Extracts Of Pituitary Glands Research Articles

Effect of Pituitary Gland (PG) Doses on Artificial Propagation of Endangered Tarabaim, <i>Macrognathus aculeatus</i> (Bloch)

Five hormone doses viz., 40, 60, 70, 85 and 90 mg of Pituitary gland (PG)/kg body endangered Tarabaim (Macrognathus aculeatus) were tested and they were designated as Treatments T1, T2, T3, T4 and T5, respectively. Significantly (p<0.05) higher fertilization (86%) and hatching rates (50%) were obtained from T5 (90 mg of PG extract/kg body weight) than those of other treatments. However, T4 (85 mg of PG extract/kg, body weight) and T3 (70 mg of PG extract/kg body weight) gave the highest (90%) ovulation rate. In May and June, the highest fertilization rate of 82% and 86% respectively and hatching rate 45% and 75%, respectively were obtained at the temperature ranged from 27.0 to 33.0ºC. In conclusion, PG extract at a dose of 90 mg/kg body weight appeared to be the suitable dose for artificial propagation of M. aculeatus, May and June are the suitable months for its artificial propagation.DOI: http://dx.doi.org/10.3329/pa.v19i2.16951 Progress. Agric. 19(2): 111 - 118, 2008

Comparative Study of Carp Pituitary Gland (PG) Extract and Synthetic Hormone Ovaprim Used in the Induced Breeding of Stinging Catfish, Heteropneustes fossilis (Siluriformes: Heteropneustidae)

The present study compared the effectiveness of the Carp pituitary gland extract (PGE) and the synthetic hormone Ovaprim on spawning success of the stinging catfish, Heteropneustes fossilis during induced breeding. The PGE hormone was administered at 6 mg/kg of body weight for females and 2 mg/kg of body weight for males. In contrast, Ovaprim was administered at 0.3 ml/kg body weight and 0.1 ml/kg of body weight for females and males, respectively. The spawning success was higher in the Ovaprim-induced individuals with better performance recorded at all stages of spawning including latency period, ovulation, fertilization, hatching and incubation period compared to the PGE-induced individuals. In the Ovaprim induced individuals, the latency period occurred within 10 hours while in PG-induced individuals, the latency was after 15 hours. Similarly, ovulation rate was 90% for Ovaprim injected fish but lower 78.7% for PGE injected fish. Higher rate of fertilization was observed in the eggs of Ovaprim treated fishes 86.7% compared to 69.2% in PGE induced fish. On the other hand, hatching rate was 76.9% in eggs spawned from Ovaprim induced individuals compared to 72.7% in PGE induced fish and the incubation period was also shorter at 3.5 h for eggs from Ovaprim-induced fish while the PGE induced fish eggs required a 5-h incubation period. Finally, the results showed that Ovaprim treated fish yielded better results compared the PGE treated fish in terms of ovulation, fertilization and hatching rates of H. fossilis.DOI: http://dx.doi.org/10.3126/on.v10i1.7755

Domestication and Observation on Induced Breeding of Spiny Eel Mastacembelus armatus

Spiny Eel, Mastacembelus armatus categorized as endangered is a highly potential species in Bangladesh. Two different experiments were conducted on domestication and induced breeding of Spiny Eel, M. armatus at mini hatchery cum breeding complex under the Department of Fisheries Biology and Genetics, Bangladesh Agricultural University. For domestication, fish were reared under three different treatments (treatment I, II and III) each having three replications (R1, R2, R3) in nine indoor cisterns (2.33 m × 1.34 m area each) for six months and fed with three different feeds namely commercial supplementary feed (Mega feed), trash fish and chicken viscera. Highest growth performance was observed upon statistical analysis in treatment II fed with trash compared with treatment I and III. Subsequently four induced breeding trials were conducted using carp pituitary gland (PG) extract. Male and female broods were induced to breed using different doses (35-100 mg.kg-1 body weight of female and 5-10 mg.kg-1 body weight of male) of PG extract. PG dose of 35 & 40 mg.kg-1 body weight precipitated ovulation but successful fertilization (82.00 ± 2.21) was observed at the dose of 40 mg.kg-1 body weight. Data were analyzed using one-way analysis of variance (ANOVA).

