Induc&on of gonadal matura&on in teleosts by recombinant gonadotropins. Ignacio Giménez RARA AVIS BIOTEC S.L.

Transcription

1 Induc&on of gondl mtur&on in teleosts by recombinnt gondotropins Igncio Giménez RARA AVIS BIOTEC S.L.

2 ENVIRONMENTAL STIMULI CENTRAL NERVOUS SYSTEM HYPOTHALAMUS GnRH HYPOPHYSIS FSH LH Testes Spermtogenesis Spermiogenesis Ovries Vitellogenesis Finl mtur&on Ovul&on

3 Full induc&on of reproduc&ve cycles cn be done Recombinnt Gondotropins?

4 Produc&on of recfsh nd reclh FSH α βfsh LH α βlh Difficul&es: Dimers Highly glycosylted proteins Smll yields

5 Produc&on of recfsh nd reclh α βfsh α βlh DIMERS SINGLE-CHAIN MONOMERS β-fsh subunit Linker α-mture subunit β-lh subunit Linker α-mture subunit cdna

6 Produc&on of recfsh nd rec LH Detection (developed ntibodies)" Purifiction" Concentrtion" Optimiztion of expression levels" Biossy" α βfsh α βfsh α βfsh CHO CELLS CHINESE HAMSTER OVARY CELLS

7 Why CHO cells? Proper glycosil&on is essen&l for c&v&on of glycoprotein hormone receptors Mmmlin glycosyl&on pvern (more silic cid cpping) Improved biocdvity Phrmcokine&cs: Longer hlf-life CHO glycosyl&ons re thougth to be less immunogenic in vertebrtes thn glycosyl&ons from other expression systems. Disvndtge: Lesser protein yields thn other expression systems. But. Glycoproteins used in humn therpeu&cs re expresssed in CHO cells (rhfsh nd rhlh)

8 Dose? Norml vlues FSH : 2 ng/ml LH : 1-1 ng/ml FSH: 4-12 ng/ml LH : 1-5 ng/ml FSH : 18-4 ng/ml LH : ng/ml Roch et l. (29) Moles et l. (212) FSH: 1-3 ng/ml LH : 2-3 ng/ml FSH : 2-4 ng/ml LH : 4-18 ng/ml FSH: 8 ng/ml LH : 85 ng/ml FSH : 5-15 ng/ml LH : 2-14 ng/ml Chuvigné F. et l. (216)

9 Dose? Humn dily dose of rhfsh = 75 IU (stndrd dose) = 5,5 μg,1 μg/kg FSH:,1 μg 1 μg/kg FSH:,15 μg,6 μg/kg 4,3 μg/kg FSH:,5 μg,5 μg/kg 17 μg/kg (weekly) 2,4 μg/kg (dily equivlent)

10 Produc&on of teleost recfsh nd rec LH by RARA AVIS M.W. Teleost recfsh nd rec LH produced Se bss Sole Europen eel FSH FSH 7X LH LH 7X -An&-lph Dicentrrchus, Dil:1: 1 -An&-Rbbit-HRP, Dil : 1: 5 M.W.:Moleculr Weight Mrker. Yield: -FSH : 1,4-2,2 mg/l -LH : 1,8-2mg/l

11 Mle eels. Experimentl Design rfsh rlh rfsh rlh µ g recombinnt gondotropin/fish µ g recombinnt gondotropin/fish C C Weeks of tretment Weeks of tretment µ g recombinnt gondotropin/fish Group I C Weeks of tretment µ g recombinnt gondotropin/fish Group II C Weeks of tretment µ g recombinnt gondotropin/fish Group III C Weeks of tretment Peñrnd S. D. et l. Fish Gmete Workshop, Ancon,Itliy. 215

12 6 5 b * 4 GSI (%) C FSH LH I II III Group I Weeks of tretment µ g recombinnt gondotropin/fish C Weeks of tretment Peñrnd S. D. et l. Fish Gmete Workshop, Ancon,Itliy. 215

