Chicken Luteinizing Hormone Research Articles

Changes in luteinizing hormone‐containing gonadotrophs after moulting induced by fasting and zinc in laying hens (Gallus domesticus)

The purpose of the present study was to investigate egg production performance, immunohistochemical (IHC) identification, localization and morphological characterization of moulted hen's pars distalis luteinizing hormone (LH) gonadotrophs. This system has been validated for chicken LH by immunodetection of purified hormones on dot blots, western blotting and by specific labelling of cells on sagittal sections of chicken pituitaries. This study showed that egg production and egg weight increased significantly after zinc-induced moult when compared with fasting-induced moult. Luteinizing hormone-gonadotrophs are of numerous shapes and distributed throughout the adenohypophysis, with densely populated regions on the margins of pars distalis. Moulting of laying hens was associated with a significant increase in immunoreactive LH gonadotroph cell count and cell size, which peaked at the subsequent production stages. Zinc-induced moulting also caused a significant increase in LH gonadotroph cell size as compared with fasting-induced moulting. The nuclear size of LH-positive cells was distinct during decreased egg production. Our results validate the use of ovine LHbeta antibodies for the IHC localization of chicken LH gonadotrophs. This technology was further applied to the characterization of the shape and morphological changes of gonadotrophs as a function of the egg production status of the birds.

Effects of exogenous testosterone on testicular luteinizing hormone and follicle-stimulating hormone receptors during aging.

During aging, the male Japanese quail exhibits a loss of fertility, increased morphological abnormalities in the testes, and a higher incidence of Sertoli cell tumors. Although there is a coincident loss of reproductive behavior, plasma androgen levels remain high until testicular regression occurs in association with senescence. The purpose of this study was to compare mean specific binding of chicken luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as a measure of testicular receptors during identified stages during aging. Males were categorized according to age (young = 9 months, middle aged = 24 months, or old = 36+ months) and sexual behavior (active or inactive). Testicular samples were collected immediately after perfusion with 4% paraformaldehyde from the following groups: young active (n = 8), young photoregressed (n = 5), young photoregressed plus testosterone implant (n = 4), middle-aged active (n = 8), middle-aged inactive (n = 4), old inactive (n = 5), and old inactive plus testosterone implant (n = 6). A crude plasma membrane fraction was prepared from the testes of each bird and an aliquot deriving from 10 mg of testicular tissue was used for binding assay. Specific binding of labeled LH or FSH was expressed as percentage of total radioactive hormone. Results showed significant (P < 0.05) age-related decreases in both FSH and LH receptor numbers. The highest FSH binding was found in young and middle-aged active males, with low binding in old inactive males. Testicular LH binding decreased during aging, with a sharp decrease in middle-aged males, which was similar to old males. Testosterone implants weakly stimulated FSH and LH binding in old males. Both LH and FSH binding decreased in photoregressed young males. However, testosterone implants stimulated increased LH binding, but did not affect FSH binding in young photoregressed males. These results provide evidence for separate regulation of testicular LH and FSH receptors, with testosterone stimulation of LH receptor, but not FSH receptor number in young males. However, during aging there appears to be a loss of this response, potentially because of the reduced efficacy of testosterone stimulation, thereby implying a diminished capacity for response with aging.

Cloning and Gene Expression of a cDNA for the Chicken Follicle-Stimulating Hormone (FSH)-β-Subunit

Follicle-stimulating hormone (FSH) is a member of pituitary glycoprotein hormones that are composed of two dissimilar subunits, α and β. Very little information is available regarding the nucleotide and amino acid sequence of FSH-β in avian species. For better understanding of the phylogenic diversity and evolution of FSH molecule, we have isolated and sequenced the complete complementary DNA (cDNA) encoding chicken FSH-β precursor molecule by reverse transcription–polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned chicken FSH-β cDNA consists of 2457-bp nucleotides, including 44-bp nucleotides of the 5′-untranslated region (UTR), 396 bp of the open reading frame, and an extraordinarily long 3′-UTR of 2001-bp nucleotides followed by a poly(A)(16) tail. It encodes a 131-amino-acid precursor molecule of FSH-β-subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within β-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the chicken FSH-β-subunit. Four proline residues, presumably responsible for changing the backbone direction of protein structure, are conserved in chicken FSH-β-subunit as well. The nucleotide sequence of chicken FSH-β cDNA shows high homology with quail FSH-β cDNA, 97% homology in the open reading frame, and 85% homology in the 3′-UTR. The deduced amino acid sequence of chicken FSH-β-subunit shows a remarkable similarity to other avian FSH-β-subunits, 98% homology with quail, and 93% homology with ostrich, whereas a lower similarity (66 to 70%) is noted when compared with mammalian FSH-β-subunits. By contrast, when comparing with the β-subunits of chicken luteinizing hormone and thyroid-stimulating hormone, the homologies are as low as 37 and 40%, respectively. FSH-β mRNA was only expressed in pituitary gland out of various tissues examined and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated by real-time quantitative PCR.

