINH-betaA) subunits from the turkey ovary using reverse transcription-polymerase chain reaction (RT-PCR). The isolated alpha subunit and betaA subunit included the entire open reading frames encoding 329 and 424 amino acids, respectively. The amino acid sequences of mature tINH-alpha subunit and tINH-betaA subunit (12.6 and 12.9 kDa proteins, respectively), established via DNA sequence analysis, were highly conserved between the chicken and various mammals. Northern blot analysis revealed that the transcripts of tINH-alpha and tINH-betaA subunits were approximately 1.7 and 8.4 kb, respectively. In various stages of follicular development, tINH-alpha mRNA was highly expressed in small white follicles as compared to postovulatory and regressed follicles, whereas tINH-betaA mRNA was predominately expressed in preovulatory F5 follicles.

Molecular cloning and characterization of alternatively spliced transcripts of the turkey pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases the release of growth hormone (GH) and prolactin (PRL) in mammals. However, the evolutionary and functional relationships of PACAP, GH, and PRL are not clear. To understand how PACAP is regulated in the turkey, a turkey PACAP (tPACAP) cDNA has been cloned by the combination of reverse transcription-polymerase chain reaction and the rapid amplification of cDNA 5'- and 3'-ends. The deduced amino acid sequence of tPACAP-38 and turkey PACAP-related peptide (tPRP) displayed 87-97 and 52-63% similarity when compared to a variety of known PACAP-38 and PRP sequences, respectively. Two major transcripts (1.3 and 3.0 kb) of tPACAP were detected by Northern blot analysis. The highest levels of tPACAP mRNA were shown to be expressed in the hypothalamus, the cerebellum, and the cerebrum. In contrast, most of the other tissues tested expressed relatively low steady-state levels of tPACAP mRNA. Alternative splicing of tPACAP resulted in the expression of two different isoforms. The smaller form of tPACAP was expressed in the hypothalamus during early embryonic development and decreased significantly in later stages.

Reproductive tract secretions and bull spermatozoa contain different clusterin isoforms that cluster cells and inhibit complement-induced cytolysis.

Clusterin from bull rete testis fluid (RTF), cauda epididymal fluid (CEF), and octyl-beta-D-glucopyranoside extract of cauda epididymal sperm (CES) was identified and characterized using monoclonal and polyclonal antibodies (Abs) developed against ram clusterin and a beta-subunit-specific oligopeptide of porcine clusterin. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting showed that bovine RTF clusterin had dimeric and monomeric molecular weights (M(r)s) of approximately 94 kDa and of 42 and 43 kDa, respectively. Clusterin in CEF and CES had similar dimeric M(r)s (74 kDa). Reduced CEF clusterin appeared as three monomers (M(r)=40, 39, and 38 kDa), whereas reduced CES clusterin appeared only at M(r)40 kDa. Enzymatic deglycosylation resulted in similar M(r)s of clusterin from RTF, CEF, and CES. The M(r) of RTF clusterin decreased from 94 kDa to 51 kDa, indicating a carbohydrate content of 45%. After deglycosylation, the M(r) of the CEF clusterin decreased from 74 kDa to two distinct bands at 51 and 50 kDa (with carbohydrate contents of 31 and 32%, respectively), suggesting that two isoforms of the heterodimeric protein are present because of the two isoforms of the alpha-subunit. Under nonreduced conditions, a beta-subunit-specific Ab reacted with M(r) of 36-38 kDa, indicating the existence of free clusterin beta-subunits in CES. RTF, CEF, and CES extracts all caused mouse fibroblastic L-cell aggregation. CEF cell aggregation was inhibited by Hyb-17 Ab but not by other Abs. Both RTF and CEF caused a dose-dependent inhibition of complement-induced cytolysis, although RTF clusterin was more potent than CEF clusterin. We conclude that several isoforms of clusterin occur in the bull reproductive tract and that the variation in carbohydrate content among these isoforms may affect the biological or functional activity of the protein.

Association of specific DNA binding and transcriptional repression with the transforming and myogenic activities of c-Ski.

The ski oncogene encodes a transcription factor that induces both transformation and muscle differentiation in avian fibroblasts. The first 304 amino acids of chicken Ski, the transformation domain, are both necessary and sufficient to mediate these biological activities. Ski's biological duality is mirrored by its transcriptional activities: it coactivates or corepresses transcription depending on its interactions with other transcription factors. Ski represses transcription through specific binding to GTCTAGAC (GTCT element) but it possesses a transferable repression activity that can function independently of this DNA element. In this study, we locate this repression domain to the NH2-terminal two-thirds and the GTCT binding region to the COOH-terminal one-third of Ski's transformation domain. Mutations in the transformation domain of c-Ski reveal a strong correlation between GTCT-mediated transcriptional repression and the biological activities of transformation and myogenesis. We also show that a dimerization domain located at the COOH terminal end of the Ski protein increases its transforming activity and its binding to GTCTAGAC.

Vasoactive intestinal peptide stimulates turkey prolactin gene expression by increasing transcription rate and enhancing mRNA stability.

This study evaluates the transcriptional and post-transcriptional regulation of prolactin (PRL) by vasoactive intestinal peptide (VIP). Pituitary nuclei from laying (control), incubating (with enhanced VIP secretion), and VIP-immunized laying turkey hens, and from pituitary cells cultured with or without VIP were used in nuclear run-on transcription assays. Cytoplasmic PRL mRNA was analyzed by slot blot hybridization. PRL transcription was greater in hyperprolactinemic incubating birds (PRL/beta-actin=3.33) than in laying birds (PRL/beta-actin=1.83). VIP-immunoneutralized birds had 47% and 51% decreases in PRL transcription and cytoplasmic PRL mRNA, respectively when compared with laying birds. In primary pituitary cell cultures, VIP significantly increased the transcription rate of PRL (3.8-fold) and cytoplasmic PRL mRNA (3.2-fold) compared with that of non-VIP-treated pituitary cells. The stability of pre-existing PRL mRNA was measured by Northern blot analysis after addition of actinomycin D. PRL mRNA half-lives were calculated using a two-component model, with a first-long component of 18.0+/-1.0 h and a second-short component of 3.7+/-0.7 h in non-VIP-treated pituitary cells. Both half-lives were significantly increased (53. 2+/-6.9 and 26.3+/-4.3 h) in VIP-treated cells. The present data show that VIP acts to stimulate PRL expression by up-regulating the transcription rate of PRL and by enhancing PRL mRNA stability.

The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses.

DF-1 is a continuous cell line of chicken embryo fibroblasts. The cells are free of endogenous sequences related to avian sarcoma and leukosis viruses and have normal fibroblastic morphology. DF-1 cells support the replication of avian retroviruses; diverse oncogenes induce foci of oncogenic transformation on monolayers of DF-1, and avian leukosis viruses of envelope subgroups B, D, and C induce cell death and form plaques. The new cell line will greatly facilitate studies on oncogenic transformation and cell killing by avian viruses.

The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors.

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis-sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has its env gene derived from the env gene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP.

Vasoactive intestinal peptide stimulates prolactin mRNA expression in turkey pituitary cells: effects of dopaminergic drugs.

It is well documented that vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor and that dopamine (DA) is an inhibitory neurotransmitter in avian species. However, the roles of VIP and DA in the regulation of PRL gene expression are unclear. In this study, primary anterior pituitary cells cultured from laying turkeys were utilized to investigate the influence of VIP and dopaminergic D1 and D2 receptors on PRL secretion, PRL mRNA, and PRL synthesis. Incubation of pituitary cells with VIP increased PRL secretion up to 3.5-fold within 3 hr. Prolactin mRNA was undetectable during the first 2 hr of pituitary cell treatment; thereafter, the PRL mRNA content response to VIP increased with 24-48 h (P < 0.05). Total PRL content (media + cellular) increased over time in the presence of VIP. The response of cells incubated in the presence of a dopaminergic D1 receptor agonist (SKF38393) was variable and inconclusive. However, cells incubated with a dopaminergic D2 receptor agonist (quinpirole) inhibited VIP-induced PRL secretion (P < 0.05) and PRL mRNA levels (P < 0.05) in a dose-related fashion without effect on the basal levels of PRL release and PRL mRNA. These observations suggest that VIP, in addition to acting as a PRL-releasing peptide, also plays a role in the regulation of PRL gene expression. Moreover, the results of this study also indicate that a drug that can selectively stimulate dopamine D2 receptors can also regulate PRL secretion and PRL mRNA in turkey pituitary cells in culture.

Tissue-specific alternative splicing of turkey preprovasoactive intestinal peptide messenger ribonucleic acid, its regulation, and correlation with prolactin secretion.

Although vasoactive intestinal peptide (VIP) is a well characterized physiological PRL-releasing factor in avian species, its regulated expression is not fully understood. We cloned complementary DNAs encoding the prepro-turkey VIP (prepro-tVIP) molecule from an adult turkey hypothalamic complementary DNA library. When the amino acid sequence of the prepro-tVIP was compared to chicken and mammalian sequence, it was found that the isolated tVIP molecules lacked the 27-amino acid peptide histidine isoleucine (PHI) portion of the precursor protein. Several tissues showed an alternatively spliced tVIP transcript that lacked the PHI sequence. Only in the hypothalamus did tVIP-specific primer pairs and reverse transcription-polymerase chain reaction produce two alternatively spliced fragments. The larger hypothalamus-specific fragment was subjected to nucleotide sequence analysis and identified as containing the alternatively spliced PHI-containing exon, which encoded a 27-amino acid PHI peptide in addition to the 8 amino acids that flanked the peptide. Hypothalamic tVIP expression was shown to be up-regulated during the incubation phase of the reproductive cycle. The increased steady state level of tVIP messenger RNA appears to be regulated by nesting behavior, because nest deprivation dramatically suppressed its expression. Levels of the minor tVIP transcript containing both the PHI- and VIP-encoding exons did not significantly change between reproductive stages and were maintained at approximately 4-6% of the total tVIP transcript level. Our findings provide further evidence that VIP is the most important PRL-releasing factor in birds. Our study should serve as a useful model for determining whether PHI contributes in any way to the physiological role of PRL regulation. Revealing the tissue distribution of VIP and PHI gene expression and tissue-specific alternative splicing could contribute to an understanding of the physiological functions of the two peptides as well as their relative roles in PRL regulation.

Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey.

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.

Sequence analysis of the turkey LH beta subunit and its regulation by gonadotrophin-releasing hormone and prolactin in cultured pituitary cells.

cDNAs encoding the precursor molecule of the turkey LH beta subunit (tLH beta) were cloned from a turkey pituitary cDNA library. The nucleotide sequence of the longest of two different tLH beta cDNA clones contained 592 bp, and included 23 bp of the 5' untranslated region (UTR) and 92 bp of the 3' UTR in addition to a 477 bp open reading frame that encoded a 39 amino acid leader polypeptide and a 120 amino acid mature apoprotein. Turkey and chicken LH beta sequences shared approximately 92 and 93% nucleotide and amino acid sequence similarities respectively. Northern blot analysis of total cellular anterior pituitary RNA showed that an approximate 800 base transcript hybridized to a 32P-labelled tLH beta cDNA probe. The gonadotrophin-releasing hormone (GnRH)- and prolactin (PRL)-regulated expression of LH and PRL in dispersed pituitary cells was determined by Northern blot analysis of tLH beta and PRL steady-state mRNA levels and by RIA analysis of secreted LH and PRL. GnRH-treated cells showed increased levels of both tLH beta mRNA and secreted LH, whereas mRNA and secreted levels of PRL did not change significantly. Cells treated with PRL showed lower levels of tLH beta and PRL mRNA as well as decreased release of LH and PRL. When cells were treated with both PRL and GnRH, increases in tLH beta mRNA and secreted levels of LH observed with GnRH alone were negated, whereas the decreases in mRNA and secreted levels of PRL observed with PRL alone were abrogated. These findings suggest that PRL can down-regulate tLH beta gene expression and spontaneous release of LH as well as autoregulate PRL gene expression and spontaneous release of PRL, while GnRH appears capable of modulating the effects of PRL-regulated LH and PRL gene expression and spontaneous release.

Theca cell cytochrome P450 17-hydroxylase and aromatase messenger ribonucleic acid abundance and serum steroid levels during follicular atresia associated with incubation behavior in the domestic turkey hen.

This study was designed to examine changes in cytochrome P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA contents in the theca layer of preovulatory follicles (POF) as turkey hens transit from egg laying to incubation. Hens were grouped into the following categories: 1) laying hens--laid one egg per day and nested 1-2 times per day; 2) transitional hens--laid one egg per day and nested > 4 times per day; and 3) Day 1, Day 3, and Day 5 incubating hens--laid no eggs for 2, 4, or 6 days, respectively, and nested > 4 times per day. Small white follicles (SWF) and the theca layer from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest POF were dispersed and challenged with testosterone (T) for 5 h. Relative levels of C17 and ARO mRNA were examined from the theca layers of F1, F3, F5, F7, and SWF. The number of atretic follicles increased from 0 (layers) to 8 (Day 5 incubating hens). Serum LH, progesterone (P), and estradiol (E), but not T, declined on Day 1 of incubation. Basal levels of P, T, and E from theca and SWF cells declined in incubating hens. Both basal and T-stimulated theca and SWF production of E decreased in incubating hens. C17 and ARO mRNA declined in SWF, F7, and F5 during follicular atresia. It is suggested that reduced gene expression of ovarian steroidogenic enzymes may be a partial determinant of reduced circulating sex steroid levels in incubating hens.(ABSTRACT TRUNCATED AT 250 WORDS)