Pituitary Homogenate Research Articles

In vitroovulation and prostaglandin synthesis by ovarian follicles ofRana dybowskii

Changes in the levels of prostaglandin F2α (PGF2α) and E2 (PGE2) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12‐O‐tetradecanoylphorbol‐13‐acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF2α to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE2 increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF2α induced ovulation but PGE2 suppressed FPH‐ or PGF2α‐induced oocyte ovulation. Basal levels of PGs increased steadily during culture. When theca/epithelium (THEP) layer and granulosa cell‐enclosed oocytes (GCEOs) were separated by microdissection and cultured independently, higher levels of both PGs were secreted by THEP than by GCEOs. Synthesis of PGs by follicle or follicular components was strongly suppressed by exogenous cAMP or indomethacin. These results suggest that: 1) PGF2α plays an important role in Rana ovulation, 2) protein kinase C is involved in PGs production, and 3) theca/ epithelium layer is responsible for the PGs production in Rana.

Expression, characterisation and immunoassay of recombinant marmoset chorionic gonadotrophin dimer and beta-subunit.

A specific and sensitive ELISA for measuring marmoset chorionic gonadotrophin (mCG) in culture medium, urine and plasma was developed using a polyclonal antibody raised against recombinant mCG, tagged with six histidine molecules (rmCG-6His), as the capture antibody. A well-characterised monoclonal antibody (518B7), which was generated against bovine luteinising hormone (bLH) and has been shown to detect CG and LH in Callithrichid monkeys, was biotinylated and used as the secondary antibody. Purified rmCG, calibrated against human CG (hCG; CR127) by bioassay, or the beta-subunit (rmCGbeta), quantified from amino acid analysis and carbohydrate analysis, was used as the standard. The assay was able to detect CG activity in medium collected from cultured marmoset embryos before attachment and through to the trophoblastic vesicle stage, plasma and urine collected from pregnant marmosets, marmoset placenta and pituitary homogenates. The assay was validated and its performance compared with a bioassay based on MA10 cell response to CG, with hCG as the standard. The sensitivity was 103 pg/ml (5 pg/well) of rmCGbeta and 476 pg/ml (24 pg/well) of the heterodimer rmCG. The mean recovery of standard added to embryo culture medium, marmoset urine and plasma was 104, 112 and 92% respectively. The intra- and interassay variation was less than 10 and 16% respectively. The low cross-reactivity with cynomolgus monkey and baboon LH, their beta-subunits, cynomolgus monkey and baboon follicle-stimulating hormone and hCG suggests that the assay is specific for mCG.

Long-Term Calorie Restriction Protects Rat Pituitary Growth Hormone-Releasing Hormone Binding Sites from Age-Related Alterations

In mammals, middle age and late adulthood is characterized by a decrease of growth hormone (GH) secretion and insulin-like growth factor 1 (IGF-1) serum levels, contributing to tissue and organ atrophy. This condition is related, at least in part, to alterations of pituitary GH-releasing hormone (GHRH) receptor-binding sites. Prevention of age-related deterioration of tissues and organs, retardation of the onset or progression of a wide range of age-related diseases and extension of both mean and maximum life span can be achieved through life-long moderate calorie restriction (CR). Because CR has been reported to positively modulate the somatotropic axis resulting in the maintenance of a youthful GH secretory pattern in aged rats, we investigated whether or not benefits of a long-term (10 months) 40% CR, started in 8-month-old male Sprague-Dawley rats, was accomplished by preventing age-related alterations of pituitary GHRH receptor binding sites. We also studied whether or not a short-term (50 days) 40% CR, started in 16-month-old rats, could revert them. Potential hormonal and metabolic modulators of the GHRH receptors were investigated as well. GHRH binding parameters were derived from saturation studies performed in pituitary homogenates with [¹²⁵I-Tyr¹⁰]hGHRH (1-44)NH₂. As previously reported, the high affinity GHRH receptor-binding sites were blunted in 18-month-old ad libitum-fed rats and the apparent concentration of total binding sites was reduced. Short-term CR neither restored high affinity GHRH binding sites nor increased the apparent concentration of total binding sites. On the contrary, long-term calorie-restricted 18-month-old rats exhibited high and low affinity GHRH binding sites (K_d1: 1.73 ± 0.35 nM; K_d2: 310 ± 41 nM; B_max1: 183 ± 55 fmol/mg protein; B_max2: 30 ± 3 pmol/mg protein) as found in 2-month-old rats (K_d1: 0.68 ± 0.15 nM; K_d2: 350 ± 47 nM; B_max1: 219 ± 53 fmol/mg protein; B_max2: 84 ± 9 pmol/mg protein). Our results imply that CR must be implemented before age-related alterations of GHRH receptor-binding sites become too severe or that CR has to be carried out for a long period of time, independently from the age at which it begins. Protection of pituitary GHRH binding sites from age-related alterations could not be attributed to changes in circulating levels of total or free T₄ or free fatty acids. Finally, the anti-aging effect of a long-term CR observed at the level of pituitary GHRH receptors does not result in a significant increase of total IGF-1 circulating levels. Identification of molecular and cellular mechanisms responsible for these actions will deserve attention in order to identify centrally and/or peripherally active classes of molecules that could preserve, in aging mammals, the functionality of the somatotropic axis through selective regulation of pituitary GHRH receptors.