Specific Anti-peptide Antibody to &amp;beta; Subunit of Chicken Thyrotropin: Production and Characterization

The unavailability of homologous antibodies to avian thyrotropin (TSH) has been a major hindrance in the study of avian reproduction and metabolism. Here we report the production and characterization of an anti-peptide antibody specific to chicken TSH β subunit. According to the cDNA sequence of chicken TSH β subunit, a synthetic peptide corresponding to the amino acid residues 35-50 (Tb3550; Asp-Ser-Asn-Gly-Lys-Lys-Leu-Leu-Leu-Lys-Ser-Ala-Leu-Ser-Gln-Asn) was prepared to produce polyclonal antibody in rabbits. This peptide sequence is highly specific to avian TSH β and was designed for the antibody to show no cross-reaction to pituitary and other chicken proteins with known amino acid sequences. Anti-Tb3550 recognized a 18.2-kDa band on immunoblot of the cephalic lobe homogenate, but not of the caudal lobe homogenate, of chicken anterior pituitary gland. Positive immunohistochemical staining with anti-Tb3550 was restricted to the cephalic lobe, whereas the cells positive to anti-chicken luteinizing hormone were scattered throughout the anterior pituitary. Anti-Tb3550 did not bind either to purified chicken luteinizing hormone or to follicle stimulating hormone preparation. These results indicate that this antibody specifically recognizes chicken TSH β subunit. This antibody should be useful for immunochemical study of avian TSH and for tracing TSH activity during the purification of the hormone.

Expression of the common alpha-subunit mRNA of glycoprotein hormones during the chick pituitary organogenesis, with special reference to the pars tuberalis.

The expression of a common alpha-subunit mRNA of glycoprotein hormones was examined in the pituitary of chick embryos at various stages of development by in situ hybridization with a digoxigenin-labeled quail alpha-subunit cRNA probe. As a comparison with the expression of alpha-subunit mRNA, the onset of luteinizing hormone (LH) immunoreactivity was examined by immunohistochemical staining with a chicken LH antiserum. Both alpha-subunit mRNA and LH immunoreactivity began to appear in the basal-posterior region of the Rathke's pouch at embryonic day (E) 3.5. At E4.5 when the cephalic and caudal lobes of the pars distalis could be distinguished in the Rathke's pouch, intense signal for alpha-subunit mRNA was restricted to the cephalic lobe, consisting of a high columnar epithelium. At E6, gonadotrophs that were ovoid in shape, expressed intense signal for alpha-subunit mRNA, and revealed intense immunoreactivity for LH, were first detected in the cephalic lobe. At this stage, alpha-subunit mRNA expression became weak in the undifferentiated columnar cells of the cephalic lobe. At E8, the pars tuberalis primordium located close to the median eminence was formed at the lateral-apical end of the cephalic lobe. The primordium expressed intense signal for alpha-subunit mRNA. Gonadotrophs showing immunoreactivity for LH were densely distributed throughout the cephalic and caudal lobes in 8-day-old embryos. The pars tuberalis primordium expressing alpha-subunit mRNA progressively extended along the median eminence with embryonal age and reached the rostoral end by E14. Thus, both primordia of the pars distalis and pars tuberalis expressed intense signal for the common alpha-subunit mRNA. This subunit may play a role in the cytodifferentiation of the adenohypophysis.

