Lamprey GnRH-III polypeptides and methods of making thereof Number:7,521,057 from the United States Patent and Trademark Office (PTO) owispatent Lamprey GnRH-III polypeptides for the four species of fish lamprey are disclosed. Also disclosed is a procedure for producing such polypeptides by recombinant techniques. Also disclosed are methods for utilizing such polypeptides for sterilizing fish.<H polypeptides, thereof, Lamprey, GnRH, II, 7521057.html, Patent, pto, 7521057

Title: Lamprey GnRH-III polypeptides and methods of making thereof

Abstract: Lamprey GnRH-III polypeptides for the four species of fish lamprey are disclosed. Also disclosed is a procedure for producing such polypeptides by recombinant techniques. Also disclosed are methods for utilizing such polypeptides for sterilizing fish.

Patent Number: 7,521,057 Issued on 04/21/2009 to Sower, et al.

Inventors: Sower; Stacia (Newmarket, NH), Silver; Matthew (Dover, NH)
Assignee: University of New Hampshire (Durham, NH)

Appl. No.: 11/172,274

Filed: June 30, 2005

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
10170096	Sep., 2005	6949365

Current U.S. Class: 424/198.1 ; 424/192.1; 530/300; 530/313; 530/333

Current International Class: A61K 39/00 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


4410514	October 1983	Vale, Jr. et al.
5076208	December 1991	Zohar et al.
5093246	March 1992	Cech et al.
5643877	July 1997	Zohar et al.
6210927	April 2001	Zohar et al.
6664369	December 2003	Lovas et al.

Foreign Patent Documents


WO 96/04927	Feb., 1996	WO

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Primary Examiner: Jiang; Dong
Attorney, Agent or Firm: Devine, Millimet & Branch PA Bruttomesso, Jr.; Raymond I. Remus; Paul A.

Government Interests

GOVERNMENT SPONSORSHIP

This Invention is funded in part by NICHD grant No. R03 HD39166-02; NSF IBN0090852; and NSF INT-981528.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of patent application Ser. No. 10/170,096 filed on Jun. 12, 2002, now U.S. Pat. No. 6,949,365 issued Sep. 27, 2005, which is incorporated herein by reference.

Claims

What is claimed is:

1. An isolated polypeptide comprising the amino acid sequence set forth by SEQ ID NO: 8.

2. A fusion protein comprising said isolated polypeptide of claim 1, and a heterologous polypeptide.

3. The isolated polypeptide of claim 1, produced by a host cell.

4. A composition comprising said isolated polypeptide of claim 1 and a pharmaceutically acceptable carrier.

5. The isolated polypeptide of claim 1, produced by a method comprising the steps of: (a) culturing a cell comprising the nucleic acid of SEQ ID NO: 7 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.

6. An isolated polypeptide produced by a method comprising the steps of: (a) synthesizing said polypeptide of claim 1 using peptide synthesis; and (b) recovering said polypeptide.

7. A method of sterilizing fish comprising administering to said fish an effective amount of a polypeptide comprising the amino acid sequence set forth by SEQ ID NO: 8.

Description

COPYRIGHT

A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the United States Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The invention relates to novel forms of GnRH in Lamprey, in particular, an isolated cDNA encoding the precursor of a novel form of gonadotropin-releasing hormone in lamprey and the isolated polypeptides encoded thereby and a method of making thereof.

BACKGROUND OF THE INVENTION

GnRH

In vertebrates, the hypothalamus and pituitary have well-defined roles in the control of reproduction. GnRH (gonadotropin-releasing hormone) is the central regulatory neurohormone controlling reproduction in all vertebrates. GnRH is a ten amino-acid peptide, synthesized in the hypothalamus and released into the hypophysial portal blood system, directly into the pituitary gland as in the case of teleost fish, or by diffusion as in the case of agnathans. Upon response to external cues (for example as environmental cues such as water temperature) and internal cues GnRH is released and acts at the pituitary gland to stimulate the synthesis and release of the gonadotropins, which in turn travel by systemic circulation to the gonads, thereby regulating steroidogenesis and gametogenesis.

GnRH has been the subject of intense research over many years because of its dual significance for understanding reproductive biology and for developing medical therapies. Aside from its importance in research for understanding reproductive biology, GnRH has many medical and other practical applications including reproductive enhancement and/or contraception in animals and fishes. In fact, GnRH and its analogs are already being used in commercial fish farming to stimulate and regulate sexual maturation and reproduction.

