Nancy M. Sherwood

The amphioxus genome illuminates vertebrate origins and cephalochordate biology

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish (Carassius auratus)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His5, Trp7, Tyr8]-GnRH (cGnRH-II) and [Trp7,Leu8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln8]-GnRH and [Tyr3,Leu5,Glu6,Trp7,Lys8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.

Evolution of GnRH: Diving deeper

Gonadotropin-releasing hormone (GnRH) plays a central role in vertebrate reproduction. The evolutionary origin of this neuropeptide and its receptor is not obvious, but the advent of genomics makes it possible to examine the roots of GnRH and delve deeper into its ancestral relationships. New peptide sequences identified in invertebrates from annelids to tunicates reveal GnRH-like peptides of 10-12 amino acids. Structural conservation suggests homology between the 15 known invertebrate peptides and the 15 known vertebrate GnRHs. The functions of the invertebrate GnRH-like peptides are not necessarily related to reproduction. We suggest that structurally related families of invertebrate peptides including corazonin and adipokinetic hormone (AKH) form a superfamily of neuropeptides with the GnRH family. GnRH receptors have also been identified in invertebrates from annelids to tunicates suggesting that the origin of GnRH and its receptor extends deep in evolution to the origin of bilaterian animals. To resolve the relationship of invertebrate and vertebrate receptors, we conducted large-scale phylogenetic analysis using maximum likelihood. The data support a superfamily that includes GnRH, AKH and corazonin receptors derived from both published sequences and unpublished gene model predictions. Closely related to the GnRHR superfamily is the vasopressin/oxytocin superfamily of receptors. Phylogenetic analysis suggests a shared ancestry with deep roots. A functional role for GnRH in vertebrates or invertebrates leads to questions about the evolutionary origin of the pituitary. Our analysis suggests a functioning pituitary was the result of genomic duplications in early vertebrates.

Exon Skipping in the Gene Encoding Pituitary Adenylate Cyclase-Activating Polypeptide in Salmon Alters the Expression of Two Hormones that Stimulate Growth Hormone Release1

In mammals, GRF and pituitary adenylate cyclase-activating polypeptide (PACAP) are encoded in separate genes. We report here that in the salmon a 4.5-kilobase gene contains five exons that encode the biologically active part of the GRF-like peptide (amino acids 1–32) on exon 4 and PACAP on exon 5. Analysis of two fish messenger RNAs reveals that a long precursor containing GRF and PACAP and a short precursor containing only PACAP are both expressed in the brain of at least five species of salmon, whereas mice express only the long precursor encoded by the PACAP gene. Synthetic salmon PACAP-38 and salmon GRF-like peptide-45 both stimulated GH release from cultured salmon pituitary cells; PACAP stimulated a concentration-dependent release of GH at both 4 and 24 h of incubation, whereas GRF-like peptide did not. Alternative splicing, resulting in the short precursor in which GRF-32 is excised, may provide a means for differential control of GH secretion with higher production of the more potent PACAP. A dupl...

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral ...

Multiple forms of gonadotropin-releasing hormone in amphibian brains.

Several forms of gonadotropin-releasing hormone (GnRH)-like molecules were found in brains of both anurans (frogs) and urodeles (salamanders). The presence of the mammalian-like GnRH molecule was confirmed by HPLC and cross-reactivity studies. Small amounts of salmonid-like GnRH molecules in the brains of frogs (Rana pipiens, Hyla regilla) and salamanders (Taricha granulosa, Ambystoma gracile) were detected by comparing the HPLC chromatographic pattern and immunological reactivity of the brain extracts with native trout and synthetic salmon GnRH. This nonmammalian form of GnRH in the amphibian brain is similar and perhaps identical, at least by indirect evidence, to a form of GnRH reported earlier to be in sympathetic ganglion, retina, chromaffin tissue, and tadpole brain. If two of the amphibian GnRH molecules prove to be mammalian and salmon GnRH, then it is likely that two separate genes in amphibians code for the distinct primary structures of the molecules. The most parsimonious interpretation of the presence of both mammalian- and salmon-like GnRH in anurans and urodeles is that a common phylogenetic ancestor also possessed the two forms of GnRH. Thus the mammalian form of GnRH may well have been present in labyrinthodont amphibians. Independent of evolutionary origin, the functions of the different GnRH molecules in amphibians are unknown.

Two populations of luteinizing hormone-releasing hormone neurons in the forebrain of the rhesus macaque during embryonic development

To investigate the possibility that a second luteinizing hormone‐releasing hormone (LHRH) population appears during development in primates, embryos and fetal brains of rhesus monkeys were immunostained with antisera specific to different LHRH forms. Two LHRH cell populations were discernible by immunoreactivity to antisera LR‐1 and GF‐6. Because one LHRH cell type migrated out from the olfactory placode several days earlier than the other, they were referred to as “early” and “late” LHRH cells, respectively. Although late LHRH neurons were immunoreactive to all anti‐mammalian LHRH antisera tested, early LHRH neurons were only detected by antiserum GF‐6. Early LHRH neurons (∼10 × 7 μm) were smaller than late LHRH neurons (∼8 × 7 μm). Early LHRH neurons were first found around the olfactory placode, in the nasal mesenchyme, and in the rostroventral forebrain on embryonic day 30 (E30), whereas late LHRH neurons were first seen in the olfactory pit on E32. Early LHRH cells were located throughout the basal forebrain on E32–E42, whereas late LHRH cells were found in the olfactory pit and along the terminal nerve on E34–E36 and were not seen in the forebrain until E38. By E51–E62, late LHRH neurons reached into the basal hypothalamus in a distribution resembling that in the older brain, while early LHRH neurons were found in the septum, preoptic region, stria terminalis, medial amygdala, claustrum, internal capsule, and globus pallidus. Based on the distribution pattern of immunopositive cells with‐antiserum LR‐1, late LHRH cells are bona fide LHRH neurons that regulate the pituitary‐gonadal axis. In contrast, the molecular form of early LHRH cells is unclear, although it is plausible that early LHRH cells may contain the molecule in which the C‐terminal epitope of LHRH is modified or absent. It is concluded that in primates there is a second population of LHRH neurons that originates from the embryonic olfactory placode before the origin of mammalian LHRH‐like neurons, and that these two populations of LHRH‐immunopositive neurons have different morphologic features and different final distributions in the brain. J. Comp. Neurol. 380:293–309, 1997.

Sequence and expression of cDNA for pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH)-like peptide in catfish.

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) are two neuropeptides that are associated with the release of pituitary growth hormone. Here a cDNA of 2501 base pairs encoding both a PACAP and a GHRH-like peptide was isolated from a brain cDNA library made from Thai catfish (Clarias macrocephalus). The organization is unlike that of the mammalian gene where PACAP and PACAP-related peptide (PRP) are encoded in one gene, and the GHRH peptide is on a separate gene. Northern analysis of catfish brain mRNA indicated that PACAP/GHRH-like mRNA has three sizes; bands of 6000, 2500, and 1000 bases suggest alternative splicing of the gene. Reverse transcriptase/PCR assay detected PACAP/GHRH-like mRNA in tissues from the brain, testis, ovary, and stomach, but not from the pancreas, pituitary, muscle, and liver. Our hypothesis that the two mammalian genes encoding GHRH or PACAP originated from a gene duplication between fish and tetrapods is supported by the present findings of similar mRNA organization and pattern of expression for the one fish gene and two mammalian genes.

Primary structures of two forms of gonadotropin-releasing hormone, one distinct and one conserved, from catfish brain

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the Thai catfish, Clarias macrocephalus, using reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of catfish GnRH (catfish GnRH-I and -II) were determined using Edman degradation. The presence of the N-terminal pGlu residue in both peptides was established by digestion with pyroglutamyl aminopeptidase. In addition, catfish GnRH-I was studied by mass spectrometry. The primary structure of catfish GnRHI is pGluHisTrpSerHisGlyLeuAsnProGlyNH2 and catfish GnRH-II is identical to chicken GnRH-II, pGluHisTrpSerHisGlyTrpTyrProGlyNH2. Functional studies showed that synthetic catfish GnRH-I released not only gonadotropin but also growth hormone from an in vitro preparation of goldfish pituitaries. Catfish GnRH-II is identical to the widely distributed and highly conserved chicken GnRH-II. Indirect evidence has suggested its presence in bony fish, but this is the first report of its primary sequence. The distinct structure of catfish GnRH-I increases the number of GnRH family members to six.

Sustained hormone release. I. Characteristics of in vitro release of gonadotropin-releasing hormone analogue (GnRH-A) from pellets

Abstract A method is described for embedding gonadotropin-releasing hormone analogue (GnRH-A) into pellets of different matrices to produce sustained release at different rates. The pellets containing GnRH-A were placed in an in vitro perfusion chamber and the release of the analogue was measured by radioimmunoassay using a specific antiserum to the analogue. Matrices containing 25, 50, 75 or 100% cellulose combined with cholesterol resulted in GnRH-A release with an initial burst at 1 h of 19–40% of the GnRH analogue contained in the pellet; an accumulated release of more than 90% of the analogue occurred by 24 h. In contrast, the release of GnRH-A from a pellet of 5% cellulose-95% cholesterol or 100% cholesterol was only 5–6% of the total amount of the analogue at 1 h with an accumulated release of 18–21% at 24 h and 36–38% by 28 days. It is suggested that these nontoxic and inexpensive pellets are suitable for aquaculture. The cholesterol-cellulose pellets that are 25–100% cellulose are potentially useful with ripe fish that require a rapid GnRH-A stimulus for final egg maturation and spawning. The 95 or 100% cholesterol pellets may be most suitable for sustained release in fish requiring days for final maturation of the ovary or in fish spawning on several successive days.