Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines.

Abstract

To establish in vivo gonadal tumor models and permanent lines of gonadal somatic cells we produced transgenic (TG) mice expressing the Simian virus (SV) 40 T-antigens (T-ag), driven by 6 or 2.1 kilobase fragments of the mouse inhibin alpha-subunit promoter. Hitherto, altogether 44 TG mice, one of which carried the shorter transgene, have produced gonadal tumors. Two founder females expressing the longer transgene, KK1 and KK3, and three established TG mouse lines were studied in detail. Penetrance of the phenotype in IT6-M and IT6-F mouse lines was 100% (tumors/TG: IT6-M 22/22, IT6-F 14/14). The T-ag mRNA was strongly expressed in the gonads, adrenal glands, pituitary, and brain. The KK-1 and KK-3 ovarian tumor cells immunostained with anti-SV40 large-T antibody. The KK-1 cells possessed high-affinity LH receptors [equilibrium association constant (Ka = 7.8 x 10(10) liters/mol] and responded to human CG by elevated cAMP and progesterone production. Also FSH slightly stimulated their cAMP and estradiol production (P < 0.01). These cells expressed cytochrome P450arom and inhibin alpha mRNA, but not cytochrome P450c17 alpha. In conclusion, the KK-1 cells are immortalized luteinizing granulosa cells expressing endogenous gonadotropin receptors, steroidogenic enzymes, and inhibin alpha. These cells will be useful in studies on the molecular aspects of granulosa cell function. The present study indicates that the 6-kilobase fragment of the inhibin alpha promoter described in this article contains the elements directing tissue-specific expression in vivo and is useful for targeted expression of other genes in the gonads.

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

“…As shown previously (Kananen et al, 1995(Kananen et al, , 1996a, these mice develop gonadal tumors originating from Leydig and granulosa cells, with 100% penetrance, at the age of 5-6 months. To find out whether the extragonadal tumors frequently found in these mice were primary or because of metastases of gonadal tumors, we gonadectomized neonatal mice (Kananen et al, 1996b).…”

“…The weights of the ovaries of double TG and bLHb-CTP mice showed no significant difference at 3 months, but the former were markedly larger at the age of 5 months (Figure 1a). At the age of 3 months double TG mice presented with highly proliferating tumors with mitotic figures (Figure 1b), akin to those seen in Inha/Tag mice at an older age and shown to be of granulosa cell origin (Kananen et al, 1995), and they had luteinized follicles, as found in bLHb-CTP mice (Risma et al, 1995) ( Figure 1b). The granulosa cell origin of these tumors was reconfirmed by aromatase-specific immunostaining (Figure 3b).…”

“…We have previously shown that tumorigenesis in Inha/ Tag mice is gonadotropin dependent (Kananen et al, 1995(Kananen et al, , 1996a(Kananen et al, , b, 1997, but it has not been clear whether LH or FSH is mainly responsible for the tumorigenesis.…”

Induction of protein growth factor systems in the ovaries of transgenic mice overexpressing human type 2 lysophosphatidic acid G protein-coupled receptor (LPA2)

“…In contrast, only a background level of staining is seen in the stromal region of non-TG ovaries (Figure 4). The human mutant LPA 2 (Edg-4) TG mice were expected to have high levels of LPA 2 expression in the sex cordstromal compartment of their ovaries based on the known targeting properties of mouse a-inhibin large promoter (Kananen et al, 1995;Hsu et al, 1996). There are also consistently many more prominent nodules composed of luteinized cells observed in the TG ovaries than in age-matched wild-type ovaries.…”

“…Human LPA 2 (Edg-4) TG mice were generated by injecting C57BL/6 oocytes with a minigene consisting of a 1053 bp cDNA encoding human mutant LPA 2 , with a C-terminal cytoplasmic tail 35 amino acids longer than that of the wildtype LPA 2 , coupled to the XbaI site of the 6 Kb a-subunit of the mouse inhibin promoter (Kananen et al, 1995;Hsu et al, 1996). Human LPA 2 TG mice were identified by slot blot and PCR analyses of tail-tissue genomic DNA (Laird et al, 1991).…”