Stress responsiveness of the pituitary-interrenal axis during early life stages of common carp (Cyprinus carpio).

Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.

An intersexual migratory (silver) longfinned New Zealand eel and its gonadal response to treatment with salmon pituitary homogenate

An intersexual migratory (silver) longfinned New Zealand eel and its gonadal response to treatment with salmon pituitary homogenate

An intersexual migratory (silver) longfinned New Zealand eel and its gonadal response to treatment with salmon pituitary homogenate

An intersexual adult New Zealand longfinned eel Anguilla dieffenbachii, displaying testicular tissue within its ovaries, is described. Gonad samples were collected at 0, 21, 42 (by biopsy) and 54 days (autopsy) after the start of an experiment that aimed to induce ovarian maturation by repeated treatment with 5 mg kg−1 salmon pituitary homogenate (SPH). Very small patches of testicular tissue (almost always single seminiferous tubules), not visible macroscopically, were observed on all sampling dates. Treatment with SPH resulted in advanced stages of spermatogenesis in these seminiferous tubules, synchronous with the development of oocytes, which reached the migratory nucleus stage after 53 days. This finding is discussed in relation to current knowledge on sexual development in anguillid eels.

Evaluation of a sensitive, in vitro bioassay for chicken thyroid stimulating hormone using FRTL-5 cells, a rat thyroid cell line

An in vitro bioassay for mammalian thyroid stimulating hormone (TSH) based on TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production in FRTL-5 cells, a rat thyroid cell line, was used to measure chicken TSH. The addition of chicken pituitary homogenate equivalent to > or = 25% of a chicken pituitary gland to cultured FRTL-5 cells increased cAMP within these cells in a dose-dependent manner. The glycoprotein fraction derived from the pituitary homogenate was further fractionated by isoelectric focusing within a pH range of 5 to 11. Analysis of the focused fractions by the bioassay detected three major components with isoelectric points of 9.30, 7.12, and 3.82, in addition to several minor ones distributed over a wide range of pH, from alkaline to acidic. The isoelectric focusing profile obtained by the bioassay was clearly different from those obtained by radioimmunoassay for chicken LH and radioreceptor assay for chicken FSH, indicating that fractions contained chicken TSH. The homogenate of the cephalic portion of the chicken anterior pituitary gland was 4.46 times more active than that of the caudal portion in the bioassay, which is consistent with previous findings on localization of TSH in the chicken pituitary. We conclude that the bioassay using FRTL-5 rat thyroid cells is a sensitive, specific, and time-saving method of measuring chicken TSH.

Pretreatment Reproductive Stage and Oocyte Development Induced by Salmon Pituitary Homogenate in the Japanese Eel <i>Anguilla japonica</i>

Pretreatment Reproductive Stage and Oocyte Development Induced by Salmon Pituitary Homogenate in the Japanese Eel <i>Anguilla japonica</i>

Isoelectric Focusing Profiles of Pituitary Homogenates of the Japanese Quail during Sexual Maturation.