Over the past 15 years or so, a considerable amount of research has been devoted to the effects of GnRH and its analogs on reproduction in fish. Many of the economically important fish do not reproduce spontaneously in captivity. Thus manipulation of their reproductive cycles is crucial to marine aquaculture. Almost all of the research to date has been focused on GnRH-based spawning induction therapy in a number of commercially important species (Zohar et al., 1989). Brood females of salmon and other valuable species will spawn in captivity, but have difficulties in their spawning and the timing of spawning. By implanting a GnRH agonist into a brood female, a fish farmer can ensure that the female will ripen at the proper time, thus preventing potentially costly guesswork. However, while there has been considerable success in achieving high yields in rearing fish, there has been only limited success in the manipulation of the reproductive cycles and spawning of the reared fish. In addition, most of the work to date has focused on or examined the ability of GnRH agonists to induce spawning in females. Few researchers have examined the ability of GnRH antagonists to sterilize male fish, due to its lack of commercial application in aquaculture. However, a new method of sterilization would be very useful in the field of sea lamprey control in the Great Lakes.

During the past few years, the Great Lakes Fisheries Commission (GLFC) has been searching for alternative methods to control sea lamprey populations. In its 1992 Strategic Plan, the GLFC stated that one of its major objectives was to suppress sea lamprey populations to target levels by reduction of the use of lampricides and by development of new control methods by 2010. A compound called Bisazir is currently being used in a sterile-male release program. This compound is extremely hazardous to humans, however, and required a special facility to be constructed at Hammond Bay Biological Station, MI in 1991 for its use. Other chemosterilants that are non-hazardous need to be developed. Although some have suggested inhibiting gonadal development by the negative regulation of GnRH, there have not been any viable methods developed. See U.S. Pat. No. 6,210,927 to Zohar, which is incorporated herein in its entirety, for brief mention of inhibition of gonadal development in fish and examples of some uses and applications for seabream GnRH.

Thus, it would be desirable to have a method of sterilizing male sea lampreys, and other fish or animals, using a lamprey or other appropriate species GnRH antagonist.

There are also many potential therapeutic human reproductive applications for GnRH. Since 1971 when the primary structure of mammalian GnRH was determined, over 7,000 analogs to GnRH have been made and tested in hundreds of studies in mammals. So far, the most active synthetic agonists are found to be those with D-amino acid substitution in position 6 of the GnRH decapeptide. The most effective GnRH antagonists to date are those that have substitutions in position 6 as well as substitution of amino acids in positions 1, 2, and 3.

As a result of these studies several mammalian GnRH analogs have been shown to be highly successful and are currently being used for sterilization, conception and other therapeutic and clinical applications. In fact, the clinical application of GnRH analogs as therapeutic drugs generates over 2 billion dollars per year in sales. Hence there is considerable interest in the function of each residue in the GnRH so that analogs can be designed with maximum efficiency as agonists or antagonists to the GnRH receptor, for use as drugs. Furthermore, the responses to GnRH and analogs are different in males compared to females, suggesting that different neuroendocrine mechanisms may be involved.

To date, many analogs have proven useful, but produce undesirable side effects, such as affecting more than just the target. For example, Lupron Depot.RTM. which is a GnRH analog and is now one of the leading chemical treatments for advanced prostate cancer and endometriosis in humans has undesirable side effects. Specifically, continuous treatment of Lupron Depot.RTM. results in decreased levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH). In males, testosterone is reduced to castrate levels. In pre-menopausal females, estrogens are reduced to post-menopausal levels.

Thus, there is still critical information that is needed for understanding the biological activity of these analogs. The potential wider use of GnRH antagonists in humans awaits the availability of potent analogs that do not have the side effects (including high histamine releasing activity) seen with currently-used analogs.

Lamprey

GnRH has also been studied in several species in the process of researching the evolution of reproductive biology, one of which species is the lamprey. Lampreys and hagfish of the Class Agnatha are of particular importance in understanding endocrinological relationships since they are the modern descendants of the most primitive vertebrates available for study. They represent the oldest lineages of extant vertebrates--which evolved over 550 million years ago. Therefore, the study of lampreys and the characterization of brain and pituitary hormones in lampreys is particularly important for understanding the molecular evolution and functional diversity of reproductive hormones, and can potentially yield valuable insight into human reproductive processes. As noted above, GnRH is the central regulatory neurohormone controlling reproduction in all vertebrates. However, until about 15 years ago, there was little evidence for neuroendocrine control of reproduction in lampreys.

