Search

To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a "hetero-antibody" RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCG beta or anti-ovine LH beta was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration.

Rat prolactin (PRL) cDNA was constructed in mammalian expression vector, pSVL. Transient expression of rat PRL was performed in COS-1 cells by the DEAE-dextran method. The production of recombinant rat PRL started within 48 h from the cells and reached the level of 1.0-1.5 micrograms/ml/5 x 10(5) cells. The molecular size of recombinant rat PRL was the same as that of standard rat PRL (Mw: 23,000), suggesting successful removal of the signal peptide. The radioimmunoassay and isoelectric focusing analysis showed that recombinant rat PRL has almost the same immunological and biochemical characteristics as those of standard rat PRL. As biological tests, receptor-binding activity, Nb 2 node lymphoma cell growth activity, and mammary gland stimulating activity were examined. The radioreceptor assay showed that recombinant rat PRL has binding activity to mammary microsomal membrane similar to that of standard rat PRL. Recombinant rat PRL also stimulated the growth of Nb 2 lymphoma cells as standard rat PRL. Finally it was shown that recombinant rat PRL promotes the synthesis of the secretory materials in the lumen of mouse mammary gland with the same potency as that of standard rat PRL. In conclusion, recombinant rat PRL, which was produced in mammalian cells in the present experiment, has immunological, biochemical and biological characteristics similar to those of standard PRL, and has full bioactivity.

The isoelectric components of LH have been isolated from the male rat pituitary glands by preparative isoelectric focusing. The biological activities of these components were measured in vitro from testosterone production in rat Leydig cells at equal doses and expressed as the equivalent of NIH-LH-S1 immunoreactivity. There were significant differences in the bioactivities among the components. The bioactivities of the components decreased with decreasing pI; components E (pI = 9.6) and F (pI = 9.8) showed about 5- to 6-fold higher activities than component A' (pI = 7.9). When the rat pituitary extracts were measured for LH by radioimmunoassay, and then measured for the ratio of biological activity to immunoreactivity (B:I), the order was as follows; intact female rats greater than intact male rats greater than orchidectomized rats. This phenomenon could be explained by the differences in the relative amounts of the LH components which changed according to the physiological states. These observations indicate that the LH components with different pIs and B:I ratio are related to the different estimation of total LH in the rat pituitary glands by bioassay and radioimmunoassay.

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.

The role of thyrotropin-releasing hormone (TRH) in the secretion of TSH from the anterior pituitary was investigated in rats by active and passive immunization with TRH. The plasma TSH response to propylthiouracil (PTU) in TRH-bovine serum albumin (BSA)-immunized rats was significantly lower than that of BSA-immunized or non-immunized rats. Similarly, the increased plasma TSH level following PTU treatment was significantly suppressed after iv injection of antiserum to TRH. However, the decline in plasma TSH levels was not complete. The results of the present study indicate, at least in part, the physiological significance of endogenous TRH in the regulation of pituitary TSH secretion.

The antiandrogenic properties of a new nonsteroidal antiandrogens, AA560 (N-2-chloromethyl-2-hydroxypropionyl)-3, 4, 5-trichloroaniline) were investigated. The ventral prostate, dorselateral prostate and coagulating gland weights in rats given AA560 at 1-9 mg/head were significantly less than those in the intact rats. The seminal vesicle weights in rats given 3-9 mg/head were significantly less than those of the intact group. In intact animals given daily 3 or 9 mg of AA560 there were significantly increases of serum FSH, LH and testosterone concentrations. In the in vivo experiment, the pretreatment with AA560 decreased the uptake of 3H-androgens in the nuclear fraction of the ventral prostate. On the other hand, a significant increase in the uptake of 3H-radioactivity in the cytosol fraction was observed. It was proved by the in vitro displacement study that AA560 inhibited the binding of 5 alpha-dihydrotestosterone with a receptor protein in the prostatic cytosol.

