Carol M. Warby

Three Forms of Gonadotropin-Releasing Hormone, Including a Novel Form, in a Basal Salmonid, Coregonus clupeaformis

Abstract Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH2. HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the α-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.

Primary structure of solitary form of gonadotropin-releasing hormone (GnRH) in cichlid pituitary; three forms of GnRH in brain of cichlid and pumpkinseed fish

GnRH is a decapeptide family with at least nine distinct structures. Vertebrates, except for most placental mammals, have more than one of these GnRH forms within the brain. We report chromatographical and immunological evidence that three forms of GnRH are in the brains of both cichlid (Haplochromis burtoni) and pumpkinseed (Lepomis gibbosus) fishes. We argue that the three forms correspond to those previously described as sea bream GnRH (sbGnRH), chicken GnRH-II and salmon GnRH. In contrast, only one GnRH form was present in the pituitary of the cichlid and is identified as sbGnRH by amino acid sequence. This is the first report in which the primary structure of GnRH is determined from pituitary tissue. The N-terminus was identified by monitoring the digestion of the peptide by pyroglutamate aminopeptidase with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The amidation of the C-terminus was established using an esterification procedure for monitoring with MALDI-MS. This report supports the idea that three forms of GnRH within one species is widespread in the order Perciformes. The present study establishes sbGnRH as the third GnRH form in H. burtoni and predicts that sbGnRH is synthesized in preoptic neurons, then transported to the pituitary in the preoptic-hypophyseal axons for the release of one or both gonadotropins.

Studies on the molecular weight and cryoprotective properties of polyvinylpyrrolidone and dextran with bacteria and erythrocytes

The distribution of molecules in various commercial samples of polyvinylpyrrolidone was studied by gel-exclusion chromatography on Sephadex columns. High-speed centrifugation on sucrose gradients was also used in this study. Unequivocal evidence is presented that the molecular weight spread in Plasdone C (PVP, K30) is much wider than the manufacturers published data. A continuous distribution of molecules associated with Plasdone C from about 1,000 to 200,000 daltons was demonstrated. This material is claimed to have an average molecular weight of 40,000 daltons with a range of 20,000–80,000 daltons. Radioactive 14C PVP gave essentially similar results. Estimates based on the elution pattern of bovine serum albumin (67,000 daltons) suggest that about 50% of the molecules in Plasdone C are larger than 67,000 daltons, and dialysis experiments indicate that about 12% of the molecules are less than about 12,000 daltons. Undialyzed PVP, not toxic per se, was toxic to Pseudomonas when this bacterium was frozen and thawed in its presence. Dialyzed PVP, however, gave good protection against freeze-thaw damage. Ultraviolet absorption spectra of PVP gave maxima at approximately 203 nm unaffected by dialysis or changes in pH from 4.0 to 7.0. Standard curves are published for the estimation of pure PVP samples by either uv spectroscopy or the iodine color reaction. Dextran samples of characterized molecular weight from 10,000 to 500,000 daltons were investigated for freeze-thaw protection with rabbit erythrocytes and Pseudomonas. Dialysis had no effect on cryoactivity. Higher concentrations of dextran were better than lower concentrations in both cell systems. With erythrocytes the influence of molecular weight on cryoprotection was negligible. With bacteria, however, there was a trend toward greater cryoprotective activity with higher molecular weight.

Low-temperature preservation of mammalian cells in tissue culture with polyvinylpyrrolidone (PVP), dextrans, and hydroxyethyl starch (HES)☆

Abstract The effects of freezing and thawing on Chinese hamster cells in tissue culture in the presence of PVP, HES, and various dextrans have been investigated. Cooling and thawing rates within a limited range (20–260 °C per min for cooling and 6–115 °C per min for thawing) were studied and best results were achieved with a cooling rate of 20 °C per min and a thawing rate of 115 °C per min in both 10% PVP, and 10% HES. Experiments demonstrated HES to be as good as, and possibly better than PVP. A number of dextrans of average molecular weight (10,000–500,000 daltons) were shown to be poor as cryoprotective agents in contrast to results obtained with this polymer with red cells and bacteria. The presence of 10% serum during all freezing and thawing procedures decreased day-to-day variability and with dextrans increased their limited effectiveness. Pinocytosis, as a mechanism by which polymers act to prevent freeze-thaw damage, is unlikely.

Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Gonadotropin-releasing hormones, including a novel form, in snook Centropomus undecimalis, in comparison with forms in black sea bass Centropristis striata

The molecular forms of gonadotropin-releasing hormone (GnRH) in brain-pituitary extracts were determined for snook Centropomus undecimalis and black sea bass Centropristis striata. The extracts were analyzed in both isocratic and gradient high performance liquid chromatography (HPLC) programs. Eluted fractions were tested in radioimmunoassays with 4 different antisera made against 3 distinct GnRH peptides. Results show that snook contain 3 forms of GnRH, all of which are present in males and females irrespective of the stage of the reproductive cycle. Larger quantities of these GnRH peptides are present in snook in the nonreproductive phase than in snook in the reproductive phase. One form of snook GnRH is immunologically and chromatographically similar to salmon GnRH, and a second form is similar to chicken GnRH-II. However, the third snook GnRH appears to be distinct from the 7 known forms of the vertebrate hormone. In contrast, sea bass contain only the salmon GnRH-like and chicken GnRH-II-like forms of GnRH and, hence, appear to match the more usual pattern of GnRH peptides in teleosts. We speculate that one of the GnRH genes was duplicated and then altered in a fish ancestral to snook but not sea bass, even though both species of fish are in the recently evolved Perciformes order.

Gonadotropin-releasing hormone from Thai catfish: Chromatographic and physiological studies

Two forms of immunoreactive gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of Thai catfish, Clarias macrocephalus and C. batrachus. The peptides were detected using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). In both the HPLC systems, catfish GnRH-I eluted earlier than catfish GnRH-II and also eluted before the synthetic standards of mammalian, lamprey, chicken I, chicken II, and salmon GnRH. Hence, catfish GnRH-I appears to be the most hydrophilic GnRH family member because of this early elution from the HPLC. Catfish GnRH-II eluted in a position similar to that of chicken GnRH-II. This study suggests that catfish GnRH-I is a novel form of GnRH, whereas catfish GnRH-II is the same as chicken GnRH-II. Indirect evidence suggests that the catfish molecule is 10 amino acids in length and has an amide at the C-terminus. Moreover, the novel catfish GnRH appears to be different within the domain of amino acids 5 to 10 compared with mammalian GnRH because it is not recognized by antiserum B-6. An injection of native chicken GnRH-II was more effective than salmon or mammalian GnRH for induced ovulation in C. macrocephalus.

Transcription and Translation of the Salmon Gonadotropin-Releasing Hormone Genes in Brain and Gonads of Sexually Maturing Rainbow Trout (Oncorhynchus mykiss)

Abstract Rainbow trout sexually mature at the end of Year 3. The form of GnRH that controls gonadotropin release in trout is salmon GnRH (sGnRH). In the tetraploid rainbow trout, two genes encode an identical sGnRH peptide. The sGnRH gene-1 produces one mRNA, whereas sGnRH gene-2 can produce more than one. This study asks whether the transcripts and their protein products are expressed in the brain and gonads and whether the pattern correlates with sexual maturity over the final year leading to first spawning. Brain sGnRH mRNA and protein were continuously present throughout the third year. We show for the first time that the long sGnRH-2 mRNA transcript is expressed in neural tissue and not exclusively in gonadal tissue. Expression of the long sGnRH-2 mRNA in the brain coincides with high levels of sGnRH peptide in the brain during a time of increased gonadal growth. Thus, the long sGnRH-2 mRNA in the brain may act to regulate sGnRH production in a stage-specific rather than a tissue-specific manner. In gonads, local sGnRH is thought to play an autocrine/paracrine role in regulating gonadal maturation and spawning. In the maturing gonads, sGnRH gene-1 and -2 are expressed intermittently. Strikingly, sGnRH peptide was not detected in the gonads at any time during Year 3. These results suggest that either the sGnRH transcripts in the gonads are not translated into protein or, if translated, the protein is rapidly released, resulting in gonadal content below 1 fM per fish.

Guinea Pig Gonadotropin-Releasing Hormone: Expression Pattern, Characterization and Biological Activity in Rodents

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.

Effect of freezing on the molecular weight of bacterial DNA

Freezing and thawing of E. coli cells does not produce single strand breaks. Young cultures of E. coli cells demonstrate a biphasic distribution of DNA molecules compared with older cultures that contain the normally expected distribution of DNA molecules.