There are approximately 40 species of lampreys that are classified as parasitic or non-parasitic. Lampreys spawn only once in their lifetimes, after which they die. All larval lampreys, called ammocoetes, live in fresh water as borrowing organisms in the bottoms of streams or lakes. In the parasitic sea lamprey, sexual maturation is a seasonal, synchronized process. The sea lampreys begin their lives as fresh water ammocoetes, which are blind, filter feeding larvae. After approximately 5-7 years in freshwater streams, metamorphosis occurs and the ammocoetes become free-swimming, sexually immature lampreys, which migrate to the sea or lakes. During the approximately 15 month-long parasitic sea phase, gametogenesis progresses. After approximately 15 months at sea, lampreys return to freshwater streams and undergo the final maturational processes resulting in mature eggs and sperm, and finally spawning.

As stated above, however, until about 15 years ago, there was a question as to whether there was brain control of reproduction in lampreys. The question of whether there is hypothalamic control over reproduction in lampreys has special significance, because lampreys are modern descendants of the one of the oldest lineages of extant vertebrates and are among the most primitive vertebrates available for study. Thus, the study of lamprey reproduction can shed light on the overall evolution of vertebrate reproduction.

Currently thirteen structures of GnRH have been determined in various vertebrate species and two in invertebrates. They have traditionally been named for the species from which they were first isolated. Table 1 summarizes the various known forms of the GnRH decapeptide. Also, the history of discovery, isolation and characterization of the various known forms of cDNA sequences encoding GnRH precursors is summarized in Table 2 which lists the characterized cDNA's of GnRH precursors.

Table 1

The 15 known GnRH isoforms, grouped together based on the regions of similarity, with differences from mammalian mGnRH underlined.

TABLE-US-00001 TABLE 1 The 15 known GnRH isoforms, grouped together based on the regions of similarity. GnRH 1 2 3 4 5 6 7 8 9 10 Vertebrate Mammal pGlu His Trp Ser Tyr Gly Leu Arg Pro Gly-NH.sub.2 Guinea Pig pGlu Tyr Trp Ser Tyr Gly Val Arg Pro Gly-NH.sub.2 Chicken - I pGlu His Trp Ser Tyr Gly Leu Gln Pro Gly-NH.sub.2 Rana pGlu His Trp Ser Tyr Gly Leu Trp Pro Gly-NH.sub.2 Seabream pGlu His Trp Ser Tyr Gly Leu Ser Pro Gly-NH.sub.2 Salmon pGlu His Trp Ser Tyr Gly Trp Leu Pro Gly-NH.sub.2 Medaka pGlu His Trp Ser Phe Gly Leu Ser Pro Gly-NH.sub.2 Catfish pGlu His Trp Ser His Gly Leu Asn Pro Gly-NH.sub.2 Herring pGlu His Trp Ser His Gly Leu Ser Pro Gly-NH.sub.2 Chicken -II pGlu His Trp Ser His Gly Trp Tyr Pro Gly-NH.sub.2 Dogfish pGlu His Trp Ser His Gly Trp Leu Pro Gly-NH.sub.2 Lamprey - III pGlu His Trp Ser His Asp Trp Lys Pro Gly-NH.sub.2 Lamprey - I pGlu His Tyr Ser Leu Glu Trp Lys Pro Gly-NH.sub.2 Invertebrate Tunicate - I pGlu His Trp Ser Asp Tyr Phe Lys Pro Gly-NH.sub.2 Tunicate - II pGlu His Trp Ser Leu Cys His Ala Pro Gly-NH.sub.2

The 15 primary structures of GnRH where originally sequenced in pig, mGnRH (Matsuo et al., 1971; Burgus et al., 1972), guinea pig, gpGnRH (Jimenez-Linan et al., 1997), chicken, two forms, chGnRH-I and chGnRH-II (King and Millar, 1982a; King and Millar, 1982b; Miyamoto et al., 1983; Miyamoto et al., 1984), salmon, sGnRH (Sherwood et al., 1983), lamprey, two forms, lGnRH-I and lGnRH-II (Sherwood et al., 1986; Sower et al., 1993), catfish, cfGnRH (Ngamvongchon et al., 1992), dogfish, dGnRH (Lovejoy et al., 1992), herring, hGnRH (Carolsfeld et al., 2000), seabream, sbGnRH (Powell et al., 1994), rana, rGnRH (Yoo et al., 2000), medaka, mdGnRH (Okubo et al., 2000), and tunicate (a protochordate), two forms, tGnRH-I and tGnRH-II (Powell et al., 1996).

Table 2

The history of discovery, isolation and characterization of the various known forms of cDNA sequences encoding GnRH precursors.