The receptor binding activity (R) and immunoreactivity (I) of serum and pituitary FSH were measured in normal, gonadectomized, and gonadal steroid-treated rats of both sexes employing radioreceptor assay and radioimmunoassay, respectively, and R/I ratios were analyzed. At 11 weeks of age, R/I ratios of serum and pituitary FSH were significantly higher in normal females than in males. From 11 to 13 weeks of age, the ratios of both FSH further increased in females, while in males, the ratio of serum FSH decreased and that of pituitary FSH increased. Orchiectomy caused an increase in the ratio of serum FSH, while ovariectomy caused a decrease, so the ratios in the gonadectomized animals of both sexes became almost equal, suggesting the secretion of a gonadectomy type FSH in these animals. Both testosterone propionate (TP) and estradiol benzoate (EB) treatments decreased the ratios of serum FSH in gonadectomized rats. However, TP and EB had an effect opposite on the ratio of pituitary FSH in these rats, i.e. TP caused a decrease, while EB caused an increase. This effect was more obvious in females than in males. These observations indicated that the presence and absence of the gonads, and the gonadal steroids influence not only the quantity but also the quality of FSH. The changes in the quality of the hormone are discussed in relation to its multiplicity.

The effects of chlorpromazine (CPZ) and estradiol benzoate (EB) on serum prolactin (PRL) levels were studied in gonadectomized male and female rats. In both sexes CPZ (25 mg/kg body weight) produced an elevation of PRL when measured 2 hr after the injection, but the elevated levels were higher in ovariectomized rats than in orchidectomized rats. These results reconfirm a sexual difference in the regulatory mechanism of PRL secretion in response to the dopamine receptor blocker. Pretreatment with 5 microgram EB 48 hr before CPZ injection abolished this sexual difference in serum PRL concentration.

Follicle stimulating hormone (FSH) in rat serum is successfully measured by a radioreceptor assay system employing PMS-treated immature rat ovary. The non-specific inhibitory effect of serum was partially overcome by the addition of merthiolate to every component, while the residual effect was compensated for by using FSH-free serum which was prepared by passing the pooled female diestrous rat sera through an immunoadsorbent column packed with anti-ovine FSH-coupled Sepharose 4B. The assay system consisted of 100 microliter of Tris-MgCl2-BSA or standard, 100 microliter of FSH-free serum or sample, 100 microliter of the receptor preparation and 100 microliter of 125I-FSH. The incubation was carried out for 4 hr at 37 degrees C and 500 microliter of cold Tris-MgCl2-BSA was used for the termination. Serum FSH could be measured within a range of 0.125-16 ng NIAMDD rat FSH I-3/tube. The mean within-assay coefficient of variation was 10.5%. The mean between-assay coefficient of variation was 11.0%. The assay values obtained by RRA showed a good correlation to those by RIA under the same physiological states of the animals. The ratio of the assay values, RRA/RIA, was found to change according to the sex and the physiological states, e.g. around 1.3 in normal males and 1.7 in orchiectomized animals and 2.21 in female rats. Serum FSH levels in female rats obtained by RRA and RIA changed almost in parallel until 20 : 00 (hr) of proestrous day, but after the first surge of serum FSH they were not parallel. These facts seem to indicate possible changes in the affinity of FSH with its receptor according to the state of animals and lead to the problem of the heterogeneity of FSH.

Anti-LH-RF serum was produced in rabbits by synthetic LH-RF-BSA conjugate prepard with bis-diazotized benzidine. Employing this antiserum, a radioimmunoassay for LH-RF was established with the assay range from 0.8 to 10 ng/ml. Acid extracts of hypothalami from 4 day cycling rats through various stages of the estrous cycle particularly in the afternoon of proestrus were measured for their LH-RF contents. The hypothalamic LH-RF content was high at 10:00 on the day of proestrus and declined until 17:00 and thereafter, the content gradually increased to reach the basal level. Serum LH showed the highest level at 17:00 on the day of proestrus and fell at 21:00 on the same day. Accompaning this change of serum LH level, pituitary LH content began to decrease at 17:00 of proestrus. These results indicate that LH-RF released in the afternoon of proestrus may participiate to stimulate the preovulatory discharge of LH in the rat.