Effects of timing and duration of feed restriction during rearing on reproductive characteristics in broiler breeder females

The objective of this study was to determine whether there exists a critical period during rearing when restricted feeding of broiler breeder hens can be most beneficial on subsequent egg production. Broiler breeder pullets were subjected to combinations of either ad libitum or restricted feeding during three periods before sexual maturity. Body weight gain, feed intake, and egg production were recorded. Ovary and oviduct weights were noted at age of first oviposition. At 15 and 18 wk of age and at age of first oviposition, chicken luteinizing hormone releasing hormone-I (cLHRH-I) in the median eminence and the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary and plasma were determined. The results demonstrated that alternation between ad libitum and restricted feeding during rearing changed the growth curves of the birds. Feed restriction from 7 to 15 wk followed by either ad libitum or restricted feeding led to improved reproductive performance, suggesting that long-term feed restriction may not be necessary to attain good reproductive performance. The birds restricted from 7 to 15 wk of age had higher proportional weights of ovary (> or = 1.7%) and oviduct (> or = 1.58%) at age of sexual maturity. The cLHRH-I levels in the median eminence and gonadotrophin contents in the pituitary followed that of growth in response to feeding levels and timing of feeding and could be related to the timing of the onset of lay. At age of first egg, all groups had similar levels of cLHRH-I, LH, and FSH. However, no clear effect of level of feed intake or time period of restriction could be observed for plasma LH or FSH concentrations.

Chicken Pituitary Glycoproteins: New Isolation Method

A new method for isolation of glycoproteins from chicken pituitaries was applied. The procedure consist of chromatography on ConA-Sepharose and by HPLC on S Hyper D and Vydac C4 columns. The hormonal activity of the glycoproteins was tested by determining their stimulatory effect on cAMP or testosterone production. Molecular weights of the products of tryptic cleavage of the hormone were determined using mass spectrometry (MALDI TOF). A comparison of the values obtained with theory shows that the protein is the β-unit of chicken luteinizing hormone.

Effect of long-term food restriction on pituitary sensitivity to cLHRH-I in broiler breeder females.

The effect of long-term food restriction on the sensitivity of the pituitary to exogenously administered chicken luteinizing hormone releasing hormone I (cLHRH-I) was investigated in three groups of broiler breeder females fed ad libitum, fed a restricted quantity of food or fed a restricted quantity of food to obtain an intermediate body weight between those of the first two groups. At 16 weeks of age, basal FSH release was higher in ad libitum fed birds, culminating in ovarian development and subsequent oestradiol production by the small follicles. At this age, LH secretion was independent of ovarian feedback factors. In all groups, cLHRH-I was most active in releasing LH in intact and ovariectomized animals and, to a lesser extent, in releasing FSH in ovariectomized birds. At 39 weeks of age, basal FSH concentrations were similar among intact animals of all groups, whereas LH concentrations differed among groups, with higher values in the restricted birds. This food effect was enhanced in ovariectomized birds. Furthermore, the high response to cLHRH-I in the ovariectomized, restricted birds compared with the ad libitum, ovariectomized group suggests an improved sensitivity of the hypothalamic-pituitary axis. In conclusion, birds fed ad libitum showed the highest responsiveness to ovarian factors and to cLHRH-I in releasing FSH in the period before sexual maturity. No effect of amount of feeding could be observed for LH. However, during the egg laying period, LH release by cLHRH-I was highly dependent on amount of feeding and on ovarian feedback regulation. This finding indicates that the amount of feeding can modify the sensitivity of the pituitary to cLHRH-I, and possibly to gonadal hormones, during the laying period.

An Electrochemical Enzyme Immunoassay for Chicken Luteinizing Hormone: Extension of the Detection Limit by Adequate Control of the Nonspecific Adsorption