TABLE-US-00002 GnRH cDNAs and Genes Isoform Organism Year Reference Mammalian Human 1984 Seeburg et al., Nature Norway Rat 1986 Adelman et al., PNAS Mouse 1986 Mason et al., Science Norway Rat 1989 Bond et al., Mol Endocrinol African Clawed Frog 1994 Hayes et al., Endo Tree Shrew 1995 White et al., Soc Neurosci Haplochromis burtoni 1998 White et al., Gen Comp Endo Japanese Eel 1999 Okubo et al., Zool Sci Bullfrog 2001 Wang et al., J Exp Zool Salmon Goldfish 1991 Bond et al., Mol Endo Atlantic Salmon 1992 Klungland et al., Mol Cell Endo Rainbow Trout 1992 Alestrom et al., Mol Marine Biol Biotechnol Cherry Salmon 1992 Suzukiet at., J Mol Endo Brook Trout 1992 Klungland et al., Mol Cell Endo Chinook Salmon 1992 Klungland et al., Mol Cell Endo Rainbow Trout 1992 Klungland et al., Mol Cell Endo Brown Trout 1992 Klungland et al., Mol Cell Endo Plainfin Midshipman 1995 Grober et al., Gen Comp Endo Sockeye Salmon 1995 Coe et al., Mol Cell Endo Medaka 2000 Okubu et al., Biochem Biophys Res Commun Australian Bonytongue 2001 Okubu and Aida, Gen Comp Endo European Sea Bass Zmora et al., (unpublished) Zebrafish Torgersen et al., (unpublished) Verasper moseri Amano (unpublished) Lamprey III Sea Lamprey 2002 Silver et al., Am Zool Pacific Sea Lamprey 2002 Silver et al., Am Zool Australian Lamprey 2002 Silver et al., Am Zool Pouched Lamprey 2002 Silver et al., Am Zool Lamprey I Sea Lamprey 2001 Suzuki et al., J Mol Endo Guinea Pig Guinea Pig 1997 Jimenez-Linan et al., Endo Chicken I Chicken 1993 Dunn et al., J Mol Endo Rana Frog 2000 Yoo et al., Mol Cell Endo Medaka Medaka 2000 Okubu et al., Biochem Biophys Res Commun Catfish African Catfish 1994 Bogerd et al., Eur J Biochem Chicken II Goldfish 1994 Bogerd et al., Eur J Biochem Haplochromis burtoni 1994 White et al., PNAS Tree Shrew 1995 White et al., Soc Neurosci Rhesus Monkey 1996 Dong et al., Mol Cell Endo Human 1998 White et al., PNAS Striped Sea-Bass 1998 Chow et al., J Mol Endo Rhesus Monkey 1998 White et al., Soc Neurosci Haplochromis burtoni 1998 White et al., Gen Comp Endo Human 1998 White et al., PNAS Japanese Eel 1999 Okubo et al., Zool Sci Medaka 2000 Okubu et al., Biochem Biophys Res Commun Australian Bonytongue 2001 Okubo and Aida, Gen Comp Endo Bullfrog 2001 Wang et al., J Exp Zool Verasper moseri Amano (unpublished) European Sea Bass Zmora et al. (unpublished) Silver-Gray Brushtail Possum Lawrence et al. (unpublished) Rio Cauca Caecilian Ebersole et al., (unpublished) House Shrew White et al. (unpublished) Seabream Sockeye Salmon 1995 Ashihara et al., J Mol Endo Striped Sea-Bass 1998 Chow et al., J Mol Endo Haplochromis burtoni 1998 White et al., Gen Comp Endo Verasper moseri Amano (unpublished) European Sea Bass Zmora et al. (unpublished) Red Sea Bream Okuzawa (unpublished)

To date, it has been believed that there is only one form of mammalian GnRH that controls the pituitary in mammals. The first GnRH was isolated and characterized from mammals in the early 1970's and is now referred to as mGnRH. However, it is now believed that there are at least two forms of GnRH in all species, which are not just alternative splice variants, but rather are encoded by separate genes (White et al., 1994). The presence of multiple forms of GnRH suggests a functional differentiation, although this has not been characterized.

For example, two main forms of GnRH have been isolated in sea lampreys: lamprey GnRH-I and lamprey GnRH-III. The cDNA (or gene sequence) of lamprey GnRH-I has also been identified, along with cDNA's of eleven of the fifteen known GnRH's in other species. Again, lampreys are studied because they are the most primitive vertebrates for which there are demonstrated functional roles for multiple GnRH neurohormones involved in pituitary-reproductive activity. Thus the study of lamprey can provide insight into higher vertebrate reproduction. Both lamprey GnRH-I and -III have been shown to induce steroidogenesis and spermiation/ovulation in adult sea lampreys (Deragon and Sower, 1994; Gazourian et al., 1997; Sower, 1990; Sower et al., 1993; Sower, 1998).

