Precocious Puberty, Elevated Steroidogenesis, and Leydig Cell Hyperplasia in Male Mice with a Gain of Function Mutation in the Luteinizing Hormone Receptor Gene.

Abstract

The activation of the luteinizing hormone receptor (LHR) in the gonads by luteinizing hormone (LH) is critical for steroidogenesis and gametogenesis. Several activating mutations in human LHR have been identified in boys with sporadic or familial male limited precocious puberty (FMPP) or testotoxicosis. Boys with this mutation present with elevated levels of testosterone in the context of prepubertal LH levels, the onset of puberty by age four or earlier and Leydig cell hyperplasia. The majority of the activating mutations are in transmembrane helix 6 of the 7 transmembrane G protein-coupled receptor, the most common being a mutation of aspartic acid 578 to glycine. We have created a knock-in mouse (KiLHR) with an activating point mutation, caused by substitution of glycine for aspartic acid at position 582 (D582G) in mouse LHR, corresponding to the common D578G mutation in human LHR. In this study, we have examined the effects of the mutated LHR on reproductive and metabolic function in male KiLHR mice. RT-PCR analysis of RNA from multiple tissues of heterozygous KiLHR mice demonstrated expression of the WT and mutant alleles only in the gonads and brain. Male KiLHR mice exhibited precocious puberty as evidenced by premature balanopreputial separation at postnatal day 14.5 compared to 22.4 days for WT mice and presence of major urinary proteins (MUPs), which are secreted at the onset of puberty, in KiLHR mice at postnatal day 15 compared to day 22 in WT mice. KiLHR mice were fertile. Male KiLHR mice were analyzed at neonatal (7 and10d), prepubertal (2 and 3 weeks), pubertal (6 weeks) and adult (12 and 24 weeks) ages. Hormonal analysis indicated that the KiLHR mice had significantly elevated serum and testicular testosterone levels as early as postnatal day 7 and remained elevated through adulthood. No significant difference was seen in serum estradiol levels; however testicular estradiol levels were elevated in KiLHR mice compared to WT mice. Gonadotropin levels were decreased in KiLHR compared to WT mice. Expression of the steroidogenic enzyme genes was upregulated as early as 7 days consistent with the elevated testosterone levels. The levels of LHR mRNA were significantly upregulated at all ages, but not those of the follicle stimulating hormone or steroid hormone receptors. Histological analysis revealed premature Leydig cell proliferation and the presence of Leydig cell hyperplasia as early as 7 days of age. Gene expression analysis suggested the precocious development of adult Leydig cells. At 24 weeks, Leydig cell adenomas were apparent is some of the KiLHR mice. However, there were no apparent differences in testicular germ cell or Sertoli cell development. KiLHR mice at 24 weeks of age exhibited impaired glucose tolerance compared to WT mice although there was no difference in fasting glucose or insulin levels. No changes in body weight or body fat were measured between the genotypes. The overall phenotype of precocious puberty, elevated testosterone and Leydig cell hyperplasia in the KiLHR male mice is similar to that seen in humans with FMPP. These studies suggest that the KiLHR mice will be a novel model to understand the effects of premature LHR activation during early Leydig cell development as well as the consequences of chronic elevated testosterone levels on adult testicular and metabolic function. Supported by NIH HD044119.