Crude extracts were prepared from the anterior pituitary glands of male and female birds of 8- and 10-week-old Japanese quails and fractionated by isoelectric focusing. Focused fractions were subjected to a radioreceptor assay using homogenized rat ovary as receptor preparation and 125 I-labeled rat follicle stimulating hormone as radioligand. This radioreceptor assay was previously characterized for measuring mammalian follicle stimulating hormone. Components having basic Isoelectric points (pI) were detected in both male and female birds of 8- and 10-week old. The component was most basic (pI 12.2) and largest (1.6μg/fraction/pituitary gland) in 10-week old female birds. The component in males was larger in 10-week-old birds than in 8-week-old birds. The present findings suggest that changes in pI and amounts of Isoelectric components may be related to sexual maturation in Japanese quails.

Inhibition of nitric oxide facilitates LH release from rat pituitaries

We examined the effects of nitric oxide (NO) modulators on rat pituitary LH content in vivo and studied their response to LHRH-stimulated LH secretion in vitro in ovariectomized adult female Sprague Dawley rats. Alzet mini pumps (flow rate 10 μl/h) delivering either normal saline (Group I), 1.2 mg nitroglycerin, a donor of NO (Group II) or 50 mg of nitro-L-Arginine methyl ester, a NO synthase (NOS) inhibitor (Group III), were subcutaneously implanted into experimental animals. Following 36 h infusion, pituitaries were removed and either frozen for LH quantitation, or fragmented and challenged in the superfusion system with 10 min pulses of LHRH (1 ng/ml) at 90 min intervals for 10 hours. LH was assayed by radio-immunoassay (RIA) in the homogenates of pituitaries and in aliquots of the superfusate collected every 10 mins. Significantly lower pituitary LH levels were noted in Group III (150.3 ± 18.6 ng) in comparison to Groups I (215.6 ± 5.5 ng; p < 0.04) or II (221.2 ± 14.9 ng; p < 0.01), suggesting that low levels of NO stimulate LH secretion in vivo. The pituitary LH contents were not significantly different in Groups I and II. In vitro studies reveal that exogenous LHRH stimulated response, measured as average pulse response (90 minute period after LHRH), and total LH released during the 10 hour perfusion, was 290 ± 23.6 ng and 1646.7 ± 270.8 ng, respectively, in Group III; 57.9 ± 3.1, and 344.7 ± 24.3 ng in Group I, and 105.3 ± 6.3, and 633.7 ± 77.1 mg in Group II. Thus, our in vitro studies demonstrate significantly enhanced (p < 0.05) LHRH- stimulated LH secretion in Group III in comparison to Groups I and II, while Group II shows higher responsiveness than Group I (p < 0.05). The results of the current studies provide evidence that NOS inhibition facilitates pituitary LH secretion. The differential responses to LHRH-stimulated LH secretion in vitro in the 3 groups suggest a possible role of NO in modulating pituitary LHRH receptor concentrations. However, this will have to be tested by further studies.

Follicle-Stimulating Hormone in the Brushtail Possum (Trichosurus vulpecula): Purification, Characterization, and Radioimmunoassay