In studying the various forms of GnRH, which is a ten (10) amino acid protein, the forms most closely related to an ancestral GnRH molecule are most likely the forms present in fishes of ancient origin, for example, lampreys. In all GnRH peptides studied to date, as can be seen from Table 1, certain regions of the molecule have been highly conserved among all species studied, including the NH2-terminal, pGlu1 and Ser4, and the COOH-terminal. The conservation of the NH2- and COOH-termini suggests that these regions are significant for conformation, receptor binding, and resistance to enzymatic degradation, and in receptor-mediated events required for gonadotropin release.

In addition, as can be seen in Table 2, the known cDNA's predict a GnRH consistent with other neuropeptides. The tripartite precursor polypeptide, called prepro-GnRH is synthesized as part of a larger protein which upon post-translational modification yields the mature decapeptide (Klungland et al. 1992). The tripartite prepro-GnRH consists of a leader peptide at the N-terminal hydrophobic signal domain in direct linkage with the GnRH decapeptide; followed by a 3 amino acid dibasic cleavage processing site (GLY-LYS-ARG); and, at the C-terminal end an additional peptide called GnRH associated peptide (GAP). The precursor is processed by cleavage at the dibasic amino acids (LYS-ARG). GnRH and GAP are then stored within the secretory granules until secreted (Wetsel et al., 1991; Endocrinol. 129: 1584-1594).

The mammalian form of GnRH was first isolated form porcine and ovine hypothalamic extracts, giving rise to the popularly held view that only a single form of GnRH is present in all mammals. However, a question that has arisen over the years with respect to mammals is: How does one GnRH differentially regulate the release of two pituitary gonadotropin hormones, LH and FSH? An answer could be found in the fact that, as noted above, in recent years it has been shown that in vertebrates, at least two different forms of GnRH are expressed within the brain, although not necessarily the hypothalamus, of a single species. Generally, where two forms of GnRH have been found, one GnRH is located in the hypothalamus and functions as a neurohormone regulating the pituitary in the control of the gonadotropin release. The second form may have a neurotransmitter or neuromodulatory function and is localized in areas outside the hypothalamus such as in the midbrain regions. In a limited number of mammals a second form of GnRH has been shown to exist, and it is generally extra-hypothalamic. Where two forms of GnRH have been found in a species, it is also believed that separate genes encode for the multiple forms of GnRH (White et al. 1994; Suzuki et al., 2000). In addition, the presence of multiple forms (and locations) of GnRH suggests a functional differentiation (such as differential regulation of FSH and LH), although this has not been characterized.

Based on studies of lampreys and other species in which two forms of GnRH are found, and the high degree of conservation of amino acid sequence between species, study of a known second form of GnRH in one species (for example lampreys) could lead to identification and isolation of a second form of GnRH in other species. If separate genes were found and isolated for multiple forms of GnRH in various species, including primate, and especially human, these findings would have a substantial impact on our understanding of the release of gonadotropins and would be of great value in clinical studies and practical applications.

In addition, the elucidation of the nucleotide sequence of the cDNA's and/or genes of GnRH and other brain hormones in the lamprey is necessary in order to answer questions concerning both comparative analysis of species and the molecular evolution of neuroendocrine hormones in vertebrates. Using such knowledge, in both humans and other species, new GnRH analogs could be developed that may not have the side effects produced with the GnRH analogs currently used in various applications. Study of a novel GnRH in lampreys, and the evolutionary insights yielded therefrom, could lead to discovery of a novel hypothalamic GnRH in mammals which could lead to the formation of new, useful GnRH analogs.

Thus, despite the knowledge of GnRH to date, there remains a need in both marine aquaculture and human medicine for greater knowledge of GnRH and the evolution of neuroendocrine hormones. This knowledge could be used to produce more effective analogs which could better manipulate reproduction in fish, and which could more effectively be used in human therapy, including reproductive and cancer therapy among others.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8. Another aspect of the present invention includes an analog of the isolated polypeptide. Another aspect of the present invention includes a fusion protein comprising the isolated polypeptide and a heterologous polypeptide.

Some aspects of this aspect of the invention include one or more of the following. The isolated polypeptide produced by a host cell. A composition comprising the isolated polypeptide and a pharmaceutically acceptable carrier.

In accordance with another aspect of the present invention, an isolated polypeptide produced by a method including the steps of: (a) culturing a cell which comprises a nucleic acid encoding the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 under conditions such that the polypeptide is expressed; and (b) recovering the polypeptide.

