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The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Prolactin (PRL) is one of the major hormones that control amphibian metamorphosis. Recently, a PRL (PRL1B) gene that is different from the known PRL (PRL1A) gene has been found in the genomes of several amphibian species. In order to ascertain whether the PRL1B gene is expressed in the bullfrog (Rana catesbeiana) pituitary, cloning of cDNA encoding PRL1B in the pituitary of the premetamorphic bullfrog tadpole was attempted. The bullfrog PRL1B amino acid sequence predicted from the obtained cDNA showed 62% identity with those of Xenopus PRL1Bs that have been presumed from the genome sequences, whereas the sequence identity between bullfrog PRL1A and PRL1B was 48%. A molecular phylogenetic tree showed that bullfrog PRL1B is most appropriately grouped with amphibian PRL1Bs. Quantitative PCR analysis revealed that the mRNA expression levels of bullfrog PRL1B in the pituitary were high during pre- and prometamorphosis, sharply declined at metamorphic climax and became undetectable after metamorphosis. In contrast, PRL1A mRNA levels were relatively low during pre- and prometamorphosis, rose at climax and remained high after metamorphosis. Immunohistochemical study using antibodies against partial peptides of PRL1A and PRL1B revealed that most of the PRL1A- and PRL1B-immunoreactive cells in the larval pituitary were distributed separately, but that some of the cells immunoreactive with both antibodies were also present. Western blot analysis with the larval pituitary extract indicated that PRL1B-immunoreactive band appeared at the position of molecular weight ca. 22.1 kDa and PRL1A-immunoreactive band at the position of ca. 22.8 kDa. The results obtained in this experiment suggest the possibility that PRL1B plays as-yet-unknown role(s) during the pre-climactic period of metamorphosis. This is the first report on the existence of PRL1B as a protein in the amphibian larval pituitary., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pituitary prolactin (PRL) shows an episodic pattern of evolution in mammals, with a slow underlying rate (near stasis) and periods of rapid change in some groups. PRL evolution in bats, the second most speciose mammalian order, has not previously been studied, and is examined here. Slow basal evolution of PRL is seen in some bats, particularly megabats, but in most microbat groups evolution of PRL is more rapid. Accelerated evolution of PRL is particularly notable in the family Vespertilionidae, where analysis of nonsynonymous and synonymous substitutions indicates that it reflects adaptive evolution/positive selection. Remarkably, vespertilionid bats also show a large sequence insertion, of variable length, into exon 4 of PRL, giving a protein sequence 18-60 amino acids longer than normal, with the longest insertions in bats of the genus Myotis. An equivalent insertion has not been reported in PRL of any other vertebrate group. In the 3-dimensional structure of the complex between PRL and the extracellular domain (ecd) of its receptor (PRL:PRLR 2 ) the inserted sequence is seen to be introduced in the short loop between helices 2 and 3 of PRL; it is far removed from the receptor-binding sites, and may not interfere with binding. The ecd of the receptor also shows variable rates of evolution, with a higher rate in the Vespertilionidae, but this is much less marked than for the hormone. The distribution of substitutions introduced into PRL during vespertilionid evolution appears to be non-random, and this and the evidence for positive selection suggests that the rapid evolution and insert sequence introduction were associated with a significant change in the biological properties of the hormone., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plasma prolactin (PRL) is released from lactotrophs in the anterior pituitary. As plasma PRL levels rise during incubation in domestic fowl, the number of lactotrophs (PRL-immunoreactive, PRL-IR cells) increases while the number of growth hormone secreting cells, somatotrophs (GH-IR cells), declines. We measured plasma PRL levels using radioimmunoassay (RIA) and examined the distribution of lactotrophs and somatotrophs in the anterior pituitary of breeding and nonbreeding zebra finches of known ages with and without prior breeding experience using fluorescent immunohistochemistry (IHC). Plasma PRL levels were higher in breeding than in nonbreeding birds, regardless of age, sex, or previous breeding history. PRL-IR cells were localized primarily, but not exclusively, to the cephalic aspect of the anterior pituitary (AP) and along the ventral margin. Birds with prior reproductive experience had more PRL-IR cells than birds with no prior reproductive experience and breeders had slightly higher PRL-IR cell counts than did nonbreeders, but there was no correlation between the number of PRL-IR cells and plasma PRL levels. GH-IR cells were concentrated in the caudal aspect of the AP with some cells in the cephalic lobe, but numbers did not differ between any of the groups studied. An increase in PRL-IR cells corresponded with an increase in GH-IR cells. An increase in lactotroph number with reproductive experience in zebra finches may facilitate future reproductive events by allowing for more robust PRL secretion and increased reproductive success., (Published by Elsevier Inc.)