Controlled Breeding of Asian Catfish Clarias batrachus using Pituitary Gland Extracts and Ovaprim at different Temperatures, Latency Periods and their Early Development

Present study was conducted on induced breeding of commercially important Clarias batrachus due to the non-availability of its quality seeds from the natural resources for environmental degradation, shrinkage of natural breeding ground and illegal killing of juveniles and brood fishes. The aim of the study was to achieve success in fertilization and hatching using various doses of different inducing agents at different temperatures and latency periods followed by stripping method. In this study the developmental stages of fish (fertilized egg to 45th day old fish) were characterized chronologically. A trial was also made to optimize the survival rate of young developing fish upto 45th day by manipulating their feeding schedule and environmental conditions. The breeding experiments were done with pituitary gland extracts (40 and 120 mg/kg. body weight for female and 25 and 50 mg/kg. body weight for male) and Ovaprim (0.8 and 2.0 ml/kg body weight for female and 0.4 and 1.0 ml/kg body weight for male) at 26o, 28o and 30oC. The highest rates of fertilization (80%) and hatching (71%) of eggs were recorded in Clarias batrachus injected with carp pituitary gland extracts @ 50 mg/kg body weight of male and 120 mg/kg body weight of female at 28oC with a latency period of 15 hours. The fertilization and hatching rates were 77% and 65% respectively at 28°C at the higher doses of Ovaprim. The highest survival rate (82.5%) of developing fish was achieved supplying zooplankton as live feed upto 12th day followed by alternate supply of zooplankton, boiled egg with vitamin C and chopped tubifex from 13th to 45th day of rearing in indoor polyvinyl chloride tray with minimum fluctuation in temperature and dissolved oxygen.

An Economically Viable Approach for Induced Breeding of Labeo Rohita by Ovatide, Ovaprim And Carp Pituitary Extract.

Indian major carp Labeo rohita was induced breeding in eco-carp hatchery with 3 different inducing agents, viz. ovaprim, ovatide, Carp pituitary extract at fish breeding center at Amloh Distt. Fathegarh Sahib (Punjab) India. Total ten trial doses of ovaprim, ovatide and Carp Pituitary Extract (CPE) used for induced breeding in Labeo rohita. The fertilization was found 61.30% with ovaprim, 58.50% with ovatide and 55.96% with carp pituitary extract treatment. The percentage hatchling was 72.20% with ovaprim, 66.37 % with ovatide and 59.25% with carp pituitary extract treatment.A comparison was carried out for fecundity, fertilization, and hatching rate during the induced spawning of Labeo rohita administered single dose of ovaprim and ovatide while carp pituitary extract double dose to female brooder. Ovaprim performed much better than Ovatide and pituitary gland extract.

The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca(2+) dynamics. We found that TRPV4 interacts with beta-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca(2+)-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca(2+) increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.

Distribution and Accumulation of L-Ascorbic acid and α-Tocopherol in Japanese Eel, Anguilla japonica during the Induced Final Maturation with Extract of Pituitary Glands

Distribution and Accumulation of L-Ascorbic acid and α-Tocopherol in Japanese Eel, Anguilla japonica during the Induced Final Maturation with Extract of Pituitary Glands