Rat epidermal growth factor (rEGF) was isolated from the submaxillary gland of male rat by reversed phase high performance liquid chromatography and ion exchange chromatography. The binding of purified rEGF to human carcinoma cells (A-431) and its tritiated thymidine uptake on rat epidermal fibroblast cells (FR) were almost the same as those of purified commercially available mouse EGF (mEGF). Antisera to rEGF was raised in rabbits and a radioimmunoassay (RIA) system was established. The assay range of the RIA was about 1.0 to 100 ng/ml. The within assay coefficients of variation were 5 to 10%, while the between assay coefficients of variation were 5 to 13%. The tissue content of rEGF of male rats (10 weeks old) was examined. As a result, the submaxillary gland was found to contain a very high concentration of rEGF (214 micrograms/g wet tissue) as predicted, and digestive tissues, stomach, intestine and duodenum contained 2.49, 3.57, 9.44 ng/g wet tissue, respectively. The amounts in prostate and seminal vesicle were relatively high, being 65.6 and 2,268 ng/g wet tissue, respectively. The amount in the submaxillary gland increased markedly after 7 weeks of age. These results suggest that EGF is an important factor in gonadal function.

Serial modifications of Bottenstein and Sato' serum-free hormone-supplemented medium resulted in a new promising medium (1 : 1 mixture of L15 and MCDB 104 containing several supplements) for culturing neonatal rat brain cells. This medium favored the morphological and biochemical differentiation of the neurons, including particular types of cholinergic and cholinoceptive neurons, obtained enzymatically from the septum, preoptic area, and hypothalamus. On the other hand, the growth of non-neuronal cells was markedly suppressed in this medium. Therefore, their effects on the neurons are minimized in this culture. Effects of triiodothyronine (T3) and estradiol (E2) on the activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), synthetic and degradative enzymes for acetylcholine, respectively, were examined in this culture. The optimal concentrations of T3 and E2 for AChE activity were around 1 nM and 10 pM, respectively. However, E2 appeared to be somewhat inhibitory at higher concentrations. Although the activity of ChAT was maximum around 10 pM of E2, the ChAT activity increased as the concentration of T3 was increased to 100 nM.

Pituitary samples were obtained from female rats at various stages of the estrous cycle, and from intact male and gonadectomized rats with and without estradiol treatment. The pituitary extracts with 60% EtOH pH 9.5, were fractionated by preparative isoelectric focusing (IEF), and immunoreactive prolactin (IR-PRL) was measured by RIA. Three types of IR-PRL molecular species were found in these IEF profiles. The first type (species A) was consistently found in an area of pH 4.5-5.4, and consisted of two main subspecies with pls 5.0 (Al) and 5.25 (A2). Species A occupied most part of pituitary IR-PRL in males, gonadectomized animals, and in females in a basal state such as diestrus (D) II 17:00. Species A was also found exclusively in the serum at proestrus (PE) 19:00. The amounts of species A decreased notably when the secretion became active from PE 15:00 to 22:00, then increased at estrus (E) 6:00 and 10:00 when the second type (species B), which was found in the area of pH 5.4-6.8 only in trace amounts at basal states, increased markedly. Species B decreased again at E 17:00, while species A fully recovered. Species B also increased when PRL biosynthesis was stimulated by estradiol in intact male and gonadectomized rats. These findings indicate that species A must be the storage and secretory type of IR-PR, and that species B must be IR-PRL in the biosynthetic process which is to be finally converted into species A. A third type (species C) was found in a region of pH 3.5-4.5 in the IEF profiles of gonadectomized animals. This species is possibly IR-PRL molecules under degradation. When the pituitary was extracted serially with 0.25 M ammonium sulfate pH 5.5 (fraction AMS) first, then with 60% EtOH pH 9.5 (fraction ET), fraction AMS contained mostly species B and C, while fraction ET contained species A almost exclusively. The results obtained with this differential extraction roughly coincided with IEF data, though some disagreements were observed.

The immunological and biological potencies of a new synthetic TRF analogue, L-pyroglutamyl-L-histidyl-L-pipecolic acid amide, were compared with those of TRF. In a radioimmunoassay system for TRF, parallel inhibition curves were obtained with TRF and the analogue, the immunological potency of the latter being approximately 50 per cent of the former. In vitro and in vivo TSH-releasing activities of the analogue were almost equal to those of TRF. As was observed with TRF, the in vitro TSH-releasing effect of the analogue was reduced in the presence of T4, and the analogue was inactivated by incubation with rat serum. The data suggest that the pyrrolidine ring of TRF can be replaced with piperidine ring without significant loss of the biological activity.

