WO2002081693A1 - Production, composition and usage of a recombinant hormones system for superovulation in vertebrates and invertebrates - Google Patents

Abstract

The present invention relates to development of a process for production, composition and usage of species-specific recombinant hormones with the feature of synergics function in the ovulation process. This composition, which is able to stimulate the secretion of Follicle Stimulating Hormone (FSH) from hypophysis and to increase FSH receptor expression in growing follicles, is used to obtain superovulation in vertebrates and invertebrates with better results than that of current commercial products.

Description

PRODUCTION, COMPOSITION AND USAGE OF A RECOMBINANT HORMONES SYSTEM FOR SUPEROVULATION IN VERTEBRATES AND INVERTEBRATES Field of the Invention The present invention relates to development of a process for production, composition and usage of species-specific recombinant hormones with the feature of synergies function in the ovulation process. This composition, which is able to stimulate the secretion of Follicle Stimulating Hormone (FSH) from hypophysis and to increase FSH receptor expression in growing follicles, is used to obtain superovulation in vertebrates and invertebrates with better results than that of current commercial products. Background of Invention

The gonadotrophins are glycoproteins hormones with structural similarities and just only two origins: pituitary (FSH and Luteinizing Hormone, LH) and placenta (CG, Chorionic Gonadotropin) . Gonadotrophins have their actions on reproductive process, and autocrine, paracrine and endocrine interactions support the female reproductive cycle. In females, FSH is essential for follicular maturation, and with other hormones, is involved in the mechanism of recruitment, growing, ovulation and atresia of follicles.

In primordial follicles the oocyte is enclosure by a single layer of granulosa cells, which represents the Go phase of cellular cycle. Under specific signals those cells start to grow slowly (Richards, 1980) . As those cells are exposed to FSH, they start to produce estrogens that will allow a quickly proliferation and consequently formation of pre-ovulatory follicles. At this stage, the granulosa cells produce LH receptors (Vilenbroek e Richards, 1979) that promote changes in follicular structure and function that will stimulate the luteinization of granulosa cells (Richards, 1994).

The embryo transfer is one of the most interesting process and biotechnology of assisted reproduction. Basically, it consists in a hormonal stimulation of ovaries from a female with high genetic quality to provoke the development and maturation of several follicles simultaneously (superovulation) . After natural conception or artificial insemination (Al) , the embryos are collected by a uterine flushing and transferred to the receptors cows, that will bring gestation until birth (Hafez, 1993) . The efficiency of this process is about 60% (Pereira, 1996) and several factors as phenotype, nutrition, environment among others are responsible. However, the most important factor is related to the quality of the hormone used to stimulate ovaries. Between comercial hormone preparation, there are those with FSH-like activity, as PMSG (Pregnant Mare Serum Gonadotrophin) and those that are swine or ovine adenohypophysis extract, where FSH is partially purified. The FSHs from swine or ovine could be utilized in bovine superovulation, caused by their structural homology of about 70%. The current methods used involves administration of lyophilized crude extract from swine or ovine hypophysis, PMSG or HCG (human corionic gonadotropin) (Hafez, 1993). Besides the risk of contamination between species (e.g. bovine spongiform encephalopathy) , the differences in the primary structure of proteins and the oligosaccharides composition could be responsible to the low efficiency of the process. It has been noted that subsequent applications of these preparations produce specific antibodies against heterologous hormones that will modify the reproductive cycle of embryo donors. Due to this, the interval between harvests of a donor is 60 to 90 days, which allows an average of 4 harvests per year per donor.

FSH is a heterodimeric glycoprotein of two non- covalently associated subunits, designated α and β, which confers the unique biological specificity for each hormone. Separate genes located on different chromosomes encode them. The alpha subunit has expression levels always high (Pierce e Parsons, 1981) . However, FSH has short half-life and low expression levels . The promoter of subunit beta gene is mainly regulated by activin.