In accordance with another aspect of the present invention, an isolated polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8; an (b) a polypeptide comprising amino acids 1 to 10 of SEQ ID NO:8; (c) an analog of the polypeptide comprising amino acids 1 to 10 of SEQ ID NO:8; and (d) an analog of the polypeptide comprising amino acids 1 to 10 of SEQ ID NO:8 wherein the analog comprising a substitution of at least 1 position of the amino acids 1 to 10, the position selected from the group consisting of: 1, 2, 3, 6, 7, and n-terminal.

Some aspects of this aspect of the invention include one or more of the following. Where the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. Where the isolated polypeptide consists of amino acids 1 to 10 of SEQ ID NO:8. Where the isolated polypeptide is produced by a host cell. Where the isolated polypeptide is produced in a recombinant host cell. Where the host cell is bacterial.

In accordance with another aspect of the present invention, an isolated polypeptide as described above produced by a method including the steps of (a) expressing the polypeptide by a cell; and (b) recovering the polypeptide.

In accordance with another aspect of the present invention, an isolated polypeptide as described above produced by a method including the steps of (a) synthesizing the polypeptide of claim 7 using peptide synthesis; and (b) recovering the polypeptide.

In accordance with another aspect of the present invention, a method of sterilizing fish including administering to the fish an effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or any portion or portions of the polypeptide, an analog to said polypeptide, or an analog to any portion or portions of the polypeptide set forth in SEQ ID NO: 8.

These aspects of the invention are not meant to be exclusive and other features, aspects, and advantages of the present invention will be readily apparent to those of ordinary skill in the art when read in conjunction with the following description, appended claims and accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

Table 1 (PRIOR ART) lists the amino acid sequences of the 15 previously known GnRH forms. The amino acid sequences of the 15 previously known GnRH forms including lamprey GnRH-III are shown along with the conventionally accepted nomenclature (GnRH peptides are usually named for the species from which they were first isolated). The different forms are grouped based on regions of similarity, with differences from mammalian mGnRH underlined (SEQ. ID. NO's 23-37);

Table 2 lists the characterized cDNA's of GnRH precursors, and their references;

Table 3 shows the antisense primers used in 5' RACE for each respective lamprey species of the present invention (SEQ. ID. NO's 9-12);

Table 4 shows the primer pairs used in full-length transcript isolation for each respective lamprey species of the present invention (SEQ. ID. NO's 13-20);

Table 5 shows the inhibition constants (K.sub.1) of lamprey GnRH-putative antagonists in the pituitary of male land-locked P. marinus;

FIGS. 1a-d list the novel cDNA's of the present invention, from 4 species of lamprey, G. australis, M. mordax, L. tridentatus, and P. marinus respectively, each encoding for the lamprey GnRH-III precursor (prepro-lGnRH-III) (SEQ. ID NO.'s: 1, 3, 5, and 7 respectively). The open reading frame of the prepro-lamprey GnRH-III from each species is underlined, while the poly-adenylation sequence is in bold. The deduced amino acid sequences of the prepro-lamprey GnRH-III peptides (hereinafter "lGnRH-III") from each respective species is below each cDNA sequence;

FIGS. 1a-d also show the deduced amino acid sequence of the prepro-lGnRH-III (SEQ. ID NO.'s 2, 4, 6, and 8 respectively) starting with the 24 amino acid (25 for G. australis) hydrophobic signal peptide which is underlined. The lGnRH-III decapeptide, is underlined and is followed by the GlyLysArg dibasic cleavage site, and the 55 amino acid GAP (GnRH Associated Peptide) region. The amino acid sequences are shown immediately below their corresponding nucleotide sequences;

FIG. 2 shows secondary structure projections for the lamprey prepro-GnRH-III's of the 4 species of lamprey, G. australis, M mordax, P. marinus, and L. tridentatus respectively;

FIG. 3 shows the results of experiments in which four groups of 12 sea lampreys each were injected two times with various compounds and behaviors of spawning activity, resting, nest building, swimming, and fanning were monitored; and

FIG. 4 is a schematic illustrating a method of the invention for sterilization of male sea lamprey.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes isolated cDNA's, and the peptides encoded thereby, encoding the lamprey GnRH-III precursor, comprising the coding regions for the lamprey GnRH-III signal peptide, the lamprey GnRH-III decapeptide, the conserved cleavage site, and an associated peptide called GnRH-III associated peptide (GAP). The cDNA of the lamprey GnRH-III were isolated from 4 species of lamprey, from the three families of lamprey: L. tridentatus, and P. marinus from Petromyzonidae; G. australis from the Geotriidae, and M. mordax from the Mordaciidae.