290 OPTIMAL OF DOSE OF OVINE PITUITARY GLAND EXTRACT OVAGEN® FOR SUPERSTIMULATION OF BEEF COW DONORS IN ARGENTINA

The success of an embryo transfer program is measured by the number of calves born alive by female donor in a given period. The success is influenced by several factors related to the number of ovulations, fertilization rate, and embryo viability (Armstrong D 1993 Theriogenology 39, 7–24). One of the main inconveniences of embryo transfer programs is the variability of superovulatory response to treatments. An experiment was designed to determine the optimal dose of ovine pituitary gland extract Ovagen® (ICPbio, Ltd., Auckland, New Zealand) for inducing superovulation in Aberdeen Angus donor cows. Sixty cycling multiparous donors with a condition score between 3.5 and 4.5 (Scale 1 to 5) were used. All donors received an intravaginal progesterone device DIB (1 g of Progesterone, Syntex, Buenos Aires, Argentina), along with 2 mg of EB (Estradiol Benzoate, Syntex, Buenos Aires, Argentina) and 50 mg of progesterone (Laboratorio Rio de Janeiro, Argentina) on Day 0. Superestimulatory treatment began on Day 4 and donors were randomly assigned to 3 treatment groups according to the total dose of NIADDK-oFSH-Z (Ovagen®) as follows: Group 1, cows received the total dose recommended by the manufacturer (100%* Group), 17.6 mg; Group 2, cows received 75% of the total dose recommended by manufacturer (75% Group), 13.2 mg and Group 3, cows received 50% of the total dose recommended by manufacturer (50% Group), 8.8 mg. All cows received two 150 μg of D+ cloprostenol IM (Ciclase, Syntex) injections on Day 6 given at 12 h interval. DIB was removed on Day 7 a.m. On Day 8 a.m., cows received 0.05 mg of GnRH IM (Gonasyn, Syntex). Fixed time AI was done on Day 8 p.m. and Day 9 a.m. with high quality frozen–thawed semen. On Day 15 embryo collection was performed by non-surgically method and evaluated by developmental stage and quality. The efficiency of superestimulatory response was evaluated by total amount of collected ova-embryos, fertilized ova and embryos Grade 1, 2, and 3, (according to IETS manual). One way AOV test was used to compare variables among groups and results are shown in Table 1. A significant increase in total CL was observed for the groups receiving 75% or 100% of the recommended dose with respect to the group receiving 50% of that dose. There was also a significant greater number of grade 1 embryos for the groups receiving 75% or 100% of the dose than in the group receiving 50% of the recommended dose. There were no differences for any of the evaluated parameters between 75% and 100% dose groups. These results suggest that acceptable superstimulatory responses can be obtained using reduced doses of Ovagen® in Aberdeen Angus donor cows. (*) the percentage makes reference to the total dose recommended by the laboratory of origin. Table 1.Embryo collection results of superstimulated donors with different dose* of NIADDK-oFSH-Z (Ovagen) Research supported by Syntex S.A. and Eolia S.A.

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse

BackgroundThe hypogonadal (hpg) mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland.MethodsInitially, Western blot and immunohistochemistry were used to analyse tissues from hpg mice to identify potential sites of action of estradiol. In the main study, hpg mice were treated for 50 days with either an estradiol implant or daily injections of recombinant human FSH, or a combination of both, to determine whether estradiol would have an additive or synergistic effect with FSH on testis development, as assessed by histological analysis and stereological quantification of Leydig, Sertoli and germ cell proliferation.ResultsWestern blot analysis revealed ERα immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERα in nuclei of anterior pituitary cells and Leydig and peritubular cells in hpg mice. Histological and morphometric analyses revealed that estradiol treatment alone was as effective as FSH in promoting Sertoli cell production and proliferation of the seminiferous epithelium, resulting in the production of elongating spermatids. Combined estradiol and FSH treatment did not produce a greater effect than either treatment alone, though an increased dose of FSH significantly increased seminiferous tubule volume and testis weight and increase Sertoli cell numbers further within the same time frame. In contrast, estradiol caused substantial increases in the wet weight of the seminal vesicles, whereas FSH was without effect on this tissue, and did not augment the actions of estradiol.ConclusionAs ERalpha receptor is abundantly expressed in the pituitary gland of hpg mice, and estradiol did not exert effects on testis development over and above those of FSH, we conclude that the action of estradiol on testis development in hpg mice is predominantly via the stimulation of pituitary FSH release.

Chromatofocusing Fails To Separate hFSH Isoforms on the Basis of Glycan Structure

Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the near total loss of receptor binding activity in several preparations could not be explained by aggregation alone, and the mechanism remains unknown.

Breed differences in clearance of porcine FSH in hypophysectomized rats

Extracts of anterior pituitary (AP) glands were infused i.v. into hypophysectomized male rats followed by sequential sampling of blood for 120 min. Determination of follicle-stimulating hormone (FSH) concentrations established that FSH from Chinese Meishan males decreased in the circulation of rats more slowly than FSH in extracts of AP from crossbred occidental pigs ( P < 0.003). Additionally, FSH from AP extracts of castrated males disappeared somewhat more slowly ( P < 0.06) than FSH from extracts of boars. Evaluation of FSH by bioassay and radioimmunoassay yielded similar concentrations in AP from Meishan and crossbred boars. Serum testosterone concentrations increased with time through 90 min after infusion of AP, but the rate of increase of testosterone was not related to amount of luteinizing hormone (LH) that was administered indicating LH receptor saturation. Unexpectedly, the rate of increase in testosterone was more rapid with AP extracts from boars than with extracts from castrated males. Observations from the current study imply structural alterations of FSH in the AP of Meishan males relative to crossbred males allowing sustained concentrations in the circulation, and this FSH possesses similar activation of the FSH receptor. The amount of LH in the AP extracts saturated the LH receptors of the hypophysectomized male rats, but some factor in extracts of boars differed from those of castrated males.