Follicle-stimulating hormone (FSH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands by using the following purification techniques: fractional ammonium sulfate precipitation, triazinyl-dye affinity chromatography, hydrophobic interaction chromatography, and gel filtration. A yield of 18 μg of FSH per gram of pituitary, with a recovery of 12%, was obtained from 1400 glands (20.3 g wet weight). The purified FSH activity per gram of protein was 1320 times more potent than the initial pituitary homogenate. Contamination with possum luteinizing hormone (LH) was <0.02%. The amino acid composition of possum FSH was similar to that of ovine FSH. Amino-terminal sequencing for 11 cycles indicated that the alpha subunit has the same sequence as ovine FSH except for residue 7, where the possum FSH alpha subunit contains isoleucine compared to the ovine subunit which contains threonine. The beta subunit has two substitutions in the first 11 residues and does not contain the N terminal serine that is found in ovine FSH. Amino acid sequencing did not detect any contaminating proteins. Possum FSH bound possum and bovine testicular receptors with similar affinities. It was also able to stimulatein vitrocAMP production by Chinese hamster ovary cells which express recombinant FSH receptors. In the receptor assays and the bioassay possum FSH has about 21% of the potency of ovine FSH (USDA-oFSH-19-SIAFP-RP2). An RIA was developed for possum FSH using125I-possum FSH and an antiserum raised against human FSH. The RIA has a sensitivity of 0.3 ng/ml, a 50% displacement of 2.7 ng/ml, and a cross reactivity of 0.05% against possum LH. Plasma FSH levels in male possums (10.4±2.4 ng/ml,n=4) were higher (P<0.05) than levels in females (1.0±0.1 ng/ml,n=4). Five days after gonadectomizing these possums the plasma FSH levels increased (P<0.05) to 27.1 ± 0.2 ng/ml in the males and to 6.6 ± 2.0 ng/ml in the females. In summary, we have purified and partially characterized possum FSH. We have also set up an RIA for the hormone and shown that males have higher levels than females and that plasma FSH increases after gonadectomy.

Differential effects of gonadotropin and orthovanadate on oocyte maturation, ovulation, and prostaglandin synthesisby Rana ovarian follicles in vitro

Effects of gonadotropin and sodium orthovanadate on oocyte maturation, ovulation, prostaglandin, and progesterone synthesis were examined during in vitro culture of Rana ovarian follicles obtained from hibernating animals. Frog pituitary homogenates (FPH, 0.05 gland/ml) effectively induced oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation (> 90%) in ovarian follicles obtained in mid-hibernation (mid-December through late January). In contrast, orthovanadate induced a limited amount (< 45%) of ovulation of some oocytes without concomitant induction of maturation during mid-hibernation. In late hibernation (February to early March), considerable spontaneous maturation and ovulation occurred in cultured ovarian follicles. During this period both FPH (0.0005-0.05 gland/ml) or orthovanadate (0.01-1 mM) treatment markedly increased the incidence of ovulation and accelerated the onset of ovulation in a dose dependent manner. When examined after 12 h of culture, few (< 5%) oocytes ovulated in response to orthovanadate had undergone GVBD whereas most (> 90%) of those ovulated in response to FPH had matured. However, the majority of oocytes ovulated (72%) within 6 h of exposure to FPH had intact GVs, indicating that GVBD is also not a prerequisite for ovulation induction in response to FPH. Orthovanadate stimulated PGF2 alpha in a dose dependent manner but failed to stimulate progesterone production whereas FPH stimulated secretion of both PGF2 alpha and progesterone. The amount and time course of PGF2 alpha secretion in response to orthovanadate were similar to results produced with FPH stimulation. Treatment of PGF2 alpha to follicles obtained in late hibernation also accelerated the ovulation of the oocytes. Taken together, the data suggest that orthovanadate enhanced ovulation of immature oocytes was mediated via enhanced PGF2 alpha production and that oocyte maturation is not essential or prerequisite for in vitro oocyte ovulation in Rana.

Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland.

A new preparative procedure without using ion-exchanger is described for the efficient purification of canine LH (cLH), FSH (cFSH) and TSH (cTSH) from the pituitary gland. The hormones were extracted from the pituitary homogenate with an ammonium sulfate solution, and were separated by Concanavalin (Con) A affinity-, hydrophobic interaction-, then immobilized metal ion affinity chromatography. In the immobilized metal ion affinity chromatography, we used copper (Cu2+) as chelated metal ion with ammonium ion gradient and pH gradient in phosphate buffer to attain separation of the hormones. High purity of cLH, cFSH and cTSH was indicated as single bands in SDS-PAGE, with apparent molecular masses of 34, 36 and 37 kDA, respectively. The purified hormones showed two bands corresponding to alpha (20 kDa) and beta subunits (cLH beta: 16 kDa, cFSH beta: 22 kDa, cTSH beta: 16 kDa) under reducing condition in SDS-PAGE. The purified hormones were prepared in good recovery (LH: 53%, FSH: 34%, TSH: 36%) with high biological activity or binding activity to the receptor. Cross-contamination of the purified hormone was less than 0.5%. Examination of the hormone fraction with isoelectric focusing showed that major peaks of isoelectric isoforms were maintained throughout the purification steps of cLH and cFSH, while a few peaks were lost in Con A affinity chromatography in cTSH purification. It was concluded that the present method could prepare highly purified cLH, cFSH and cTSH which retained isoforms of the hormones and biological activity or binding affinity to the receptor.