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.

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 approximately 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 (approximately 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 microg 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 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h 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 microg) 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.

A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA library constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) and cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert encoding the C-terminal amino acid sequence of the mature hormone molecule. Using this clone as a probe, the full-length newt PRL cDNA was screened from the cDNA library. The PRL cDNA clone thus obtained consisted of 1024 bp encoding the entire sequence of the mature PRL molecule in addition to its signal peptide. The amino acid sequence of newt PRL deduced from its nucleotide sequence showed higher homologies with those PRL sequences of tetrapod animals than with those of teleosts. Northern blot analysis revealed the newt PRL mRNA size to be approximately 1 kb. In situ hybridization using the newt PRL cDNA as a probe revealed that the pituitary region expressing PRL mRNA corresponded to that immunoreactive with antiserum against PRL. PRL mRNA levels in the pituitary of newts subjected to room and low temperatures were determined by Northern analysis employing the PRL cDNA as a probe. PRL mRNA levels were significantly higher in the pituitaries of newts subjected to 10 degrees than in those of newts kept at 23 degrees. Likewise, immunoassayable plasma PRL levels were higher in animals subjected to 10 degrees than in those kept at 23 degrees., (Copyright 2001 Academic Press.)

Prolactin (PRL) and two variants of growth hormone (GH), purified from pituitaries of striped bass (Morone saxatilis) and its hybrid with white bass (M. saxatilis x M. chrysops) by gel filtration chromatography under alkaline conditions followed by reversed-phase high pressure liquid chromatography, appear similar between species. Both the minor (GH I) and the major (GH II) forms of purified GH appeared as single bands (M(r) approximately 23,000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as did the purified PRL (M(r) approximately 24,000). The molecular weights of GH II and PRL determined by MALDI TOF mass spectroscopy were 21.2 and 21.3 kDa, respectively. In Western blotting experiments, an antiserum against tilapia (Oreochromis mossambicus) 24K PRL specifically recognized Morone PRL, while an antiserum against tilapia GH specifically recognized Morone GH I and II. Chemical identities of the putative PRL and GH I were further confirmed by N-terminal peptide sequencing, while internal sequence analysis was performed on GH II because it was blocked at its N-terminus. Over a stretch of 29 amino acids, Morone PRL was found to be 76% identical to tilapia 24K PRL, 72% identical to tilapia 20K PRL, 72% identical to chum salmon (Oncorhynchus keta) PRL I, and 69% identical to eel (Anguilla japonica) PRL I. Alignment of the hybrid striped bass GH sequences with those of several other advanced marine teleosts indicated 75-85% sequence identity for GH I (40 amino acids) and 95-98% identity for GH II (45 amino acids). Biological activity of striped bass GH II was confirmed using a heterologous in vitro assay of insulin-like growth factor I mRNA production by coho salmon (On. kisutch) hepatocytes. An in vivo bioassay, involving hypophysectomy of hybrid striped bass and treatment of the fish maintained in fresh water with homologous PRL, confirmed that the purified striped bass PRL was also bioactive., (Copyright 2000 Academic Press.)