Receptor fractions were prepared from follicle-rich ovaries (for FSH), luteal cell-rich ovaries (for LH and PRL), and adrenals (for PRL) of rats. Divalent metal ions, Mg++, Ca++, and Mn++ showed inhibitory effects on the binding of LH and FSH to their receptors. The binding of the former was more sensitive to these ions than the latter. On the other hand they showed bell-shaped promotive effects on PRL-ovarian receptor binding, the maximal effects being observed at 10-20 mM. Besides these ions, Ba++ also had a promotive effect, while other divalent metal ions such as Zn++, Cd++, Ni++, and Co++ showed inhibitory effects on PRL-ovarian receptor binding at 5 mM. Mg++ and Ca++ also promoted PRL-adrenal receptor binding, while Mn++ promoted the binding at 10 mM but inhibited it at higher concentrations. Association constant (Ka) and binding capacity (Bmax) of PRL receptors of the ovary and the adrenal were significantly different (ovary: Ka = 0.69 X 10(10) M-1, Bmax = 62 fmol/mg protein, adrenal: Ka = 0.21 X 10(10) M-1, Bmax = 99 fmol/mg protein). Ka of the ovarian PRL receptor was not influenced by these divalent ions, while that of the adrenal receptor was doubled by Ca and Mn ions, Bmax of the latter was also increased. A cooperative effect of Mg and Ca ions was observed on Ka and Bmax of the adrenal receptor. The sizes of the PRL binding sites of these organs revealed by affinity labelling were 17K and 40K in the ovary, and 40K and 110K in the adrenal. These results indicate the different properties of receptors in these different target organs.

We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15 x 10(-10) M. The standard curve was obtained with 0.2-25.6 ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the non-specific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intra-assay and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.

In order to elucidate the presence of free FSH-beta and LH-beta and their relation to the native hormones, immunoreactive FSH (IR-FSH), IR-LH, IR-FSH-beta, and IR-LH-beta subunits in the rat anterior pituitary and serum from 1200hr of proestrous day to 0600hr of estrous day were measured by radioimmunoassay. The amounts of IR-FSH-beta found were always more than those explained by the cross-reaction between FSH, and IR-FSH and IR-FSH-beta. On the other hand, IR-LH-beta was always less than the amount explained by the cross-reaction with LH, and changes in IR-LH and IR-LH-beta were parallel. There was also a difference between IR-FSH and IR-FSH-beta gel-filtration patterns in the rat anterior pituitary extracts. IR-FSH was eluted as a single peak, while IR-FSH-beta was found in three different regions. The first peak appeared near the void volume, the second corresponded to that of FSH, and the third was in the region of smaller molecules which was thought to be free FSH-beta. The first and the third peaks ofI R-FSH-beta changed characteristically during the estrous cycle. Radioiodinated FSH-beta, when incubated with the anterior pituitary extracts and applied to gel-filtration on Sephadex G-100, appeared also in three major peaks corresponding to those of IR-FSH-beta in the extracts. The first peak seemed to be aggregated or non-specifically bound FSH-beta and the second seemed to be FSH, while the third was thought to be free FSH-beta. These results indicated the presence of free FSH-beta in the rat anterior pituitary and serum, and raise a doubt about free LH-beta.

The seasonal variation in pituitary gonadotropin in the adult male newt, Cynops pyrrhogaster pyrrhogaster was investigated by means of isoelectric focusing (IEF) coupled with radioreceptor assay (RRA), which employed Anolis or Xenopus testicular homogenates as receptors and 125I-rat FSH as radioligand. In the Anolis RRA system, the standard curve was obtained with 0.125-16 ng/tube of NIAMDD rat FSH I-3. Purified preparations, chicken LH IEF-1, chicken FSH AGCHD11113A and bullfrog basic gonadotropin-IV competitively inhibited the binding of the radioligand, but NIAMDD rat LH I-4 and human chorionic gonadotropin did not crossreact. The autoradiographic study revealed that 125I-rat FSH bound to the constituent cells of the seminiferous tubules in the Anolis testis, but scarcely to Leydig cells. In the IEF pattern of gonadotropin in February obtained by Anolis RRA, distinct peaks were observed at pH 9.05 (component B) and 8.55 (component C), and less distinct peaks were observed at pH 9.80 (component A), 7.55 (component D) and 7.05 (component E). When the same fractions were assayed by Xenopus RRA, five components were found in the alkaline region, which corresponded to those observed with Anolis RRA. Similar results were obtained with pituitary extracts in May. In July, the IEF pattern obtained by Anolis RRA indicated two additional components at pH 6.30 (component F) and 5.27 (component G) in the acidic region, which were not found by Xenopus RRA. The relationship between the testicular function and the nature of pituitary gonadotropin in the reproductive cycle was discussed.