Activin is a homodimer of two-beta subunit (different from that of FSH) linked by a disulfide bond, with no oligosaccharide attached (Vale et al, 1986) . In pituitary and ovary activin modulates control of expression of several hormones as FSH and LH and also act on differentiation and proliferation of embryo and different kinds of cells. The complementary action of activin and FSH results in a significant increase in the FSH receptor expression (Kishi et al, 1998) . Li et al (1997) showed that activin supports survival of granulosa cells and cell proliferation. Moreover, in culture, it keeps the FSH receptors working without FSH. Katayama et al, (1990) found that activin increases the number of gonadotropes in anterior pituitary cell culture. Activin with or without FSH has an autocrine action on granulosa cells, stimulating differentiation in the pre-antral stage of folliculogenesis and prevents premature luteinization of antral follicles in vitro (Findlay, 1993) .

The distribution of activin on spermatocyte, oocyte, fertilized or not and in several organs during embryogenesis points out important action of this hormone on embryo development (Vale et al , 1994) . Although the activin action on FSH receptors is known, there is no citation about the combined action of these two hormones upon embryo viability and quality. Information that embryo quality is affected by FSH preparations with high concentrations of LH is known since eighty-decade (Moor et al, 1984) . FSH preparations without LH increase the number of embryos with quality (Donaldson e Ward, 1986) . Every commercial product used today for superovulation has difference proportions of FSH and LH. The patent EP 839831 proposes antibodies against LH to diminish negative effect of LH. FSH production is inhibit by inhibin that belongs to the superfamily TGF-β (transforming growth factor-β) (Rivier et al , 1985) . Inhibin is a heterodi er of alpha and beta subunits (also different from that of FSH) , where beta is the same of activin. In the organism, the expression of alpha subunit is higher than that of beta because the promoter region of alpha subunit has several cAMP cis-active elements (Pei et al , 1991) . Although activin has a powerful biological activity, the expression levels in the organism are very low. Inhibin may be used as an antagonist of activin when there is an activin overstimulation, like in ovarian tumors (Ling et al, 1986) .

The patents WO 8600078, US 5102807 and EP 222491, described purification, from follicular fluids, and production of recombinant inhibin, in mammal cells, to be used in the control of mammal's fertility.

In human assisted reproduction programs, it has been used FSH purified from urine or recombinant FSH to stimulating follicular maturation in women with chronic anovulatory syndrome or luteal phase deficiency

(Loumaye et al , 1998) . The recombinant bovine FSH was first produced by Chappel, et al (1988), and showed to be efficient to stimulate ovaries. However, there were variations in ovarian response like those produced by commercial gonadotrophins (Bellows et al , 1991) . Patent WO 9013627 proposed in vitro oocyte maturation by co- cultivation of oviduct cells and native or recombinant gonadotrophins . The efficiency in the development of morula or blastocyst range from 16 to 32%. This proposal is very expensive due to the structure for cultivation.

The results obtained with recombinant FSH, that doesn't allow good responses in comparison with that of purified FSH, demonstrate the necessity of other factors .

The patent US 4717670 describes, for the first time, the production of FSH beta chain of swine through DNA recombinant technology. The authors claim the sequence code region and the plasmid. The expression system was mammal's cells and the authors suggest the utilization of only beta chain to produce superovulation in mammals or in association with alpha chain produced by other company. In vivo, the chains can associate spontaneously to form dimeric structure. However, the biological activity of glycoprotein hormones is dependent on the presence of intact di ers with specific levels of glycosilation. Newly synthesized alpha and beta subunits are rapidly assembled in the endoplas ic reticulum, and the oligosaccharides in the dimers undergo hormone specific post-translational modifications. Oligosaccharides have been implicated in intracellular events such as folding, subunit assembly and secretion. Differences in the ratio sialylated/sulfated can affect the bioavailability and bioactivity of glycohormones (Boime et al , 1999) . Other approaches to produce alpha and beta subunits have been proposed. The WO 9002812 describes production of hybrid gonadotrophins composed by equine alpha subunit and a beta subunit from different species. The hormones were expressed in C127 epitelioid cells from mice co-transfected with cDNAs of both subunits. The purification process is constituted by three very hard stages, with no references to biological activity. WO 9002757 and EP 404546 patents describes similar methodologies, with the subunits from the same specie. The first proposed virus or plasmid as vector containing the code region of subunits, flanked by strong promoter like mice metallothionein and sequences of DNA known to be stimulators of expression like SV40 intron. The second one proposed instead of cDNA of alpha and beta subunits, genomic sequences corresponding to the subunits . In both proposals the expression system was mammal cells. No references to biological activity are cited in those patents. The expression levels are very low when compared with yeast, and it is necessary expensive mediums and long periods of cultivation.

