Development of an Homologous Radioimmunoassay for Secreted Prolactin from the California Ground Squirrel (Spermophilus Beecheyi)1 (original) (raw)
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Biology of Reproduction, 1987
Prolactin (Prl) secreted by cultured ground squirrel (Spermophilus beecheyi) pituitaries (SbPrl) was purified b y gel filtration on Sephadex G-1 0 0 and ion-exchange chromatography on Polybuffer Exchanger 94. Purification f r o m culture medium from 190 pituitaries yielded 1.1 mg of purified SbPrl. The SbPrl has an apparent molecular weight of 27,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 6. 3 , and does not contain any asparagine-linked carbohydrate. Purified SbPrl displaces '251-labeled ovine Prl from binding sites on lactating rabbit mammary gland membranes and stimulates secretion of a-lactalbumin b y cultured mouse mammary gland epithelial cells.
General and Comparative Endocrinology, 1998
The aim of this study was to test the hypothesis that prolactin may up-and down-regulate prolactin receptor gene expression in the anterior pituitary gland and hypothalamus respectively. Experiments were carried out in bantams (Gallus domesticus). Comparisons were made of concentrations of PRLR mRNA in the anterior pituitary gland and basal and preoptic hypothalamus in adult males and females held on long days (low vs high plasma prolactin); in 3-week-old juvenile male and females on short days (high vs low plasma prolactin); in 8-week-old juvenile male and females on short days (both low plasma prolactin); in adult laying, incubating, and out-of-lay (high, very high, and low plasma prolactin, respectively); in adult cockerels exposed to long or short days (high vs low prolactin); and in adult hens exposed to long or short days (high vs low prolactin).
Annual changes in serum concentrations of prolactin in captive male black bears (Ursus americanus)
Reproduction, 1995
Prolactin may be involved in the regulation of reproduction in black bears (Ursus americanus) as it is a mediator of photoperiodic changes in a number of species. The objectives of this study were to validate a radioimmunoassay to measure prolactin in bear serum and to describe seasonal changes in serum prolactin concentrations in captive male bears. Serum samples were obtained nine times during a year from three captive male black bears that were denning between November and March and active during the other months. The heterologous prolactin radioimmunoassay, using pig 125I-labelled prolactin and goat anti-pig prolactin as a primary antibody, was validated. Injection of thyrotrophin-releasing hormone into the three male bears in June resulted in a rapid increase in serum concentrations of prolactin (t = 0, 11.4\p=n-\14.8 ng ml \m=-\1; t = 15\p=n-\30min, 18.4\p=n-\28.7 ng ml \m=-\1).
Biology of Reproduction, 1988
During a long-term field study of a free-living population of California ground squirrels (Spermophilus beecheyi), blood samples were drawn at regular intervals from marked females via femoral venipuncture, and plasma progesterone (P) and prolactin (PRL) were measured by radioimmunoassay. Marked fluctuations with season and reproductive condition occurred in circulating levels of both hormones, with peak levels occurring during the spring breeding season. Two peaks in P concentration were observed each spring, the first occurring during pregnancy, and the second during lactation. Peak PRL levels in females were also reached during the lactation interval, midway between the two P peaks. Analysis of repeated measures from individual females showed a marked decline in circulating P around the time of parturition. Juveniles had lower mean P levels than adults, and yearlings had lower peak levels during their initial reproductive episodes than older females did. The observed pattern of P secretion in S. beecheyi differs from that known for most mammals, but resembles those reported for other ground-dwelling sciurid rodents.
Radioimmunoassay of prolactin in plasma of bullfrog tadpoles
Endocrinologia japonica, 1982
Antiserum to prolactin (PRL) of bullfrog (f), Rana catesbeiana, was produced by immunizing rabbits with the highly purified fPRL obtained from adenohypophyses of adult bullfrogs. In Ouchterlony's agar double diffusion test, a single precipitin line was produced between the fPRL antiserum and fPRL or the bullfrog pituitary extract. No precipitin line was produced between the antiserum and ovine PRL, bovine PRL, ovine growth hormone (GH) or fGH. The acetone-dried powder of bullfrog pituitary glands which had been incubated with the fPRL antiserum had much less potency in promoting collagen synthesis of the tail fin of bullfrog tadpoles than that incubated with normal rabbit serum. Histological studies on bullfrog adenohypophyses revealed that the cells that immunologically reacted with the antiserum to fPRL were erythrosinophilic. The antiserum to fPRL was used to develop a radioimmunoassay (RIA) in which fPRL and 125I-fPRL were employed as the standard and the radioligand, respectively. Several dilutions of plasma of both adult and larval bullfrogs yielded dose-response curves which were parallel to the standard curve. Bovine PRL, ovine PRL, ovine GH and fGH did not react in this assay. Plasma from hypophysectomized bullfrogs had no detectable immunoreactive prolactin. Pituitary homogenates of Rana catesbeiana and Bufo bufo japonicus gave inhibition curves which were parallel to the standard. Pituitary homogenates of Xenopus laevis and Hynobius tokyoensis gave inhibition curves which did not parallel the standard. RIA of plasma PRL in larval bullfrogs of various developmental stages were performed. Average concentrations of prolactin during premetamorphosis (st. X), prometamorphosis (st. XVI-XIX) and at early climax stage (st. XX-XXII) were 18, 21-25 and 27-35ng/ml, respectively. At advanced climax stage PRL levels were quite high. Average values at stages XXIII, XXIV and XXV were 98, 169 and 116ng/ml, respectively. The significance of PRL levels in relation to metamorphosis is discussed.