Female rats were irradiated with 190R of X-rays at 10 days of age and sacrificed 4, 7 or 12 months later. Their ovaries were histologically examined and serum levels and pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. Both serum levels and pituitary contents of LH and FSH rose significantly 4 and 7 months after irradiation, although the ovaries were markedly reduced in weight. On the contrary, 12 months after irradiation, the ovaries increased in weight and consisted mostly of polyhedral, hyperplastic interstitial cell masses, and both LH and FSH in the serum and pituitary were reduced to normal levels. These characteristic changes in the ovarian weight and histological appearance could not be observed in the similarly irradiated animals which were received daily injections of estrone for the first 30 days of postnatal life, i.e., daily injections of 50 mug for the first 10 days, 100 mug for the middle 10 days and 200 mug for the last 10 days. Serum LH levels of the estrogenized irradiated rats at 7 or 12 months of age did not elevate although those of FSH were significantly higher than the non-irradiated intact levels. From these results, a rise in the blood levels of LH and the FSH may be attributed to the increase in weight and the histological changes in the ovaries of the irradiated female rats, and the elevation of only FSh level may not result in the abnormal growth of the irradiated ovaries.

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.

We studied the effects of shakuyaku-kanzo-toh (a Chinese herbal medicine) and its components on testosterone production by rat gonads. We used paeoniflorin as a main component of shakuyaku (paeoniae radix), glycyrrhizin as a main component of kanzo (glycyrrhizae radix) and glycyrrhetinic acid as a main metabolite of glycyrrhizin. Oral administration of shakuyaku-kanzo-toh, glycyrrhizin, and glycyrrhetinic acid decreased in vitro basal testosterone production in Leydig cells by LH stimulation. Glycyrrhizin and glycyrrhetinic acid caused a significant decrease in testosterone production with an accumulation of 17 alpha-hydroxyprogesterone when incubated with isolated Leydig cells, while paeoniflorin showed no such effect. The inhibitory effect of glycyrrhetinic acid was far more potent than that of glycyrrhizin, causing about 90% inhibition at 10 micrograms/ml. Glycyrrhizin and glycyrrhetinic acid did not change the cyclic AMP or progesterone level in the Leydig cells. When 14C-labeled androstenedione was incubated with microsomal fraction of testicular or ovarian tissue, glycyrrhizin and glycyrrhetinic acid inhibited the conversion of androstenedione to testosterone, indicating that these compounds inhibit the activity of 17 beta-hydroxysteroid dehydrogenase (EC. 1.1.1.64). The ED50 of glycyrrhetinic acid was about 4 microM.

Synthetic LHRH, at a dose of 5 mug/kg, induced a marked increase in the serum LH level but a marked decrease in the serum prolactin level when injected iv to normal adult female rats on the day of estrus. In adrogenized adult rats which had been showing persistent estrous vaginal smears, a less marked increase in the serum LH level was shown following injection of LHRH. Prolactin level decreased only slightly in these animals. Control level of serum prolactin in normal estrous and androgenized rats were 4.95 +/- 0.35 and 5.42 +/- 0.65 mIU/ml, respectively. Thyroidectomized-normal cycling rats responded to LHRH with a slight decrease in the serum prolactin level when injected on the day of diestrus. Prolactin level increased, however, 5 min after LHRH injection in thyroidectomized-androgenized adult rats, which were thyroidectomized at the age 4, 6 or 10-12 weeks and have retained a low prolactin level similar to that in the diestrous stage of normal cycling rats thereafter. The present results suggest that regulatory system of prolactin secretion became labile by androgenization and this status led to the release of prolactin by LHRH treatment after thyroidectomy.