13 Spermiting mles (%) Volume (ml) Density (1 8 cells/ml) Totl Motility (%) b b b * b b Weeks of Tretment * b * FSH LH GI GII GIII µ g recombinnt µ g gondotropin/fish µ g recombinnt gondotropin/fish µ g recombinnt gondotropin/fish µ g recombinnt gondotropin/fish rfsh rlh C C Weeks of tretment C 1 Weeks 3 of tretment Group I C Weeks of tretment Group II Group III Weeks of tretment C Weeks of tretment Peñrnd S. D. et l. Fish Gmete Workshop, Ancon,Itliy. 215

14 Progresive Motility (%) VSL ( µ m/s) VCL ( µ m/s) b b b* b* c* bc* b bc FSH LH GI GII GIII µ g recombinnt gondotropin/fish Group I C Weeks of tretment 4 * b b* VAP ( µ m/s) b Weeks of tretment b Actully, we re gelng bever results with higher doses Peñrnd S. D. et l. Fish Gmete Workshop, Ancon,Itliy. 215

15 Hlf - life Recombinnt Sole FSH nd LH Dose = 17 μg/kg (IM) NDve Sole FSH nd LH (Mles) ELISA: Chuvigné F. et l. Comp. Biochem. Physiol., Prt A Mol. Integr. Physiol. 216

16 EXPERIMENT 1 SOLE MALES EXPERIMENTAL DESIGN rlh STEROIDOGENESIS HISTOLOGY

17 EXPERIMENT 2 SPERM PRODUCTION Sline 6 μg LH biweekly 6 μg FSH weekly 6 μg FSH weekly + 6μg biweekly WEIGHT: 354 ± 7 g

18 INSTITUTO ESPAÑOL DE OCEANOGRAFÍA CENTRO OCEANOGRÁFICO DE MURCIA PLANTA DE CULTIVOS DE MAZARRÓN RESTARTING VITELLOGENESIS Dicentrrchus lbrx Weigth: 24,2 ± 17,1 g 22 months Nturl condi&ons of photoperiod nd T. FSH Molés G. et l. 211 LH

19 Group A: Sline Group B: 1 μg rsbfsh (weekly) x 8 weeks Group C: 2 μg rsbfsh (weekly) x 8 weeks Group D: 4 μg rsbfsh (biweekly) x 8 weeks + 1 μg rsblh weekly (lst 4 weeks) Group A Group B Gruop C Group D Men 146,8 317,1 374,3 352,2 SD 12,5 58,6 35,7 25,2 1% 216% 255% 24% OOCYTE DIAMETER (1 greter oocytes/slide)

20 Experimentl extension Individuls of Groups A (n=1) nd C (n=1) were treted for 12 more weeks. Group A: Sline 4 μg rsbfsh Group C: 2 μg rsbfsh 1 μg rsblh 2 μg rsblh 4w 8w 2w Februry Índice Gondosomático (%) A C b b b M M1 M2 M3 GONADOSOMATIC INDEX July

21 End of the experiment. Mcroscopy Group A. Control Group C

22 End of the experiment. Microscopy Group A. Control Group C Mesurement of 25 bigger oocyte dimeters (μm) /slide. Group A Group C Men 137,1 79,1 SD 11,5 55,5 Gimenez I. et l. First Iberin Congress of Aquculture. Huelv, Spin. 215

23 Wreckfish : First Vempt Se bss recombinnt FSH nd LH + 2 GnRH IMPLANTS Eggs lid nd fer&lized in july 216

24 Conclusions - Single-chin homologous recombinnt gondotropins hve been expressed in CHO cells for : - Anguill nguill - Sole seneglensis - Dicentrrchus lbrx - The single-chin recombinnt gondotropins hve biologicl c&vity in vivo t the doses tested. - The effec&ve doses re in the rnge of doses used on birds nd mmmls - The hlf-life of the hormones llows weekly (t lest) tretments - These recombinnt gondotropins re ble to induce full gmetogenesis from immture gonds nd possibly, from prepubertl individuls. Also, they could be used to induce «out of seson» cycles - These recombinnt gondotropins could be useful in quculture to induce reproduc&ve cycles nd for reserch purposes

25 Thnks for your Ven&on