American Journal of Primatology, 1992
The immunological and biological activities of growth hormone (GH) and prolactin (PRL) in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliuiensis) have been studied. Proteins in pituitary homogenates were solubilized in 1% SDS, electrophoresed on 12% polyacrylamide gels, and transferred to nitrocellulose. Squirrel monkey GH and PRL were identified by immunoblotting with anti-human GH antibodies and a monoclonal antibody to ovine PRL, 6Fl1, respectively. Squirrel monkey GH appeared predominantly as two proteins of apparent molecular weight 22 and 20 kD, corresponding to native and variant forms of human GH. Squirrel monkey PRL appeared as two proteins of apparent molecular weight 24 and 26.5 kD, which co-migrated with native and glycosylated forms of ovine PRL. The cross-reactivity of neutralizing antibodies to human GH and PRL with squirrel monkey GH and PRL were examined using the Nb2 lymphoma bioassay. One of three monoclonal antibodies to human GH (2A1) neutralized squirrel monkey GH with a n apparent affinity for squirrel monkey GH = 70 ng IgG/ml) which was fourfold lower than for human GH (IC5,, = 15 ng IgG/ml). Both polyclonal [AR38-5(1)1 and monoclonal (9C3) antibodies to human PRL inhibited the activity of squirrel monkey PRL, although their affinities for squirrel monkey PRL were four-and twentyfold lower than for human PRL. The activities of antibodies 2A1 and 9C3 on GH and PRL in squirrel monkey serum were also examined by the Nb2 bioassay. The anti-glucocorticoid RU486 was used in all incubations with squirrel monkey serum to eliminate the effect of high glucocorticoid levels on Nb2 cell growth. The mitogenic activity of squirrel monkey serum in the Nb2 assay was completely eliminated in the presence of 2A1 and 9C3. This study represents the first description of the biochemistry of GH and PRL in the squirrel monkey.
General and Comparative Endocrinology, 1992
Bennett's wallaby prolactin (wPRL) and growth hormone (wGH) were purified from an aqueous extract of pituitary glands. The extract from 202 glands (6.5 g wet wt) was processed by gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and then anion-exchange chromatography on DEAE-Sepharose CL-6B. The yields of wPRL and wGH were 5.2 and 15.7 mg, respectively. Since recovery of wPRL from the anion exchange column was 10%, anion exchange was performed in the presence of 20% acetonitrile in a subsequent purification. Recovery from this column was markedly increased to 42%. The purified hormones each gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 21,000 and 23,000 for GH and PRL, respectively. Each hormone was positively identified by its N-terminal amino acid sequence, which showed high sequence identity with the equivalent eutherian hormone. Semianalytical gel filtration of purified hormone was used to demonstrate that each hormone remained as a monomer in aqueous solution. Each purified hormone was tested in the heterologous PRL radioimmunoassay (RIA) which has been used in many earlier studies to measure marsupial PRL. Highly purified wPRL was less potent than ovine prolactin (5.3 compared with 1.5 ng/ml at 50% displacement) and the cross-reaction of wGH was < 0.01%. Antibodies were raised against wPRL and wGH and a homologous RIA was developed for each hormone. The sensitivity of the wPRL assay was 0.8 ng/ml which is similar to that of the heterologous PRL assay. Cross-reaction with a number of eutherian pituitary hormones or wGH was < 0.07%. The wGH assay detected 0.8 ng/ml which is similar to that of the heterologous PRL assay. Cross-reaction with a number of eutherian pituitary hormones or wGH was < 0.07%. The wGH assay detected 0.8 ng/ml, cross-reacted with GH from several eutherian species, and showed low cross-reaction with wPRL (< 0.5). In both the wPRL and wGH assays, pituitary homogenates from several species of marsupial diluted in parallel with the wallaby standard, suggesting that these assays will be of use in studies of a number of marsupial species.
Homologous radioimmunoassay for plasma and pituitary prolactin in the toad, Bufo japonicus
General and Comparative Endocrinology, 1989
A specific and sensitive homologous radioimmunoassay (RIA) was developed for the measurement of toad (Bufo japonicus) prolactin (PRL). PRL isolated from toad pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and a homogenate of the anterior lobe of the pituitary gland of toads yielded dose-response curves which paralleled the standard. Plasma from hypophysectomized toads showed the least amount of cross-reaction. Bovine PRL, ovine PRL, ovine growth hormone, and a homogenate of toad neurointermediate lobes showed no inhibition of binding even at relatively high doses in this RIA. A preliminary application of homologous RIA for toad PRL was performed by determining plasma and pituitary PRL levels in adult toads of both sexes captured in spring and late summer. Plasma PRL levels of male toads remaining in the pond for breeding were significantly higher than those of the males feeding in the bush or hibernating under the ground. The pituitary PRL content in spring toads of both sexes was higher than those in summer toads. The pituitary PRL content in females was invariably lower than those in simultaneously captured males.