Radioimmunoassay of rat luteinizing hormone (LH) in the sera and the anterior pituitaries of normal and castrated males and proestrous females employing different batches of anti-LH sera gave the results suggesting heterogeneity of immunoreactive LH (IR-LH). Gel filtration analyses of anterior pituitary extract and pooled sera failed to give positive evidence for the heterogeneity. But when the extracts of normal rat anterior pituitaries were electrofocused and the fractions were assayed, the pituitary glycoprotein hormones, LH, FSH and TSH, were found to consist of several components with different isoelectric points. In electrofocusing patterns of IR-LH, distinct peaks were observed at pH 8.5 (component A), 8.8 (component B), 9.1 (component C) and 9.3-9.4 (component D), and less distinct peaks were observed at pH 7.9 (component A'), 9.6 (component E) and 9.8 (component F). There seemed to be some sexual difference in the relative amounts of these components. Orchiectomy caused marked changes in electrofocusing patterns of IR-LH, i.e., increase of components A', A, B, C and D. The blockade of LH release by estradiol benzoate and progesterone treatment in orchiectomized rats caused a marked increase of component A. At the proestrous LH surge, IR-LH components were evenly released. Radioimmunoassay of IR-LH components with multiple antisera indicated that these antisera had different affinities to these components.

Anterior pituitaries were removed from female rats at various stages during the estrous cycle and FSH was fractionated by isoelectric focusing (IEF). Fourteen FSH components were observed during the estrous cycle and twelve of them were distributed between pH 3.71 and 6.66. IEF profiles of FSH in the pituitaries varied with the stage in the estrous cycle. Especially at the time of serum FSH surge on the day of proestrus, most of the components decreased, while only a highly alkaline component showed an increase. When these FSH components were separated and their nature was examined by radioreceptor assay (RRA), radioimmunoassay (RIA) and gel filtration, differences were observed among these components in the RRA/RIA ratio and gel filtration profile. As a general tendency, the RRA/RIA ratio of the components became greater while the apparent molecular size became smaller, as their pI became higher. However, some highly acidic components showed a biphasic elution pattern and the most acidic one eluted the latest on gel filtration, suggesting that these components may be heterogeneous in terms of molecular size. The FSH concentration in sera collected at different stages in the estrous cycle was measured by both RRA and RIA. The RRA/RIA ratio was high when the serum immunoreactive FSH was low, and low during the FSH surge. From these findings, it is concluded that the quality of FSH molecules present in the anterior pituitary gland changes dynamically throughout the estrous cycle, especially during the period of serum FSH surge. Furthermore it is suggested that the type of FSH secreted from it also varies according to the stage in the estrous cycle.

Five-day-old female rats were androgenized with 1,000 or 100 microgram testosterone propionate and were examined regarding the response to LHRH at 4, 7 and 12 weeks of age by measuring peripheral LH concentrations. The order of magnitude in LH release was 7 greater than 4 greater than 12 weeks old, whereas in normal rats, 4 greater than 12 greater than 7 weeks old. LH release in 4- and 7-week-old rats was higher than that in normal controls at the respective age, but was much lower than that in normal controls 12 weeks old. The LH release by Des-Gly10-(D-Ala6)-LHRH-ethylamide (TAP127) was greater than that by natural LHRH both in normal and androgenized rats at 7 or 12 weeks old. The results indicate that the pituitary gland in androgenized rats responds to LHRH to a much larger extent during the premature period and its responsiveness declines during the course of maturation. A marked hypersensitivity was observed in 7-week-old rats androgenized with 100 microgram testosterone propionate. The process of androgenization may include the induction of alterations in the sensitivity of the pituitary to LHRH and probably in the LH synthesizing ability of the pituitary.

After a single PMS (50 IU) injection to 25-day-old rats, FSH receptor content of the ovarian tissue increased progressively for 4 days, then showed a tendency to decrease, while LH receptor content remained unchanged for 3 days, then gradually increased. From these facts, we established a radioreceptor assay system, employing 3,000 rpm precipitates of homogenates of the ovaries obtained 3 days after PMS injection as the receptor preparation. The dissociahe standard curve was obtained with 0.125--16 ng/tube of NIAMDD rat FSH I-3. Purified preparations, NIAMDD rat LH I-4 and NIAMDD rat TSH I-4 influenced the binding only at high concentrations possibly owing to FSH contamination. When the anterior pituitary homogenates obtained from rats in the various physiological states were assayed by this system, the intra-assay coefficient of variation and inter-assay coefficient of variation were 7.5% and 13.7%, respectively, and the assay values were well correlated with those obtained by radioimmunoassay (r = 0.988, the slope of the regression line = 1.14).

The hormone levels in the anterior pituitary gland and serum in Nagase analbuminemia rats (NAR), a mutant strain established from Sprague-Dawley rats with hyperlipidemia, were examined. For the anterior pituitary gland, the prolactin, TSH, GH, LH and FSH contents in male NAR were significantly lower than those of normal rats. In female NAR, prolactin, TSH and LH levels were also lower than those in normal rats, whereas FSH and GH were normal. For the serum, the concentrations of TSH, total T3, total and free T4, estradiol-17 beta and testosterone were examined. The serum testosterone concentration in NAR was lower than that of normal rats. Histochemical examination of the hydroxysteroid dehydrogenase (HSD) activity of testes was made in relation to the serum testosterone level. NAR testes, which are rather small compared with those of normal rats, have lower HSD activity. A higher level of serum TSH was seen in NAR. Total and free T4 concentrations were low in the male NAR only. Estradiol-17 beta and T3 concentrations in NAR were unchanged. Changes in serum LH and FSH levels during the estrous cycle in NAR were also studied. Their patterns of change are normal.

Chicken LH components from the glycoprotein fraction of the anterior pituitary glands have been separated to isoelectric homogeneity by means of isoelectric focusing, and investigated for their biological activities in vitro. The activities of these components were measured with LH receptor binding, cyclic AMP accumulation and testosterone production in rat Leydig cells at equal doses expressed as immunoreactivity of IRC-2 (Gunma). All the components were active in the heterologous assay systems, although the relative potency expressed as the ratios of biological activity to immunoreactivity (B:I) differed significantly among the components. Component I, the amount of which is the largest (40-50%), with the most alkaline isoelectric point (pI) showed the highest B:I ratio, and the B:I ratio decreased with decreasing pI in the same way as in rat LH components (Wakabayashi, 1980; Hattori et al., 1983). Therefore, pituitary LH from photostimulated male quail, where the relative amount of component I was increased, was estimated to have higher B:I ratios than that from short-day (SD) treated male quail. Furthermore, the differences in activities among the components obtained by the three assays were in the following order: testosterone production greater than cyclic AMP accumulation greater than receptor binding, suggesting that the hormonal actions of components with higher B:I ratios (I, II and III) are efficiently amplified in the biological response to the final step. In the incubation of pituitary glands with hypothalamic extracts, component I in the pituitary glands from the long-day (LD) treated group was mostly decreased after the incubation, while all the components were decreased in parallel in the SD-treated group. The results suggest that the LH component releasable to GnRH changes in endocrine status though component I with the highest B:I ratio is relatively releasable in both SD- and LD-treated groups.

In order to prepare an antibody directed at the common amino acid sequence of mammalian, avian, and fish luteinizing hormone-releasing hormones (LHRHs), C-terminal free LHRH was conjugated with bovine thyroglobulin, and was used as the antigen. A monoclonal antibody (LRH13) was obtained as an ascitic fluid by fusing the spleen cells of a BALB/c donor mouse immunized with the antigen to X63.Ag8.653 mouse myeloma cells followed by limiting dilution cloning and transplanting a positive clone to BALB/c mice. This monoclonal antibody seems to belong to IgG2b as it was eluted from protein A-Sepharose CL-4B with citrate buffer pH 3.5. competitive binding experiment using fragment peptides of LHRH indicated the binding site of LRH13 was a region around serine and tyrosine, and modification of mammalian LHRH by radioiodination caused a marked decrease in the binding activity. LRH13 has an affinity constant of 0.134 X 10(9) M-1 to native mammalian LHRH, and binds C-terminal free LHRH with a similar affinity (1.6X), however, it binds with higher affinities to N- and C-terminal free LHRH (12.9X), N-terminal free LHRH (10.4X), salmon LHRH (8.3X) and chicken LHRH-I (6.0X). Chicken LHRH-II, where tyrosine is replaced for histidine, has a lower affinity (0.3X) than that of mammalian LHRH. From its high affinity to N-, C-terminal free LHRH, LRH13 is also expected to bind possible precursor peptides of LHRH. Immunohistochemical staining of the brain sections obtained from rats, mice, chickens, Japanese quail, and rainbow trout successfully visualized cell bodies and fibers distributed from the olfactory bulb to the median eminence, indicating high LHRH specificity and wide crossreactivity in animal classes of this monoclonal antibody. With this antibody, LHRH-like immunoreactive substance in the pineal gland was also stained with fixation at neutral pH.