Profiles of α-melanocyte-stimulating hormone in the Japanese flounder as revealed by a newly developed time-resolved fluoroimmunoassay and immunohistochemistry

Profiles of α-melanocyte-stimulating hormone (α-MSH) in the Japanese flounder were examined by a newly developed time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. A TR-FIA for α-MSH was newly developed, and its levels in the pituitary gland and plasma of Japanese flounder reared in a white or black tank for 5 months were compared. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, α-MSH antibodies, samples, and europium-labeled Des-Ac-α-MSH. The sensitivity of the assay, defined as twice the standard deviation at a zero dose, was 0.98 ng/ml (49 pg/well). The intra- and interassay coefficients of variation of the assay were 8.8% ( n = 8) and 17.3% ( n = 5), respectively, at about 50% binding. Cross-reactivities of Des-Ac-α-MSH and Di-Ac-α-MSH were about 100%. Cross-reactivities of adrenocorticotropic hormone, salmon gonadotropin-releasing hormone (sGnRH), and chicken GnRH-II were less than 0.2%, and that of melanin-concentrating hormone was less than 2.0% at 50% binding. Displacement curves of serially twofold-diluted hypothalamus extract, pituitary gland extract, and plasma extract of Japanese flounder with the assay buffer were parallel to the α-MSH standard curve. Moreover, displacement curves of serially twofold-diluted hypothalamus and/or pituitary gland extract of masu salmon, goldfish, red seabream, Japanese eel, tiger puffer, and barfin flounder with the assay buffer were also parallel to the α-MSH standard. In Japanese flounder, total immunoreactive (ir)-α-MSH levels in the pituitary gland were lower in the black tank, whereas those in the plasma tended to be higher in the black tank, suggesting that the synthesis and release of α-MSH are higher in the black tank. α-MSH-ir cells were detected in the pars intermedia and a small part of the pars distalis of the pituitary gland. α-MSH-ir cell bodies were located in the basal hypothalamus and α-MSH-ir fibers were distributed not only in the hypothalamus but also in the telencephalon, midbrain, cerebellum, and medulla oblongata, suggesting that α-MSH functions as a neuromodulator in the brain.

Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors

Freshly isolated hepatocytes rapidly lose their differentiated properties when placed in culture. Therefore, production of a simple culture system for maintaining the phenotype of hepatocytes in culture would greatly facilitate their study. Our aim was to identify conditions that could maintain the differentiated properties of hepatocytes for up to 28 days of culture. Adult rat hepatocytes were isolated and attached in Williams’ medium E containing 10% serum. The medium was changed to either fresh Williams’ medium E or keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract. The hepatic phenotype was then analysed using RT-PCR, immunohistochemistry, Western blotting and assays of liver function. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their phenotype for 3–4 weeks, based on expression of liver proteins, ureagensis and response to xenobiotics. In contrast, hepatocytes cultured in Williams’ medium E rapidly lost the expression of liver proteins after 3 days. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their expression of liver-enriched transcription factors (C/EBPα and β, HNF4α and RXRα) while expression was either lost or reduced in cells cultured in Williams’ medium E. These results suggest that keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract can maintain the hepatic phenotype for a prolonged period and that this is probably related to the continued expression of the liver-enriched transcription factors.

Experimental data supporting the expression of the highly conserved GnRH-II in the brain and pituitary gland of rats

