158 PRODUCTION OF RECOMBINANT LH FOR USE IN TIGER (TIGRIS ALTAICA) ASSISTED REPRODUCTION:A PRELIMINARY REPORT

Abstract

Genetically, porcine LH is the closest commercially available gonadotropin analog to tiger LH (93% homologous); however, its use may lead to possible autoimmune reactions, lessening ovarian responses in stimulated tigresses over time (Crichton et al. 2005 Biol. Reprod. 68, 105–113). To overcome this problem for use in assisted reproduction, we produced recombinant tiger LH (tLH), and tested the bioactivity of several tLH constructs using heterologous (rat) and homologous (cat) Leydig cell assays. To clone tLH, mRNA was isolated from an Amur tiger pituitary by TRIzol extraction (Invitrogen, Carlsbad, CA). DNA was synthesized from the mRNA using reverse transcriptase (Stratagene, La Jolla, CA) and PCR was performed using tiger-specific primers for glycoprotein hormone α subunit or LH β subunit. The α subunit was cloned into the double-expression vector pIRES (Invitrogen). The tLH β subunit was cloned into the second site of pIRES and also into the plasmid pGS. Chinese hamster ovary (CHO) K1 and human granulosa cell tumor (COV 434) cells were transfected with plasmid DNA by calcium-phosphate precipitation: (1) pIRES containing α and pGS containing LH β, or (2) pIRES containing α and LH β. Cells were grown in selection media (250 µg mL^–1 geneticine for pIRES, or 25 µm methionine sulfoximine for pGS). Media was collected and clarified at 1500g for 30 min. An immature rat Leydig cell assay protocol (Bousfield et al. 2001 Biol. Reprod. 64, 136–147) detected biological activity (testosterone production) by RIA. Of 14 tLH constructs created, 1 wild-type construct (LH WTCHO8) had LH activity 3 times greater than any other. A domestic kitten Leydig cell assay was performed in order to assess comparative sensitivities and specificities. Domestic kitten testicles, obtained from a local spay clinic, were disassociated with collagenase (225 U mg^–1, Worthington Biochemical, Lakewood, NJ); however, the cells were more difficult to disperse than rat testicles, leading to low Leydig cell yields as determined histologically. Modification of the Leydig cell collagenase protocol for the cat was achieved by increasing the temperature and surface area, and agitating the minced tissue in medium on a stir plate. Samples of the 14 tLH constructs were run in parallel using rat and cat Leydig cell assays. Although rat Leydig cell testosterone concentrations (3.4 ng mL^–1) were nearly 10-fold greater, the same trend for the different constructs was found in the cat Leydig cells with the same wild-type construct (LH WTCHO8, 0.37 ng mL^–1) having greater LH activity than any other. The lower testosterone concentrations in the cat bioassay may be explained by insufficient Leydig cell numbers, age (sensitivity), damaging effect(s) of collagenase, or felid specificity. Still, these results lend validity to the use of the heterologous rat Leydig cell bioassay for recombinant tiger LH.