Overlapping cDNA partial clones of pituitary prolactin from the marsupial brushtail possum (Trichosurus vulpecula) were isolated and sequenced. The nucleotide and deduced amino acid sequences showed high sequence identity with pig prolactin (84.3 and 92.5%, respectively) and all of the expected structural features of a quadruped prolactin. A prolactin gene tree was constructed and rates of evolution calculated for possum along with several mammalian and nonmammalian prolactins. Possum prolactin was most closely linked to the prolactins of eutherian mammals but branched from the main mammalian line well before the eutherian prolactins. The prolactin/GH family shows variable rates of evolution ranging from 0.3 substitutions/amino acid site/year x 10(9) for pig prolactin to 7.0 substitutions/ amino acid site/year x 10(9) for the mouse. Since divergence from the eutherian mammals, possum prolactin has shown a slow rate of evolution (0.2 substitutions/ amino acid site/year x 10(9)). As expected, the prolactin gene was expressed in the possum pituitary gland but not in the liver, lung, kidney, heart, or mammary gland.

The complete amino acid sequence of prolactin (PRL) from a chondrostean species, the sturgeon (Acipenser gueldenstaedti), has been determined. Sturgeon PRL was isolated from the pituitary glands by gel filtration on a Sephadex G-25 column and high-performance liquid chromatography on a reverse-phase column following acid-acetone extraction. Sturgeon PRL was identified by immunoblot reactivity using antisera against salmon and ovine PRL. It consists of 204 amino acid residues, which is the largest among known PRLs, and contains three disulfide bonds corresponding to those of tetrapod PRLs. Sequence comparison with PRLs from other vertebrates revealed that sturgeon PRL has slightly higher sequence identities (35-46%) with teleost PRLs than with tetrapod PRLs (30-40%). These structural characteristics imply that an ancestor of the ray-finned fishes had PRL with three disulfide bonds and at some point after divergence of Chondrostei, the disulfide bond in the amino-terminus of PRL was lost.

The tilapia pituitary secretes two forms of prolactin (tPRL) and a single growth hormone (tGH). The tPRLs share only 69% sequence identity and are designated tPRL177 and tPRL188 to indicate the number of amino acid residues in each isoform. Our aim was to develop specific antisera for detection of these three related polypeptides. Ten peptides corresponding to unique epitopes on the tPRLs and two peptides of tGH were synthesized using solid-phase methods, conjugated to carrier proteins, and used as immunogens for antibody production in rabbits. Select antisera for the tPRLs were highly specific, exhibiting only 1% cross-reactivity to the alternate tPRL under noncompetitive ELISA conditions at dilutions used in immunocytochemical analysis. The anti-tGH specifically bound to cells in the proximal pars distalis. Production of both tPRLs by a single cell type was indicated by the binding of both anti-tPRL177 and anti-tPRL188 to the same cells in the rostral pars distalis. Ultrastructural analysis of PRL-producing cells stained sequentially using the two different anti-tPRL antibodies labeled with immunogold of two size classes indicated that both tPRLs appear in the same granules. These findings suggest that the biological significance of two forms of PRL in the adult tilapia is not a function of differential regulation of two different classes of PRL cells or differential release of unique secretory granules.

A major obstacle in the production of specific antibodies toward chicken prolactin (PRL) has been overcome by mimicking a putative epitope of the molecule using the synthetic decapeptide Lys-chPRL 59-67. This peptide represents the highest hydrophilicity peak of the amino acid sequence of chPRL that was recently derived from the nucleotide sequence. Polyclonal mouse antisera against the fragment specifically recognized the lactotropes in the cephalic lobe of the chicken pars distalis as illustrated by immunocytochemical double staining experiments. Monoclonal antibody production yielded antibodies that specifically labeled purified turkey PRL upon SDS-PAGE separation and immunoblotting. Turkey and chicken PRL showed a very similar polymorphism with respect to their apparent molecular weights, including the occurrence of a glycosylated variant of chicken PRL. The monoclonal antibodies were finally used to demonstrate the presence of PRL-like immunoreactivity both in the pituitary gland and in the brain of the quail. In the brain, immunoreactive neurons were in the nucleus accumbens and in the lateral parts of the ventro-medial hypothalamus, partly similar to those described in the rat.