The present invention also provides methods for the manipulation of ovulation and spawning in female and male fish using the lamprey GnRH peptide and analogs thereof, and methods for using the cDNA's encoding lamprey GnRH-III. Additionally, the present invention is method of producing the isolated GnRH-III polypeptides.

To date, two molecular forms of GnRH have been identified and sequenced in the sea lamprey: lamprey GnRH-I and lamprey GnRH-III (Sherwood et al., 1986; Sower et al., 1993). In addition, the cDNA of lamprey GnRH-I has been identified (Suzuki et al., 2000). In lampreys undergoing metamorphosis, there is an increase of brain lamprey GnRH-I and -III that coincides with the acceleration of gonadal maturation (Youson and Sower, 1991). In immunocytochemical studies, both immunoreactive (ir)-lamprey GnRH-I and -III can be found in the cell bodies of the rostral hypothalamus and pre-optic area in larval and adult sea lamprey (King et al., 1988; Nozaki et al., 2000; Tobet et al., 1995; Wright et al., 1994). Most of the ir-GnRH in the brain of larval stage lampreys has been shown to be lamprey GnRH-III. Thus lamprey GnRH-III may be the more active form during gonadal maturation. In addition, in females, it has been demonstrated that lamprey GnRH-III is present in higher concentrations than lamprey GnRH-I during the final stages of the reproductive season in lamprey (MacIntyre et al., 1997). Lamprey GnRH-I concentrations do not change significantly during the reproductive season, whereas lamprey GnRH-III undergoes significant increases during the same period. These results suggest also that lamprey GnRH-III may be the major form regulating reproductive processes in the female sea lamprey during the period of final reproductive maturation.

Such information, comparing GnRH of various species, also suggests that the structure and function of the GnRH's in vertebrates are highly conserved throughout vertebrate evolution.

Thus, in addition to the novel cDNA sequences of the present invention, the present invention also includes methods of manipulation of maturation and spawning of lamprey, using the cDNA's encoding lamprey GnRH-III, lamprey GnRH-III and analogs thereof, including methods of sterilization for male lamprey, and especially methods that sterilize but do not affect the spawning behavior of the males.

In addition, the present invention furthers the process of researching the evolution of reproductive biology. As noted above, lampreys and hagfish of the Class Agnatha are of particular importance in understanding endocrinological relationships since they are the modern descendants of the most primitive vertebrates available for study. They represent the oldest lineages of extant vertebrates--which evolved over 550 million years ago. Therefore, the study of lampreys and the characterization of brain and pituitary hormones in lampreys is particularly important for understanding the molecular evolution and functional diversity of reproductive hormones. In addition, understanding the function of GnRH in lamprey could have a substantial impact on our understanding of the release of gonadotropins in mammals and would also be of great value for clinical studies, thus potentially yielding further valuable insight into human reproductive processes.

This invention relates to a form of GnRH, particularly lamprey GnRH-III, and its novel cDNA in four species of three families of lamprey. The invention is based on the isolation and sequencing of cDNA for lamprey GnRH-III from members of the three families of lamprey in order to assess their phylogenetic relationship and provide insight into the evolution of neuroendocrine hormones, specifically the evolution of the GnRH decapeptide, and its function and regulation in lamprey and other animals.

The DNA molecules of the present invention and the endogenous GnRH peptides encoded thereby as described in FIGS. 1a-d, analogs and/or fragments thereof, and/or any combination of such endogenous and/or analog peptides and/or fragments thereof, including the signal and GAP peptides and/or analogs and/or fragments thereof (hereinafter referred to as "active compound") may be used to induce or inhibit gonadal development, and to induce and synchronize ovulation, spawning, sperm production, and spermiation. Additionally, Applicant's lamprey GnRH-I and III, and the cDNA of lamprey GnRH-I and III can be used to develop additional analogs for use in reproductive management of lampreys and therapies for other animals, including humans.

As has been shown, the lamprey l-GnRH-III has a similar amidated decapeptide structure to other GnRH's, but has unique (vs. mammalian GnRH) residues at positions 5-8. (See Table 1). Note also that while Table 1 shows the first amino acid of the l-GnRH-III peptide as pGlu, and FIGS. 1a-d show the first amino acid of the peptide as Gln, the Gln becomes pGlu in post-translational processing to result in the mature peptide. Thus, Table 1 lists the mature peptides, after processing. Note also, as shown in FIGS. 1a-d, that the 10 amino acid sequence of the decapeptide is the same for all 4 species of lamprey studied with respect to the present invention.