A noncompetitive heterogeneous enzyme immunoassay for the determination of chicken luteinizing hormone (LH) was equipped with an electrochemical endpoint in order to further enhance its sensitivity. The immunological principle of the original ELISA remained essentially unchanged, except for the fact that the peroxidase label was replaced by alkaline phosphatase, since in the upgraded version of the assay,p-aminophenyl phosphate was to be used as the substrate of alkaline phosphatase. Enzyme-generatedp-aminophenol was injected into a flow-injection system and detected amperometrically in a thin-layer flow cell with a glassy carbon electrode at 0.325 V vs Ag/AgCl. A classical problem associated with this type of solid-phase immunoassay is the adsorption of proteins other than the capture antibody to the solid phase. The detection sensitivity is therefore often limited by a large background signal observed in the absence of antigen. In the present study, an experiment was designed to examine in each step of the assay the contribution of each of the potential sources of background current. It was shown that the major contribution to the background current was caused by the nonspecific adsorption of biotinylated secondary antibody. Adsorption of the secondary antibody (biotinylated goat anti-rabbit IgG) to the capture antibody (mouse anti-chicken LHβ) was clearly a case of specific aspecificity, whereas adsorption to the solid phase itself had to be treated as a nonspecific aspecificity. Addition of 0.25% mouse serum to the secondary antibody as a source of mouse immunoglobulin could overcome the cross-reaction and markedly reduced adsorption to capture antibody. The second part of nonspecific adsorption was eliminated by using combinations of Tween 20 and bovine serum albumin as blocking agents. Controlling the adsorption of the biotinylated secondary antibody in this way decreased the detection limit from 39 pg/ml in the original assay to 2.5 pg/ml in the electrochemical version. This way, the plasma volume of samples containing on the order of 1 ng/ml LH was reduced to less than 10 μl. The linear range was 2.5–625 pg/ml. The method allowed us to measure LH in buffer and in adult and juvenile chicken plasma.

Molecular cloning and functional expression of chicken luteinizing hormone receptor

A complementary DNA for chicken luteinizing hormone (LH) receptor containing the entire coding region was isolated from chicken F1 granulosa cell cDNA library. Nucleotide sequence analysis revealed that there are characteristic GC-rich regions around the N-terminal part. Chicken LH receptor consists of a 19-residue signal peptide, a 366-residue extracellular domain, a 267-residue region containing seven transmembrane segments, and a 76-residue cytoplasmic C-terminal tail. The deduced amino acid sequence of the chicken LH receptor shares 67%, 69%, and 69% identity with the human, rat and porcine LH receptor sequences, respectively, and 51% with chicken FSH receptor. However, an insertion of about 30 amino acid residues is found in chicken LH receptor in the extracellular domain about 44 amino acid residues upstream of the first transmembrane segment. In addition, alternative splicing seems likely to occur at the point where the insertion starts (nucleotide position 933), resulting in the truncated forms of chicken LH receptor with only the extracellular domain. Northern blot analysis revealed the presence of multiple transcripts of LH receptor, a major 3.0-kb and minor 7-kb and 1.5-kb bands, in chicken F1 to F3 granulosa cells. The full length chicken LH receptor cDNA was transiently expressed in COS-7 cells and the transfected cells displayed a concentration-dependent increase in cAMP production when exposed to varying concentrations of chicken LH. This clearly indicates that the cloned cDNA encodes a functional chicken LH receptor protein.

Immunoreactive luteinizing hormone (ir-LH) cells in the lung and stomach of chick embryos.

Luteinizing hormone (LH) immunoreactivity was detected in the lung and stomach of chick embryos by the immunoperoxidase staining technique using specific antiserum to chicken LH. Immunoreactive LH (ir-LH) cells first appeared in the primordial cells of the epithelial layer of lung bud and foregut as well as of Rathke s pouch in the 3-day-old embryo, Hamburger and Hamilton stage 21. Ir-LH cells increased in number with advancing age of embryos in the lung, stomach, and pituitary gland. In the lung of 7-day-old embryos, stage 31, the ir-LH cells were distributed in the epithelium of primary, secondary, and tertiary bronchi, and their shapes were pseudostratified columnar, simple columnar, and simple cuboidal, depending on their sites in the intrapulmonary airway. Ir-LH cells were more numerous in the median part than in the lateral part of the lung, and the population in the epithelial layer of entobronchi of the secondary bronchi was 4 times higher than that in ectobronchi and laterobronchi of the secondary bronchi and in the primary bronchi. The immunoreactive products were found, either in the entire cell or in the apical part, facing the lumina of bronchi. In the stomach, ir-LH cells were found in the epithelial layer of gastric glands. No ir-LH cells were observed in interstitial regions, which consisted of mesenchymal cells and blood vessels, in the lung and stomach tissues. With advancing age, ir-LH cells changed their shapes to flat or squamous, coincident with the formation of parabronchi. Other pituitary hormones were not observed immunohistochemically in either the lung or stomach before hatching. Preabsorption of the antiserum against avian LH with the purified chicken LH or the extract of pituitaries from 10-day-old embryos completely destroyed the immunoreactivity to the cells in the lung and the pituitary. A single band of the immunoreaction products, whose molecular weight was around 25 K daltons, was shown by the immunostaining of nitrocellulose membrane transblotted after sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified pituitary LH, extracts of pituitaries from 10-day-old embryos, and the extracts of lungs from 7-, 10-, and 14-day-old chick embryos. These results demonstrated that ir-LH cells are present in extrapituitary tissues, and may play an important role during the development of chick embryonic lung and stomach.

Effect of Day Length on Luteinizing Hormone β-Subunit mRNA and Subsequent Gonadal Growth in the White-Crowned Sparrow, Zonotrichia leucophrys gambelii

We have applied a cDNA probe for chicken luteinizing hormone β-subunit to adenohypophysial preparations collected from Gambell's white-crowned sparrows. In situ hybridization showed luteinizing hormone (LH) β-subunit mRNA distributed throughout the adenohypophysis in a manner identical to that of LH visualized by immunocytochemistry. Northern blot analysis showed a single band (0.8 kbp) identical to that from Japanese quail. Dot blot analysis was used to quantify changes in LH β-subunit mRNA during a photoperiodically induced gonadal cycle in white-crowned sparrows. Transfer of adult males from short days (8L 16D) to long days (20L 4D) resulted in rapid testicular growth accompanied by increases in plasma levels of LH and body and liver mass. By Day 30 of photostimulation, mRNA levels in the adenohypophysis were significantly higher than those in birds on short days. In photorefractory birds, i.e., when gonads were regressed (60 days of long days), mRNA levels were significantly lower than on Day 30 of photostimulation. The increase in mRNA during photostimulatlon was accompanied by a highly significant increase in plasma concentrations of LH. The binding of iodinated LH to testes peaked at Day 15 of photostimulation and declined by Day 30. Curiously, peak production of testosterone and cAMP (induced by incubation with gonadotropins in vitro) were highest at Day 30. By 60 days of photostimulation, when birds were photorefractory, plasma levels of LH, binding of LH to the testes, and testosterone and cAMP production were greatly reduced. These data suggest that photoperiodically induced gonadal development and regression is mediated in part by regulation of mRNA levels for LH β-subunit in the adenohypophysis.

Purification and partial characterization of isoforms of luteinizing hormone from the chicken pituitary gland

Isoforms of chicken luteinizing hormone (cLH) were isolated from chicken pituitaries by differential extraction, sequential chromatography on HPLC cation- and anion-exchange columns, and gel-filtration chromatography. Three purified isoforms of the cLH had high biological potency in the chicken granulosa cell LH bioassay (4.21–7.4 × NIH-LH-S1 U/mg). One cLH isoform without detectable biological activity showed substantial immunological activity in a homologous cLH radioimmunoassay. The cLH isoforms contained negligible follicle-stimulating hormone activity, but some contained thyroid-stimulating hormone activity. The apparent molecular weight of cLH was 37,000 Da, and the holoprotein consisted of subunits with a molecular weight of approximately 17,000 Da.

The Molecular Biology of Avian Gonadotropin

Complimentary DNA for precursor molecules of chicken and quail luteinizing hormone (LH) β subunits have been cloned. From nucleotide sequences of the cDNA, the primary structures of the LH β subunit molecules of these avian species were deduced. The primary structures were similar, the amino acid sequence homology being 91.6%. For the α subunit, cloning of cDNA has been performed also in these two avian species. Predicted primary structures of the a subunit molecules of these birds were completely identical, although nucleotide sequences of their cDNA were slightly different. The LH β subunit molecules of both birds had 15 Pro residues at the same positions. Ten of them shared the same positions with Pro residues in the mammalian LH β subunits, in which about 20 Pro residues exist. Prediction of the secondary structure and exposure of each amino acid residue in the chicken LH β subunit molecule were performed with the aid of a computer program for protein engineering. It was revealed that most of 15 Pro residues did not exist in regions where the β structure was predicted but were distributed in regions where a turn or loop was predicted. In addition, three of four Tyr residues were predicted to be located inside the molecule. These results suggest that the predicted presence of a number of Pro residues in the loop of the secondary structure is a cause of high animal group and hormone specificities in the LH-LH receptor interaction. The predicted internal localization of Tyr residues is considered to cause loss of receptor binding activity by conventional radioiodination procedures.

Second Messenger Pathways Mediating Chicken Luteinizing Hormone Secretion from Dispersed Pituitary Cells1

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioiodination of Chicken Luteinizing Hormone without Affecting Receptor Binding Potency1

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of 1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; 2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and 3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final 125I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. 125I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

Steroidogenic relationships of gonadotrophin hormones in the ovary of the hen ( Gallus domesticus)

The effects of chicken luteinizing hormone (cLH: IRC-2 and PRC AE1-1), turkey LH (B221B and HS-5-18), bovine follicle-stimulating hormone (bFSH: HS-2-17), chicken FSH (cFSH: PRC DC3(2) and AGCQSQ113445C), and turkey FSH (B150A and HS-1-153) on steroid output were evaluated by in vitro incubation of various ovarian tissues with the gonadotrophins. Output of androstenedione and estradiol was determined by 3-hr incubations of individual whole small follicles, classified by size and color as follows: small white (SWF, <1 mm), large white (LWF, 2–3 mm), and small yellow follicles (SYF, 5–10 mm). The effects of gonadotrophin preparations were also evaluated in large preovulatory follicles (F1–F5). Androstenedione and estradiol output was measured in incubation media from 100,000 theca cells and progesterone content was determined in the incubation media of 10,000 granulosa cells. All incubations were conducted in 1 ml of Medium 199 at 37°. Steroid output was quantitated by radioimmunoassay of incubation media. Potency estimates were derived by calculation of a peak stimulation index. The standard reference preparation was bLH (NIAMDD-LH-B4). Steroidogenesis was stimulated by three avian LH preparations. PRC AE1-1 was the most potent, with IRC-2 and B150A showing approximately 50% of the biological activity of PRC AE1-1 in most tissues. Turkey LH HS-5-18 was generally not potent. The presence of multiple isohormones of LH was implied, as various LH preparations exhibited different potency estimates in different tissues. The effects of FSH on steroidogenesis were not significant in most cases. Although the addition of cFSH AGCQSQ113445C failed to significantly increase output of estradiol from small follicles, potency estimates of this preparation were 0.15, 0.20, and 0.13 relative to NIAMDD-LH-B4 for SWF, LWF, and SYF, respectively. However, estrogen production by these follicles was more highly stimulated by LH than by FSH, and thus it would seem that FSH does not play a significant role in steroidogenesis in the hen's ovary. The results of this study suggest that steroid biosynthesis in the hen's ovary may be regulated by multiple forms of LH.

Presence and Hormonal Control of Plasminogen Activator in Granulosa Cells of the Domestic Hen1

Plasminogen activator (PA) has been implicated in the control of ovarian cell differentiation and in the process of follicular rupture at the time of ovulation in rats. This study was conducted to evaluate whether PA activity is present in granulosa cells of the largest preovulatory (F1) follicle in the ovary of the domestic hen, and if so, to examine its hormonal regulation. An in vitro assay system, which measures the plasmin-mediated lysis of a chromogenic substrate, S-2251, was validated for use with granulosa cells of the hen to assess levels of both cell-associated and secreted PA. Chicken luteinizing hormone (cLH) suppressed PA activity in a dose-dependent fashion, with the highest dose (100 ng/ml incubation medium) resulting in levels that were 21.8% (cell-associated) and 50.9% (secreted) those of basal values (overall mean of 5 separate assays). Similarly, the ovarian steroid, testosterone, at the lowest dose administered (2 ng/ml medium), was found to inhibit both cell-associated and secreted PA activity (29.0% and 73.0% versus basal, respectively; N = 5 assays), whereas progesterone at the highest dose (50 ng/ml media) significantly depressed only cell-associated PA activity (71.9%), but not secreted PA, compared to basal levels (N = 5). By contrast, prostaglandin (PG) E1 or PGE2 at 10(-6) M, stimulated both cell-associated (a 20.9% and a 32.1% increase, respectively, compared to basal levels) and secreted (a 17.1% and a 28.3% increase, respectively, compared to basal) PA activity (N = 5); however, PGE2 at 10(-6) M, when combined with cLH at 100 ng/ml, was only partially effective in reversing the inhibitory action of cLH.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone releasing activity of [Gln 8]-LHRH and [His 5, Trp 7, Tyr 8]-LHRH in the cockerel, [formula omitted][formula omitted] and [formula omitted][formula omitted

The luteinizing hormone (LH)-releasing activity of two distinct chicken luteinizing hormone releasing hormones ([Gln 8]-LHRH and [His 5, Trp 7, Tyr 8]-LHRH) were evaluated in white Leghorn cockerels. In the first study, thirty birds were randomly allotted to five groups and injected, i.v., with 0.9% saline, [Gln 8]-LHRH (cLHRH I, 1 μM or 10 μM) or [His 5, Trp 7, Tyr 8]-LHRH, (cLHRH II; 1 μM or 10 μM). Blood samples were drawn prior to and through 60 min following the injection, and plasma was collected for LH determination. In the second study, anterior pituitary cells from cockerels were dispersed and preincubated for 1 hr. Approximately 1.5 × 10 5 cells per tube were incubated with either Medium 199 buffer (control), 8-bromo-cAMP or various doses of cLHRH I or cLHRH II at final concentrations ranging from 0.02 to 100.0 nM. At the end of a two hour incubation, supernatant was collected and the concentration of LH determined. Injection of cLHRH I or cLHRH II at 1 μM and 10 μM levels caused a significant increase in blood LH concentrations which peaked 5 min following injection. There were, however, no differences between the stimulatory effect of cLHRH I compared to cLHRH II at either dose. On the other hand, cLHRH II was found to be 4.7 times more potent than cLHRH I in stimulating LH release from dispersed pituitary cells. It is suggested that cLHRH II may have greater affinity for the gonadotroph receptor, greater uptake by the cell, and/or that it may be more resistant to in vitro degradation than cLHRH I. On the other hand, an extra pituitary site of degradation may be more effective in metabolizing cLHRH II, resulting in its equipotency with cLHRH I, in vivo .

Purification and properties of chicken growth hormone and the development of a homologous radioimmunoassay

Highly purified growth hormone (GH) has been isolated from pituitary glands of chicken ( Gallus domesticus), and a specific homologous radioimmunoassay (RIA) has also been developed. The purified chicken GH was active in the rat tibia bioassay and it gave a dose-dependent response which paralleled that of the bovine GH standard. High pressure liquid chromatography revealed that the purified chicken GH was homogenous. Chicken GH had an R f value of 0.2 in disc electrophoresis, and a MW of 26,000 from sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was estimated to be 7.6 by gel isoelectric focusing. The amino acid composition of chicken GH was found to be similar to that of mammalian GH, and the NH 2-terminal amino acid was threonine. Partial sequencing (114 amino acids) of the chicken GH showed 79% homology with bovine GH. An antiserum was developed to the purified chicken GH in a rabbit, and it was used to develop a homologous RIA using 125I-labeled chicken GH as the ligand. The purified chicken GH was iodinated via the lactoperoxidase method to a specific activity of approximately 100 μCi/μg. Plasma from chickens, medium from incubation of pituitary glands, and homogenates of pituitary glands gave parallel dilution-response curves with the chicken GH standard. Mammalian GH, prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) showed no cross-reaction with the 125I-labeled chicken GH. Purified turkey GH showed parallel dose response with the chicken GH, but purified turkey PRL did not cross-react. Chicken FSH and LH also showed no inhibition of binding. The minimum detectable concentration of the assay was 0.93 ng/tube, and the intraassay and interassay coefficients of variation were 9 and 16%, respectively. The specific binding of 125I-labeled chicken GH to a microsomal fraction isolated from chicken liver was identified, and the specific binding was generally low (1–4%). Turkey PRL, and chicken LH and FSH showed no inhibition of the 125I-labeled chicken GH hepatic binding and the ontogeny of the hepatic GH receptor binding sites in male and female chickens was examined.