Between several expressions systems CHO and insects cells are mainly utilized. They are characterized by low productions (20 ng to 20 ug/ L) and differences in the structural glycosilation, which could compromises the biological activity of the product. Taking into account that the recommended dose of FSH for superovulation is 1500 to 3000 UI (160 to 400 mg FSH) the limitations of proposed expression systems are obvious.

Another approach is proposed by the patent WO 9712038, which utilize monoclonal antibodies that bind to regions of hormone, which are responsible to FSH receptor interaction. They are small fragments of about 10 aminoacids . Peptides with those sequences are used to obtain monoclonal antibodies, which are utilized in bovine with increase in biological activity of hormone. However, with long periods of utilization those antibodies in other species could produce important side effects like donors cycle alteration or even sterilization.

Another approach with antibody was proposed against follistatin, a glycoprotein of molecular mass between 32 and 43 kDa (Ying et al , 1987), which binds to activin with high affinity, neutralizing their biological activity (Nakamura et al, 1990) . Follistatin was first isolated from follicular fluid and is broadly distributed on the organism (DePaolo et al, 1991) . In Canada, Singh et al (1999) utilized active immunization against follistatin and showed an increase in the number of growing follicles and a good superovulatory response . Patent WO 9303162 proposed the use of ecdysteroyds hormones (e.g. β-ecdysone) that are responsible by stimulation of receptor promoter steroid hormones in mammal cells transformed with heterologous genes, like gonadotrophins . There is no mention about the results from FSH gene promoter or even to the efficiency on superovulatio .

The FSH half-life is short and there is a necessity to use large or frequent doses for clinical utilization in humans (Soers et al , 1979) . The same situation is observed in bovine whose protocols for superovulation utilize large doses (» 400 mg) divided in two daily doses during four days to a satisfactory response. New strategies to diminish the frequency and quantity of FSH application have been tried.

The association with activin, which potentiates FSH action, without necessity of structural changes, is the strategy proposal by this invention. Summary of the Invention The objectives of this invention are: the development of a process for production of specie-specific recombinant hormones presenting synergic functions in the ovulation process. - development of a mixture of recombinant or natural specie-specific hormones presenting synergic functions in the ovulation process. define the scheme for usage of a mixture of recombinant or natural specie-specific hormones presenting synergic functions in the ovulation process. The present invention is characterized by the utilization of a mixture of recombinant specie-specific hormones, FSH and activin, which have several advantages to the comercial products utilized today, which are: A. Specificity

By the fact that recombinant hormones are specie- specific, they do not produce immune response, which permit to diminish rest intervals to about 35 days, allowing increase in the number of harvest per year, increasing efficiency of donors and receptors cows . This idea is shared by PI 9704515-2 A patent, where the author claim the maximization of donors only by reduction of harvest intervals with no considerations about hormones preparation utilized and antibodies production. These were considered in this invention.

B. Embryo quality

This invention contributes to embryo quality.

Activin acts on embryogenesis and potentiates FSH. Otherwise, recombinant hormones are free of contaminants like LH, which produce embryos with better quality.

C . Economy

One of the best advantages of utilization of recombinant techniques is the final price of product. The expression system utilized in this invention allows production of large quantity of biologically active protein (2 to 10 g/L) . The expression levels and glycosilation assembly are constant in the strain of yeast which is not observed for the hormone obtained by purification from hypophysis, due to animal cycle phase at the moment of sacrifice, resulting in variable biological activity (Braileanu et al, 1998) . On the other hand, our strategy utilizing pMULT vector, by which both subunits are produced simultaneously, allows the right dimers formation with right structure and glycosilation .

Brief Description of the Invention Figuras 1A-1F show the primers and cDNAs sequences corresponding to the mature alpha and beta chains.

Figure 1A shows alpha chain sequence of bovine FSH.

Figure IB shows beta chain sequence of bovine FSH. Figure 1C shows alpha chain sequence of equine FSH.

Figure ID shows beta chain sequence of equine FSH.

Figure IE shows beta chain sequence of activin.

Figure IF shows primers sequences. Figure 2 shows pGEMAOX vector.

Figure 3 shows pGEMAOXα vector.

Figure 4 shows pPIC9β vector.

Figure 5 shows pMULT vector.

Figures 6 and 7 are pictures of histological laminas from ovaries of treated and control rats . Preparation of expression vectors

The techniques utilized here are describe on Molecular cloning: a laboratory manual (Sambrook et al, 1989) . The hormone subunit gene sequences are obtained from literature (GenBank) . From the cDNA sequences of mature protein, specific primers to N- and C-terminals containing restriction sites of EcoRI and Notl were synthesized. The genomic version of FSH alpha and beta subunits contain introns, that explain why we amplified the subunit sequences from cDNA, obtained by reverse transcription (RT) from total mRNA of hypophysis. Activin gene doesn't have introns and we amplified from genomic DNA. The DNAs were amplified by PCR (poli erase chain reaction) and sequenced.

The chosen expression system was the yeast Pichia pastoris and the vector pPIC9 from Invitrogen. This vector contains an expression cassette composed by alcoohol-oxidase (AOX1) gene and the histidinol- dehydrogenase enzyme gene (HIS4). The alcoohol-oxidase enzyme allows Pichia utilize methanol- like only carbon source. The complete gene of alcohol-oxidase is composed, in sequence, by promoter region (1-948 bases) , Sacharomyces cerevisae α factor secretion signal (949-1218), multiple cloning site (1192-1241) and 3" AOXl (1253-1586) with transcription termination fragment with approximately 1.6 kb. In the multiple cloning site region there are hydrolysis site for EcoRI and Notl that make possible cDNAs insertion. The mature protein obtained through this genetic construction has a peptide segment of 8 aminoacids from vector on the N terminal portion.

By PCR, utilizing specific primers, we amplified the segment of 1.6 kb, correspondent to complete alcohol-oxidase gene. In this fragment is located at 5 "AOXl a site for Sphl enzyme. The PCR product containing all cassette expression (1.6 kb) was cloned on pGEM-T (Promega) vector that was modified as follow: first, pGEM-T lost thymidines of their extremities and extremities were religated. The resultant plasmid was submitted to digestion with Notl and treated for destruction of Notl site. Again, the extremities were religated generating a plasmid derived from pGEM-T, closed and without Notl site. This new vector was digested with Sphl for insertion of 1.6 kb fragment from pPIC9, generating pGEMAOX vector. The FSH alpha subunit was inserted to pGEMAOX through EcoRI and Notl resulting in the plasmid pGEMAOXα with an insert of approximately 2.0 kb. The β subunits of FSH and activin were cloned in pPIC9 without modifications through EcoRI and Notl resulting in the plasmids pPIC9βFSH and pPIC9βact with approximately 8.4 kb each. The pGEMAOXα vector was digested with Sphl to remove the expression cassette of 2.0 kb containing alpha subunit. This fragment was inserted into the pPIC9βFSH vector on the Sphl site, resulting in a vector designated pMULT, with approximately 10.4 kb. Two AOXl promoters characterize this vector. By insertion of alpha and beta subunits the gene of alcohol-oxidase is disrupted causing to positives clones a poor capacity to grow in that medium whose methanol is the unique carbon source. The presence of methanol however stimulates AOXl promoter in the expression of cloned alpha and beta subunits. For transformation of P. pastoris by eletroporation, pMULT and pPIC9βact vectors were digested with Bglll to liberate region from 5 "AOXl promoter until 3'AOXl. This procedure is utilized since it is possible to get the insertion of the fragment into yeast genome by homologous recombination. These clones were selected like Muts or Mut+ from medium with dextrose without methanol (MDH) or methanol without dextrose (MMH). Both mediums contain histidine. The Muts clones grow very slowly or don't grow in the MMH medium, because the alcohol-oxidase gene was disrupted with insertion of the interest gene. The phenotype Mut+ grows well in medium with methanol, just because the alcohol-oxidase gene was not altered. The Muts clones were grown in specific medium with methanol to study kinetic induction of expression. As a control it was utilized yeast transformed with expression cassette of pPIC9 digested with Bglll but without insert. On the beginning of induction (tO) and after each 24 hours, methanol was added to the medium to a final concentration of 0,5%. On the times 0, 24, 36, 48, 60 , 72 and 96 hours, aliquots were collected to determine the optical density at 600-nm and protein precipitation. Aliquots were analyzed by SDS-PAGE. For the FSH expression product, three proteins presenting 14, 18 and 32 kDa, were observed. These bands correspond to α, β and FSH (dimer α plus β) , respectively. In the activin expression product, were observed bands of 14 and 28 kDa, which correspond to β subunit and the dimer, respectively.

In vitro assays with sexually mature rats were performed with the following combination: FSH (50 ug - 5 mg) , activin (50 ug - 5 mg) , FSH:activin association and vice-versa ranging from 10:1 to 1:1 rates, with 50 ug to 5 mg each. The results showed that only FSH wasn't sufficient to promote a significant follicular growing within a 48 hours period. Activin alone showed already on the 24 hours, in a 50-ug dose, a low follicular development. The best results were obtained with the mixture of both hormones, in rates from 1 to 3 part of FSH to 6 to 8 part of activin, provoking a follicular development in both ovaries. The rate utilized above showed best results than PMSG (10 UI) . Hence, this invention define the best rate of hormones responsible to a better response without frequent negative effects like precocious luteinization of follicles, which is observed in the commercial preparations that contain LH. The pictures of histological laminas of rat ovaries treated and control (figures 6 and 7), two days after administration of hormones (annexed), show these results. In bovine, the rate proposed not only produced superovulation but also the embryo obtained was of excellent quality (spherical, symmetric with uniform size, texture and color) as described by Lindner and Wright, 1983.

This invention proposal the follow protocol to application: estrus identification (day 0) and application of a dose on the 10^th day after estrus. On 12^nd day, utilize prostaglandin F2α or analogs. After prostaglandin, new estrus identification and proceed with two artificial insemination 12 and 24 hours after. The embryo harvest is carried out on the 7 day after estrus . Although the present invention has been described in terms of preferred embodiments, any modifications and variations together with others that would be obvious for a person of ordinary skill in the art are deemed to be within the scope of the present invention whose nature is to be determined from the above description and the depended claims.

Claims

1. Process of production, composition and usage of a system characterized by association of recombinants hormones specie-specific activin and FSH, for superovulation.

2. Development of a hormone system, like claim 1, characterized by the fact that the hormones could be of recombinant or natural origins .

3. Development of a hormone system, like claims 1 and 2, characterized by construct of a double promoter

(pMULT) to simultaneously expression of alpha and beta subunits of follicle stimulating hormone.

4. Development of a hormone system, like claim 3, characterized by the fact that pMULT vector is generated by fusion of SPHAOX fragment containing one subunit, to the second one from pPIC9 vector.

5. Development of a hormone system, like claim 1, characterized by the mixture of substances in specific proportions regarding to embryo development and maturation.

6. Development of a hormone system, like claim 1 to 5, characterized by the fact that, in rats, the proportion is, (preferentially) , of 6 to 8 parts of activin to 1 to 3 parts of FSH.

7. Development of a double action hormone system, like claim 1 to 6, characterized to be a injectable drug to produce superovulation.

8. Composition like claim 1, characterized by the use of this composition to produce superovulation in vertebrates .

9. Composition like claim 1, characterized by the use of this composition to produce superovulation in invertebrates .

10. Development of a hormone system comprising the sequences ID SEQ No. 1 and ID SEQ No. 6 or sequences funcionaly equivalentes.