In addition, lampreys are among the few vertebrates to clearly demonstrate roles for multiple GnRH molecules as neurohormones involved in pituitary-gonadal function. Because lampreys have two GnRHs that act as neurohormones controlling the pituitary-gonadal axis and act in a differential manner, it is proposed that an analog to lamprey GnRH-III can be developed in which the spawning behavior would not be affected, yet the lampreys would be sterilized. Such analogs of GnRH may potentially be used to replace Bisazir in the Great Lakes lamprey sterilization program mentioned above. Thus GnRH analogs have the potential to provide a much easier, less expensive and safer method and system for controlling or regulating the lamprey reproduction. Example analogs and methods for sterilizing lamprey are described below

Endogenous lGnRH-III peptide may be isolated from lamprey brains using standard techniques as described below. In the alternative, active compound may be chemically synthesized using standard automated laboratory techniques. In accordance with the invention, active compound may be formulated for use in any of a variety of methods well known in the art and active compound of the invention may be administered by any of a variety of methods known in the art. Examples of various formulations and methods will be described below.

For example, the compositions of the present invention, as would be used on lamprey or other fish or animals, are preferably administered in a "sustained release" method. The term "sustained release" is understood to mean a gradual release of active compound in a controlled manner. Such sustained release formulations of active compound may be solid and may be prepared in any suitable form such as pellets, discs or rods, or encapsulated in microspheres. Active compound may be administered by methods including implantation of a unit of active compound in the form of pellet, disc, rod, or microsphere, or injection of active compound--either intramuscular, subcutaneous, or intraperitoneal in the form of a suspension of mini-rods or microspheres. Injectable formulations in accordance with the invention in the form of mini-rods or microspheres should be sufficiently small to pass through a syringe. Injectable formulations would be suspended in an injectable solution such as saline or various buffers prior to injection. Certain methods and formulations such as microspheres for microencapsulation are covered by various U.S. patents to Zohar.

Implantable compositions usable with the present invention may preferably comprise about 300 ug of the active compound per unit. When administering an injectable composition in accordance with the invention, the administered composition will preferably comprise about 5-200 ug of the active compound per kg of body weight of the injected fish or other animal. However, the amount of the active compound may, in some cases, be reduced if a very active analog is used.

As will be discussed in more detail later, Table 5 shows various l-GnRH-III analogs used in lamprey sterilization experiments. As noted, the DNA molecules of the present invention may be used to either induce or inhibit gonadal development, and to induce and synchronize ovulation, spawning, sperm production, and spermiation. In order to induce or inhibit various sexually reproductive activities, active compound comprised of other than the mature peptide or fragments or analogs thereof, may be made using the precursor cDNA, or a portion thereof (for example, a portion encoding the signal peptide, the l-GnRH-III decapeptide and/or the GAP) and may also, or in the alternative, be administered into fish. One method of such administration may be transfection. Transfection may be achieved, for example, by microinjection, retroviral-mediated integration, electroporation, liposome-mediated delivery, and by high velocity microprojectiles. For a review of such transgenic systems in fish, see Chen et al., 1990, Tibtech 8:209-215 which is incorporated herein by reference in its entirety.

In addition, such a transfected coding sequence may be operatively linked to an inducible promoter using standard laboratory techniques routinely practiced in the art, such that expression may be controlled experimentally. See for example, Ausubel F. M. et al., eds., 1989 Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York; and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories Press, Cold Spring Harbor, N.Y., which are incorporated herein by reference in their entireties. Controlled induction may result in an increase in expression at the appropriate stage of development. Such controlled induction of expression is particularly useful in the production of brute stock for fish breeding, or even use for fertility and reproductive treatments in other animals.

As noted above, the inhibition of gonadal development may be used in order to produce sterile fish and may be achieved by the negative regulation of GnRH. In addition to GnRH analogs that have the potential to provide a much easier, less expensive and safer method and system for controlling or regulating lamprey (or other species) reproduction, other compounds and methods may be useful to inhibit gonadal development. Among the compounds which may exhibit the ability to negatively regulate GnRH are: antisense, ribozyme, and triple helix molecules. Such molecules may be designed to reduce or inhibit either wild type, or if appropriate, mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.

Anti-sense RNA and DNA molecules act directly to block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest, would be preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well-known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated herein by reference in its entirety. As such, within the scope of the invention are various engineered ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.

The anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for synthesis of DNA and RNA molecules. These methods include well-known techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides such as for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors w