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.

Application of the Sleeping Beauty transposon system to avian cells.

We have for the first time assessed the ability of the Sleeping Beauty (SB) transposon system to enhance transgenesis in chicken and turkey cells. The efficiency of transgenesis with a transposon encoding an antibiotic resistance gene was dramatically enhanced 15- to 35-fold when transposase was supplied by co-transfection of immortalized chicken and turkey cells with a construct encoding SB. In contrast, transgenesis of primary chicken embryo fibroblast (CEF) cells was not significantly increased by providing transposase, suggesting that the benefits of transposon-transgenesis in primary avian cells will require the application of more efficient transfection methods, further enhanced SB transposase or an alternative transposon system.

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.

Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri.

Seminal plasma (SP) is known to have immunosuppressive properties in several species. Equine SP has been reported to reduce or inhibit chemotaxis, phagocytosis and complement activity in vitro. The type and amount of the SP component that suppresses sperm-polymorphonuclear neutrophil (PMN) binding in vitro was determined, and the effect of such suppression on the fertility of mares inseminated in the presence of uterine inflammation, was analyzed. Sperm cells were suspended in either SP, semen extender or a mixture of both, and each was mixed with PMN-rich uterine secretions collected at 12 h after artificial insemination (AI). SP reduced binding between spermatozoa and PMNs significantly (P < 0.05). Fertile spermatozoa were suspended in SP or semen extender and used to inseminate mares 12 h after the induction of uterine inflammation. The pregnancy rate was normal (77%) when spermatozoa were suspended in SP, but was dramatically reduced to only 5% when spermatozoa were suspended in extender. The proteins from SP, blood plasma (BP) and a skim-milk-based semen extender (skim milk extender, SME) were precipitated by ammonium sulfate, resuspended in PBS and dialyzed. The effect of the precipitated proteins on sperm-PMN binding was compared with fresh, untreated SP. Both fresh SP, and isolated SP proteins reduced sperm-PMN binding (P < 0.001). Conversely, proteins isolated from either BP or SME did not reduce sperm-PMN binding. The different concentrations of SP proteins used showed a dose-dependent suppression of sperm-PMN binding. Concentrations of 1 mg/ml SP protein significantly reduced sperm-PMN binding and 6 mg/ml reduced the binding to a level similar to that observed with fresh whole SP (P < 0.001). Finally, SP protein digested with proteinase K resulted in the complete loss of SP suppressive activity confirming that the effective component is a proteinaceous substance.

Molecular cloning and expression analysis of the turkey vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor whose activity in avian species is believed to be mediated by a specific VIP receptor (VIP-R). Circulating PRL levels are closely related to hypothalamic VIP immunoreactivity, hypothalamic VIP mRNA content, and hypophysial-portal blood VIP concentrations in turkeys. In the present study, a turkey VIP-R (tVIP-R) cDNA was cloned and its mRNA abundance was quantified in various tissues during different reproductive stages. The 2347-bp tVIP-R cDNA encoded a 457 amino acid protein, with a predicted Mr of 52 kDa. The full-length cDNA shares approximately 55% similarity with the mammalian VIP receptor-1. Northern blot analysis revealed that a major 2.7-kb transcript was expressed in laying hen pituitaries. Furthermore, two minor tVIP-R transcripts of 3.7 and 3.4 kb were observed. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using RNA from various turkey brain and peripheral tissues throughout the reproductive cycle. The steady-state levels of pituitary tVIP-R mRNA changed during the reproductive cycle, whereas mRNA expression in other tissues was not affected. The steady-state levels of tVIP-R mRNA were only affected in the pituitary, whereas mRNA expression in any of the other tissues examined following the immunization of turkeys against VIP were not affected.

Localization of clusterin on freeze-preserved bull spermatozoa before and after glass wool-sephadex filtration.

Clusterin is a major protein in bull reproductive tract secretions and sperm membrane extract. A polyclonal antibody was produced against clusterin from bull cauda epididymal fluid (CEF) and used for the localization of the protein on bull spermatozoa. Immunoblotting of unreduced bovine samples showed that the anticlusterin antibody reacted with a protein of approximately 94- to 100-kd in rete testis fluid (RTF), a approximately 57- to 76-kd protein in CEF, and with a approximately 57- to 60-kd protein from cauda epididymal sperm membrane extract. The antibody also reacted with stallion RTF and both ram CEF and RTF at relative molecular weights (Mr) that were consistent with the anticipated size of clusterin in these species. Less intense immunostaining was observed for a protein of about 2 times the predicted size of clusterin in unreduced ovine RTF, suggesting the presence of multimers of clusterin in ovine RTF. Also, a dimeric clusterin-sized protein was detected in reduced bovine CEF, suggesting the presence of unprocessed clusterin in bovine CEF. By immunofluorescence, clusterin was detected on only a small fraction of bull spermatozoa, which were morphologically abnormal. Neither permeabilization nor the method of dilution affected the reactivity of the antibody with spermatozoa (P > .05). Average clusterin-positive spermatozoa (CPS) in unpermeabilized, permeabilized, abruptly diluted, and gradually diluted semen were 10.1%, 11.3%, 15.0%, and 14.4%, respectively. CPS were eliminated from semen after filtration through glass wool-Sephadex (GWS) columns. Average CPS in unfiltered and filtered semen were 14.3% and 1.1%, respectively. We conclude that sperm clusterin in bull semen is associated with morphologically abnormal spermatozoa and that clusterin is implicated in the process of abnormal spermatozoa trapping in GWS columns. We suggest that the fraction of CPS in bull semen is a potential marker for poor semen quality.

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.

Chick ciliary neurotrophic factor is secreted via a nonclassical pathway.

In contrast to mammalian ciliary neurotrophic factors (CNTFs), chick CNTF is secreted, although it lacks an N-terminal signal. We determined that a 52 aa region of chick CNTF containing an internal hydrophobic domain could direct secretion of rat CNTF. Using a stable cell line that overexpressed chick CNTF, we found that chick CNTF immunoreactivity was punctate throughout the cytosol. Cellular fractionation confirmed chick CNTF to be protected by vesicles. Chick CNTF did not colocalize with fibronectin, calreticulin, wheat germ agglutinin binding sites, or with transferrin receptor. The distribution of chick CNTF was altered neither by brefeldin A nor by chloroquine treatment. Although the punctate pattern of chick CNTF immunoreactivity was not due to reuptake, chick CNTF could be found in a cellular compartment labeled after a brief incubation with dextran microbeads. When synthesized in vitro, chick CNTF did not translocate into microsomes. We conclude that chick CNTF is secreted via a nonclassical pathway.

Gonad-specific expression of two novel chicken complementary DNA isoforms.

Differential display reverse transcription polymerase chain reaction was used to isolate a novel cDNA clone (C47) that was initially shown to be downregulated in senescent chicken embryo fibroblast cells. In a tissue environment, C47 transcripts were only detected in gonadal tissue. The expression of the larger isoform (C47L) was essentially restricted to the ovary, and the smaller isoform (C47S) was predominately expressed in the testis. Although levels of the C47L mRNA were relatively high in both the small white and the developing larger follicles, there was very low expression in regressed and postovulated follicles. Nucleotide sequence analysis indicated that two different transcripts of the single-copy C47 gene were generated by differential polyadenylation in the 3' untranslated region. As a result of a single nucleotide deletion, the C47L mRNA produced a smaller 48-kDa protein, and the C47S mRNA generated a larger 57-kDa protein when both were translated in vitro. Both protein isoforms were shown to contain conserved C2H2 Zn finger motifs and nuclear localization signals suggestive of being putative transcription factors. These results suggest that the C47L and C47S isoforms might play an important role in the regulation and maintenance of ovarian and testicular functions, respectively, in the chicken.

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.

Molecular characterization of a new member of the immunoglobulin superfamily that potentially functions in T-cell activation and development.

Differential hybridization cloning has been used to isolate a novel chicken thymic activation and developmental sequence (cTADS). The nucleotide sequence of the cTADS cDNA predicts an open reading frame of 439 amino acids. The inferred cTADS protein possesses a hydrophobic membrane-spanning domain and putative intracellular kinase activation domains. Its extracellular domain shares similarities with the immunoglobulin protein superfamily, featuring two conserved immunoglobulin folds that resemble C1 and C2 constant regions. The cTADS sequence shows similarity to a subfamily of proteins involved in cellular adhesion: chicken neural cell adhesion molecule and human opioid-binding adhesion molecule, and to proteins that have a biological role in intracellular signaling: mouse platelet-derived growth factor receptor and human fibroblast growth factor receptor. cTADS is differentially expressed in chicken thymic cells during embryonic development and during activation through the T-cell receptor. Sequence similarities and expression patterns suggest that cTADS could be involved in cell recognition and adhesion, and/or peptide ligand binding.

Evidence for multiple prolactin receptor transcripts in the turkey.

Multiple prolactin receptor (PRL-R) mRNA transcript isoforms have been identified in mammals, but there are conflicting reports concerning the number of avian PRL-R isoforms. We hypothesized that multiple turkey PRL-R transcript isoforms exist and that PRL-R mRNA abundance may be related to reproductive status. Two turkey PRL-R cDNA fragments were generated using reverse transcriptase polymerase chain reaction (RT-PCR) that displayed a high degree of similarity to mammalian and avian PRL-R. Northern blot analysis of poly A+ mRNA hybridized to a turkey PRL-R riboprobe revealed a 3.1-kb band in the liver, oviduct, and testes. Additional 1.5- and 10.7-kb transcripts were found in the liver and testes, respectively. Hybridization of the same Northern blot to a chicken PRL-R probe verified the presence of a 3.1-kb transcript in all three tissues. A Northern blot was used to examine turkey PRL-R transcript isoform expression in laying hens. A 3.1-kb band was found in the pineal, infundibulum, magnum, isthmus, kidney, and intestine. In addition, 10.7- and 7.3-kb bands were detected in the pineal, magnum, isthmus, and intestine. Turkey PRL-R transcript isoforms were also examined throughout the reproductive cycle. The 10.7-, 7.3-, and 3.1-kb isoforms were detected in the oviduct, intestine, and pineal during each reproductive state. Turkey PRL-R mRNA levels were also compared during the reproductive cycle. Turkey PRL-R mRNA levels were greatest in laying hen pineal glands (P<0.05) and in incubating hen oviducts. This study provides the first evidence for multiple PRL-R mRNA transcript isoforms in turkeys.

Three different turkey luteinizing hormone receptor (tLH-R) isoforms I: characterization of alternatively spliced tLH-R isoforms and their regulated expression in diverse tissues.

Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-R(intact)) showed 98% and 72-75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-R(insert)) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-R(insert) isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-R(trunc)) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.