The complete amino acid sequence of prolactin (PRL) from an amphibian species, the bullfrog (Rana catesbeiana), has been determined and conserved residues and domains were analyzed by sequence comparison of PRLs from 15 species of five major vertebrate classes. The bullfrog PRL consists of 197 amino acid residues with three disulfide linkages formed between residues 4-11, 58-172, and 189-197. The bullfrog PRL shows the highest identity with sea turtle PRL (75%); lower identities with chicken PRL (72%), pig, horse, and fin whale PRLs (68%), human, cattle, sheep, and elephant PRLs (60-58%), and rat and mouse PRLs (50%); and significantly lower identity with teleost PRLs (about 30%). It is apparent that all tetrapod PRLs characterized so far contain three disulfide bonds in homologous positions and differ from teleost PRLs which lack the N-terminal disulfide loop. The tetrapod and teleost PRLs share 34 common residues and these conserved residues are clustered in six domains (PD1 to PD6), suggesting that these common residues, or at least part of them, are responsible for the activities common to all PRLs. On the other hand, PD5 is conserved significantly within tetrapod PRLs, but to a lesser extent in teleost PRLs, suggesting that the PD5 contributes to the activities specific to tetrapod PRLs.

A highly purified prolactin (PRL) was isolated from the pituitary of the Japanese eel (Anguilla japonica) by extraction with acid-acetone, gel filtration on Sephadex G-75, and reversed-phase HPLC on TSK-gel ODS 120T and on TSK-gel TMS 250. Eel PRL is comprised of two variants (ePRL I and II), which were separated by HPLC on an ODS column. The two PRLs were also secreted by organ-cultured pituitaries in a defined medium. After being dialyzed against distilled water and lyophilized, the medium was dissolved in 0.01 M ammonium acetate (pH 9.0), and then the insoluble fraction was subjected to HPLC on an ODS column to isolate the secreted PRLs. The ePRL I and II were equipotent in retaining plasma Na in the hypophysectomized killifish, Fundulus heteroclitus, transferred from sea-water to fresh water. The putative PRL-producing cells in the rostral pars distalis of the eel pituitary were specifically stained with the antiserum against the mixture of ePRL I and II. Both PRLs had a molecular weight of 22 kDa in SDS-PAGE, an isoelectric point of 10.1 by gel electrofocusing, and an N-terminal residue of valine. Amino acid compositions and the partial amino acid sequences of ePRL I and II show that they are highly homologous with a limited number of substitutions, and that they are more closely related to those of teleostean PRLs than to those of mammalian PRLs.

The complete amino acid sequence of prolactin (PRL) from a reptile, the sea turtle (Chelonia mydas), was determined for the first time. Sequence analysis was performed on fragments obtained from cleavage of intact and performic acid-oxidized hormone with lysyl endopeptidase, Staphylococcus aureus protease, and o-iodosobenzoic acid employing manual Edman degradation. The sea turtle PRL consists of 198 amino acid residues with three disulfide linkages formed between residues 4-11, 58-173, and 190-198 and possesses heterogeneity indicated by four replacements at positions 55, 145, 148, and 171. Sequence comparison with other vertebrate PRLs revealed that the degree of sequence identity conforms well to expectations based on phylogeny except for the rodent PRLs; sea turtle PRL has 86% identity with chicken PRL; 81% with horse, pig, and fin whale PRLs; 75-71% with cattle, sheep, and human PRLs; 60-56% with mouse and rat PRLs; and 35-31% with carp, salmon, and tilapia PRLs.