トラフグ養成親魚からの採卵技法の開発

We developed an effective method for the induction of oocyte maturation and ovulation in the cultured tiger puffer Takifugu rubripes by hormonal treatments, in order to supply the fertilized eggs for seedling production. Ovarian biopsy just prior to the hormonal treatments revealed the oocyte diameter and developmental stages of the oocytes.A luteinizing hormone-releasing hormone analogue (LHRH-a) cholesterol pellet (400μg/kg) implantation successfully induced oocyte maturation for three-year-old cultured tiger puffer which had yolk accumulated ovaries. Thereafter, single injection of HCG (500IU/kg) combined with chum salmon pituitary (SP) homogenate (7mg/kg) was applied to the fish to synchronize ovulation among fish. Ovulation mainly occurred between 96 and 120 hours after HCG+SP injection. Thus, a technique for obtaining eggs of high quality from the cultured tiger puffer was developed.

Changes in transcription of pituitary glycoprotein hormone α and gonadotropin IIβ subunits during ovarian development induced by repeated injections of salmon pituitary homogenate in the Japanese eel, Anguilla japonica

To investigate transcription of pituitary glycoprotein hormone a and gonadotropin IIβ (GTH IIβ) subunits in female Japanese eel, gene quantitation systems using reverse transcription-polymerase chain reaction (RT-PCR) were developed, using β-actin as an internal control gene. These systems were not only sensitive, but also precise, with low coefficients of variation within and between assays (below 20 and 30%, respectively). Using these quantitation systems, we investigated how transcription of both subunits changed in Japanese eel during ovarian development induced by repeated injections of salmon pituitary homogenate (SPH). Transcriptional level of pituitary glycoprotein hormone α was similar to that of s-actin before SPH injection, and increased slowly during ovarian development, reaching a maximum (3-fold increase over initial levels) at the migratory nucleus stage. In contrast, the level of GTH IIβ mRNA was very low (about one-tenth that of β-actin) before SPH injection, but expression increased markedly during ovarian development with an approximate 120-fold increase over initial levels (12 times that of β-actin) at the migratory nucleus stage. These results suggest that GTH II synthesis in female eel was activated during ovarian development induced by repeated SPH injections. The increase in mRNA levels of both subunits mRNA is probably due to sex steroids produced in eel ovarian follicles. Unexpectedly, transcriptional level of GTH IIβ was abnormally high compared to that of pituitary glycoprotein hormone α at the migratory nucleus stage. These results suggest that repeated SPH injections induced aberrant endocrine conditions in female Japanese eels.

Heterogeneity in buffalo pituitary prolactin.

The Ellis procedure of serial extraction of gonadotropins and growth hormone (GH) followed by alkaline ethanol extraction was adopted to process freshly frozen buffalo pituitaries. The procedure after slight modification was found very useful as more than 2 mg of GH free immunoreactive prolactin (PRL) could be isolated from each gram of wet pituitary tissue. Further, the biochemical purity and immunobiological potency of the extracted PRL, designated as P-I, was comparable with that of the highly purified samples of homologous and heterologous PRLs. No non-PRL protein was detectable in P-I. Micro-heterogeneity with regard to size, charge, co- and post-translational modifications was also investigated under different conditions of extraction and at different stages of purification. Immunological and biological potencies were compared in homologous competitive enzyme linked immunosorbent assay (ELISA) developed for buffalo PRL and in rat Nb2 lymphoma proliferation assay respectively. Structural heterogeneity was observed in all the preparations checked including fresh pituitary homogenate and highly purified hormone. Nevertheless a 25 K species corresponding to the hormone monomer was always the only paramount form comprising more than 90% of the total PRL protein in all the samples including P-I. Similar size forms were observed in all preparations and were found to be equivalents of monomers, dimers, covalent-and non-covalent multimers, disulphide bridged forms and cleaved fragments. Other sibling species identified were glycosylated PRL, charge isoforms and forms that perhaps differed in their extractability from the pituitary tissue. Strong apparent size heterogeneity was displayed by the monomeric buffalo PRL. In light of these observations and the information on the structural and functional significance and the consequences of polymeric forms, the use of a heterogeneous PRL (P-I) as a reference hormone is recommended for a valid assay.

Changes in Bioactive Prolactin-like Activity in Plasma and Its Relationship to Incubation Behavior in Breeding Ring Doves

Previous radioimmunoassay (RIA) data indicate that plasma prolactin (PRL) is elevated during the late incubation and the early posthatching periods of the ring dove breeding cycle. Although these changes are temporally associated with changes in PRL-dependent crop sac growth, the precise relationship between immunoreactive and bioactive PRL has not been directly examined. To investigate this question and to further explore the relationship between sitting behavior and PRL secretion, we used rat Nb2 lymphoma cell proliferation to estimate the concentration of bioactive PRL-like activity (PLA) in the plasma of breeding ring doves. Serial dilutions of dove pituitary homogenate and dove plasma stimulated mitogenic responses that were parallel to those observed with purified ovine PRL. Changes in plasma PLA during the breeding cycle closely resembled changes in PRL that have been previously reported by RIA, although the relative changes in PLA were more pronounced. In both sexes, PLA remained at basal levels prior to egg laying and during early incubation (Day 4–5) but then abruptly increased to reach peak values near the time of hatching (Day 14–15). Activity remained high for 3–4 days after hatching, declined gradually thereafter, and returned to baseline values by Posthatching Days 14–17. Plasma PLA levels of birds sampled at the end of incubation were correlated with those of their breeding partners. In the majority of pairs, females had higher PLA levels than their mates at this stage even though no significant overall sex differences in PLA levels were observed. Plasma PLA declined precipitously in birds that were nest deprived on the last day of the incubation period. Nevertheless, plasma PLA levels of normally breeding birds at the end of incubation were not correlated with the average time spent in the nest during the incubation period. However, day-to-day variability in time spent in the nest correlated negatively with plasma PLA in incubating males, and females exhibited a similar trend that approached significance. These data suggest (1) that published RIA estimates of PRL are reasonably accurate reflections of changes in bioactive PLA in dove plasma and (2) that while sitting duration itself is not strongly related to plasma PLA, large day-to-day fluctuations in nest occupation time are associated with reduced PLA levels in incubating doves.

Progress in controlled breeding of Nassau grouper ( Epinephelus striatus) broodstock by hormone induction

Human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogue (LHRH-a), and carp pituitary homogenate (CPH), used alone or in various combinations, were tested as spawning agents in captive E. striatus broodstock. Fifty hormone-induced strip-spawning trials were attempted using a two-injection sequence in which a priming dose (PD) was followed 24 h later by a resolving dose (RD). Hormone treatment strategies tested included, HCG alone (PD = 1000 IU kg −1 bw, RD = 500 IU kg −1 bw), LHRH-a alone (PD = 50–100 μg kg −1 bw, RD = 100–200 μg kg −1 bw), HCG (500 IU kg −1 bw) in combination with LHRH-a (50–100 μg kg −1 bw) as priming or resolving doses, and CPH (PD = 10 mg kg −1 bw) in combination with LHRH-a (RD = 50–100 μg kg −1 bw). As an alternative to hormone injection, intramuscular implantation of a cholesterol pellet containing LHRH-a (200–250 μg) was tested in two females. Females with mean oocyte diameters ranging from 482 to 561 μm were suitable for hormone-induced spawning. Oocyte diameter increased to 524–708 μm within 24 h of the priming dose and to 852–945 μm within 9–16.4 h following the resolving dose. Average diameter of spawned (water hardened) eggs ranged from 879–978 μm. Although fertilization rate (0–94.7%) varied widely among trials, successful spawnings (fertilization rate ≥ 50%) were obtained in all of the hormone strategies tested. Use of different hormones in combination showed no advantage over a single-hormone strategy. As HCG appeared to cause an immune response, LHRH-a is recommended for repeated application. Implantation of LHRH-a produced variable results, inducing ovulation in one female, but apparently inducing sex reversal in the other.

Hepatic Estrogen Receptors in the Japanese Eel, Anguilla japonica: Characterization and Changes in Binding Capacity during Artificially-induced Sexual Maturation

Estrogen receptors were identified in cytosolic and nuclear extracts of livers of female Japanese eel, Anguilla japonica. A single class of high affinity binding sites was found, with a Kd=0.97 nM for the cytosolic estrogen receptor (cER) and Kd=0.85 nM for the nuclear estrogen receptor (nER). Binding of both the cER and the nER was specific for estrogens (diethylstilbestrol: DES>estradiol-17β: E2>>estriol: E3>estrone: E1). These binding characteristics of ERs were quite different from those of the serum estrogen-binding component; [3H]E2 binding to serum was not saturable, and was displaced by testosterone but not by DES, E1 or E3. The relationships between the levels of hepatic ERs, circulating E2 and vitellogenin (VTG) during artificial maturation of cultivated female eels were examined, using eels injected weekly with chum salmon pituitary homogenate at a dose of 20 μg/g-body weight. Serum E2 levels were constantly low during pre- to midvitellogenesis, and dramatically increased in the migratory nucleus stage. However, VTG levels gradually increased from early to midvitellogenesis, and were greatly elevated in the migratory nucleus stage. Hepatic cER levels slightly increased in early vitellogenesis, and then increased significantly from midvitellogenesis to the migratory nucleus stage. In contrast, nER levels did not change significantly, although nER levels in the migratory nucleus stage were higher than those at other stages. The changes in cER levels represent increased hepatic responsiveness to estrogenic stimuli during artificial maturation. Lack of change in nER levels may be a feature of artificial maturation compared to sexual maturation in nature.

Changes in serum steroid hormones and steroidogenic ability of ovarian follicles during artificial maturation of cultivated Japanese eel, Anguilla japonica

Serum levels of oestradiol-17β (E 2), testosterone (T) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) were assessed by radioimmunoassay during induction of sexual maturation of female Japanese eel ( Anguilla japonica) by chum salmon pituitary homogenate (SPH) treatment. SPH stimulated vitellogenic growth in about 50% of the eels. Serum E 2 levels in eels which responded to SPH remained low for the first 10 weeks after the start of SPH injections, and tended to increase after 12 weeks. Levels increased further after completion of vitellogenesis. Serum levels of T increased gradually as SPH treatment progressed, and remained higher than E 2 levels during the first 12 weeks. A further increase in T levels occurred after completion of vitellogenesis. 17α,20β-DHP was not detected in serum during the experimental period. In vitro production of E 2, T and 17α,20β-DHP by ovarian follicles in response to forskolin (an activator of adenylate cyclase), and changes in the ability of follicles to convert exogenous pregnenolone or 17α-hydroxyprogesterone (17α-OHP) to E 2, T and 17α,20β-DHP, and exogenous T to E 2, were examined using 18 h incubations. Forskolin had no effect on the production of any measured steroid by follicles at any stage of development. Production of E 2, T and 17α,20β-DHP in the absence of exogenous substrates was low. The ability of follicles to produce E 2 from pregnenolone, 17α-OHP or T was low during vitellogenesis, followed by an increase during the migratory nucleus stage. The ability of follicles to produce T from pregnenolone and 17α-OHP gradually increased during vitellogenesis, followed by a further increase or a slight decrease during the migratory nucleus stage. The conversion of pregnenolone or 17α-OHP to 17α,20β-DHP increased during vitellogenesis, followed by either a further increase or a decrease during the migratory nucleus stage. A good correlation existed between serum levels of E 2 and aromatase activity (the ability of follicles to convert T to E 2). E 2 production by follicles was shown to depend largely on aromatase activity, while T production by follicles appeared to be depend largely on SPH-induced pregnenolone production. Increased aromatase activity at the migratory nucleus stage may inhibit 17α,20β-DHP production and spontaneous final oocyte maturation and ovulation.