Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors.

Abstract

Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We (7)], it is difficult to devise protocols for chronic administration of exogenous LH that mimic endogenous pulse patterns of LH. To circumvent this limitation, we report a transgenic mouse model in which elevated hormone levels are maintained chronically, without requiring multiple injections,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. supraphysiologic dosing, or dampening of the hypothalamicpituitary-gonadal axis.We utilized a two-pronged approach to achieve elevated levels of serum LH. (i) Increased secretion of hormone from the pituitary was achieved by expression of an LH(3 subunit transgene, targeted to the pituitary by a previously characterized bovine a-subunit promoter (8,9). (ii) The LH,B transgene was modified to encode a peptide extension that we proposed would slow the elimination of LH from the serum. This peptide is normally found at the C terminus of the 3 subunit of human chorionic gonadotropin (hCG) (hCG,B) and is referred to as the C-terminal peptide (CTP) (10). The CTP is thought to be a major determinant of the serum t,'2 of hCG and has been shown to increase the til2 of FSH 2-to 3-fold when fused to its (3 subunit (11). Accordingly, we constructed a transgene with the coding region of bovine (b) LH,B fused in-frame to the coding region of CTP (bLH3-CTP). MATERIALS AND METHODSConstruction of the bLH3-CTP Transgene. The bLH,B-CTP minigene was engineered by a two-step PCR. (i) A short fragment containing the C terminus of bLH,B (30 bp) fused in-frame to the CTP (last 87 bp of the hCGj3 gene) was generated. (ii) This fragment was lengthened to contain the entire bLHf3 cDNA fused in-frame to the CTP. This fusion gene was utilized for transfection experiments but was modified to contain the first intron of bLH3 for the transgene construct. The resulting insert was ligated into a BSK-plasmid already containing the bovine a-subunit promoter (positions -315 to +45) and the simian virus 40 late polyadenylylation signal. Transgenic mice were generated by microinjecting the bLH3-CTP insert into fertilized oocytes as described (12). Mice were genotyped by PCR analysis of tail DNA using primers specific to the a-subunit promoter and (3-subunit gene.Analysis of t,,2. Recombinant bLH and bLH-CTP heterodimers were generated by stably cotransfecting CHO cells with viral expression vectors containing the bovine a-subunit gene and either the bLH( or bLH,3-CTP genes as described (13). Serum-free medium was collected and ammonium sulfate-precipitated or concentrated with an Amicon ultrafiltration cell. Female rats were pretreated with 50 ,tg of antide (Sigma) in 20% (vol/vol) propylene glycol, injected subcutaneously 2 h prior to hormone injections t...

Extragonadal LH/hCG action—Not yet time to rewrite textbooks

“…Although there are some differences between ovarian steroidogenesis and ovarian tumor formation, many similarities exist between bLH-CTP overexpressing female mice and our mice, including the formation of pituitary and mammary gland tumors, obesity and infertility (Risma et al, 1995;Rulli et al, 2002;Kero et al, 2003;Mann et al, 2003;Mohammad et al, 2003). The ovarian tumor formation in bLHβ-CTP mice was found to be dependent on mouse strain (Keri et al, 2000), and we have not yet breed our mice to those strains susceptible to formation of ovarian tumors (Keri et al, 2000).…”

“…The male mice were infertile without elevated bioactive LH/hCG and without obvious testicular phenotype, and the authors speculate that high circulating hCGβ-subunit could bind to the LH/hCG-R and antagonize the effect of normal circulating LH, thus interfering with spermatogenesis in a quantitative manner (Matzuk et al, 2003). In the other TG mouse model, which expresses a fusion protein of the bovine LHβ subunit and the hCGβ C-repminal peptide (bLHβ-CTP) under common α-subunit promoter, males did not show elevated LH/hCG-bioactivity and presented with no apparent phenotype (Risma et al, 1995).…”

High levels of luteinizing hormone analog stimulate gonadal and adrenal tumorigenesis in mice transgenic for the mouse inhibin-α-subunit promoter/Simian virus 40 T-antigen fusion gene

“…Studies on experimental animals have also supported the gonadotropin theory. In inhibin-a-deficient mice, gonadotropins are essential for gonadal and adrenal tumorigenesis , and chronically elevated circulating levels of LH or human chorionic gonadotropin (hCG) can cause ovarian tumors in certain strains of mice (Risma et al, 1995;Rulli et al, 2002). Furthermore, in male rats, chronic treatment with LH produces Leydig cell hyperplasia (Neumann, 1991).…”

Clearing the way to implantation

“…Even a mild diminution of LH clearance would be predicted to affect the pulsatility of circulating LH and to lead to an increased concentration of the hormone during the interpulse period. The most extreme form of such a disruption, the complete elimination of LH pulsatility and the continuous exposure of the ovary to elevated circulating levels of the hormone, has been modeled by overexpressing LH in transgenic mice (6). The phenotype seen in these animals includes ovarian cyst formation, ovarian tumorigenesis, and sterility, markedly different effects from those reported by Mi and associates.…”

“…Structural analyses from Baenziger's group indicate that LH is modified by GalNAc-4-SO 4 , a specific and somewhat unusual glycan now known to bind the endothelial form of the Man/GalNAc-4-SO 4 receptor. They have suggested that binding of this receptor to the glycan-bearing hormone accounts for rapid clearance of LH, which is crucial to explain the pulsatile delivery of plasma LH (6). In their present article (1), this group has now explored the postulated role of the receptor in female reproduction.…”