The second GnRH form, originally identified in chickens (cGnRH-II or GnRH-II), is the most ubiquitous peptide of the GnRH neuropeptide family, being present from jawed fish to human beings. However, the presence of GnRH-II in such an important experimental model as the rat is still an object of discussion. Here we present chromatographic, immunologic and biologic activity evidence supporting the expression of GnRH-II in the rat. Olfactory bulb, hypothalamus, remnant brain and anterior pituitary from a pool of 50 female adult rats were extracted and subjected to RP-HPLC on a C-18 column. The fractions were collected and evaluated by using two different RIA systems, specific for GnRH-I and GnRH-II respectively. Under these conditions the GnRH-I standard eluted in fraction 21 (f21) was only detected with the GnRH-I RIA system, whereas the GnRH-II standard was only detected in the fraction 27 (f27) by using a GnRH-II RIA system. In the olfactory bulbs extract, the fractions analyzed by the GnRH-I RIA systems showed a single peak in f21, whereas by using the GnRH-II RIA system a single peak at f27 was observed. In the hypothalamus GnRH-I was detected in f21 meanwhile GnRH-II could not be detected. When the remnant brain and pituitary gland extracts were analyzed, both GnRH forms were detected. To the best of our knowledge, this is the first report concerning GnRH-II detection in a mammalian pituitary. Serial dilutions of f27 and GnRH-II presented similar displacement of radioiodinated-GnRH-II, demonstrating that both molecules share immunological properties. Moreover, after 60 min stimulation, both f27 and GnRH-II had similar LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures. However, we failed to characterize the GnRH-II gene in this model. These results provide strong evidence for the expression of GnRH-II in the rat brain and pituitary gland.

Purification of bubaline luteinizing hormone by gel filtration chromatography in the presence of blue dextran

A luteinizing hormone (LH) enriched fraction from the whole pituitary glands of buffaloes has been obtained by a simple, economical and scalable protocol using two gel filtration steps. An extract of pituitary glands was mixed with blue dextran and applied to the first Sephacryl S-200 gel filtration column. Blue dextran fractions eluting in the void volume of the column together with bound LH were pooled, reduced in volume and re-loaded on to the second gel filtration column, pre-equilibrated with 50 mM phosphate buffer pH 7.3 containing 1 M KCl. In the presence of 1 M KCl, LH dissociated from the blue dextran and eluted in the same elution volume as in the case of dimeric native form of buffalo LH, while blue dextran eluted in the void volume of the column. The protein obtained from the peak in the second gel filtration was found to be highly immunorective against bovine LH β subunit specific antiserum and it was 46-folds purer over the starting material as indicated by the direct binding ELISA. SDS-PAGE of the purified LH showed two major bands of LH, which was confirmed by western blot analysis. The yield of LH was found to be 262 mg LH/kg of whole pituitary glands.

Prolactin in the brushtail possum ( Trichosurus vulpecula): Development of homologous radioimmunoassay using recombinant possum prolactin

We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight ∼23 kDa. The potency of posPrl was 45.3 ± 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 ± 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (∼30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 μg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase ( P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 μg) reduced ( P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 × 75 μg at 12 h intervals) suppressed ( P < 0.01) plasma Prl concentrations until 24 h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked ( P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 μg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.

Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.

Analysis of mitogenic activity of proteins after separation by gel electrophoresis.

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions.

Radioimmunoassays of Follicle Stimulating Hormone and Luteinizing Hormone in the Dog

Heterologous radioimmunoassays for canine FSH and LH were developed and used to quantitate these hormones during proestrus, estrus and diestrus of the bitch. The assay for FSH consists of a rabbit anti-human FSH serum, radioiodinated rat FSH and dog FSH-LER-l685-3A as standard. Inhibition curves produced by the standard, crude extracts of canine pituitary glands and various canine plasma were parallel. Specificity of FSH assay was confirmed by testing for binding inhibition with canine LH (LER-l685-l). Binding was hardly affected by canine LH. The intra- and interassay coefficients of variation was 3.1% and 10.8%. Sensitivity of the FSH assay was 0.20 ng/tube which was adequate to quantify the concentration of FSH in 100 μl or less of canine plasma. The LH assay consists of a rabbit anti-ovine LH serum, radioiodinated rat LH and dog LH-LER-l685-l as standard. Crude extracts of canine pituitary glands and various canine plasma p roduced inhibition curves which were parallel to the standard curve. Binding inhibition was no affected by canine FSH(LER-l685-3A). The intra- and interassay coefficients of variation was 5.8% and 14.3%. Sensitivity of the LH assay was 10.7 pg/tube which allowed estimation of LH concentrations in 100 μl or less of canine plasma. During the reproductive cycle, levels of FSH were lowest during proestrus (51.1 ± 7.4 ng/ml) and increased to 305.5 ± 39.6 ng/ml on a day after LH peak. Levels of FSH declined by Day 4 (63.5 ± 10.4 ng/ml) then increased to 117.4 ± 11.9 ng/ml on Day 18 after LH peak. Levels of LH increased only during preovulatory LH surge, during proestrus and diestrus, were less than 0.5 ng/ml.