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. 2015 Jul;93(1):16.
doi: 10.1095/biolreprod.115.129072. Epub 2015 Jun 3.

Infertility in Female Mice With a Gain-of-Function Mutation in the Luteinizing Hormone Receptor Is Due to Irregular Estrous Cyclicity, Anovulation, Hormonal Alterations, and Polycystic Ovaries

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Free PMC article

Infertility in Female Mice With a Gain-of-Function Mutation in the Luteinizing Hormone Receptor Is Due to Irregular Estrous Cyclicity, Anovulation, Hormonal Alterations, and Polycystic Ovaries

Lan Hai et al. Biol Reprod. .
Free PMC article

Abstract

The luteinizing hormone receptor, LHCGR, is essential for fertility in males and females, and genetic mutations in the receptor have been identified that result in developmental and reproductive defects. We have previously generated and characterized a mouse model (KiLHR(D582G)) for familial male-limited precocious puberty caused by an activating mutation in the receptor. We demonstrated that the phenotype of the KiLHR(D582G) male mice is an accurate phenocopy of male patients with activating LHCGR mutations. In this study, we observed that unlike women with activating LHCGR mutations who are normal, female KiLHR(D582G) mice are infertile. Mice exhibit irregular estrous cyclicity, anovulation, and precocious puberty. A temporal study from 2-24 wk of age indicated elevated levels of progesterone, androstenedione, testosterone, and estradiol and upregulation of several steroidogenic enzyme genes. Ovaries of KiLHR(D582G) mice exhibited significant pathology with the development of large hemorrhagic cysts as early as 3 wk of age, extensive stromal cell hyperplasia and hypertrophy with luteinization, numerous atretic follicles, and granulosa cell tumors. Ovulation could not be rescued by the addition of exogenous gonadotropins. The body weights of the KiLHR(D582G) mice were higher than wild-type counterparts, but there was no increase in the body fat composition or metabolic abnormalities such as impaired glucose tolerance and insulin resistance. These studies demonstrate that activating LHCGR mutations do not produce the same phenotype in female mice as in humans and clearly illustrate species differences in the expression and regulation of LHCGR in the ovary, but not in the testis.

Keywords: activating mutations; granulosa cell tumor; infertility; luteinizing hormone receptor; ovarian cysts; precocious puberty.

Figures

FIG. 1
FIG. 1
Irregular estrous cyclicity in KiLHRD582G mice. Representative examples of estrous cycle of (A) WT (n = 9) and (B, C) KiLHRD582G (n = 8) mice. D, diestrus; P, proestrus; E, estrus; M, metestrus; O, other.
FIG. 2
FIG. 2
Enlarged reproductive tract in KiLHRD582G mice. A) Gross morphology of the reproductive tracts of 6-wk-old WT and KiLHRD582G mice showing the enlarged ovaries and uterus of KiLHRD582G mice. B) Ovaries from 24-wk-old mice illustrating the grossly enlarged ovaries with large cysts in KiLHRD582G mice compared to WT mice. The two ovaries are from the same KiLHRD582G mouse. C, D) Ovary and uterine weights corrected for body weight from 2- to 24-wk-old mice. Data are mean ± SEM (n = 7–32). E, F) Representative photomicrographs of H&E stained uterine cross-sections from WT (E) and KiLHRD582G (F) mice at 3 wk of age. Note the enlarged lumen and attenuated stroma in the KiLHRD582G uterus. Bars = 100 μm. ***P < 0.001 compared to age-matched WT mice.
FIG. 3
FIG. 3
Elevated steroid hormones and decreased gonadotropin levels in KiLHRD582G mice. Progesterone, androstenedione, testosterone, and estradiol levels were measured in the serum of mice from 2 to 24 wk of age by enzyme immunoassay (n = 5–8). Serum levels of LH and FSH were measured by radioimmunoassay (n = 6). Levels of LH and FSH were below the limits of detection in KiLHRD582G mice at all ages, except at 2 wk for FSH, and were assigned the value of the lowest standard (0.08 ng/ml for LH and 1.8 ng/ml for FSH). At 6 and 12 wk of age, the LH levels in some of the WT mice were below the level of detection because randomly cycling mice were used. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to age-matched WT mice.
FIG. 4
FIG. 4
Upregulation of several steroidogenic enzyme genes in KiLHRD582G ovaries. Relative quantity represents expression normalized to the internal control Rpl19 and relative to the calibrator sample prepared by mixing equal amounts of cDNA from all the samples. Data are expressed as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared to age-matched WT mice.
FIG. 5
FIG. 5
Ovarian pathology in KiLHRD582G mice. Representative photomicrographs of H&E stained ovary sections of WT and KiLHRD582G mice. At least three animals per age and genotype were examined. Higher magnification of the boxed areas in sections of KiLHRD582G mice at 4, 6, 12, and 24 wk are shown in the last column. C, hemorrhagic cyst, GCT, granulosa cell tumor, AF, atretic follicle. Arrow indicates tubulostromal hyperplasia. Bars = 100 μm.
FIG. 6
FIG. 6
Stromal cell luteinization and granulosa cell tumors in KiLHRD582G mice. Representative photomicrographs of immunohistochemical analysis of LHCGR, StAR, HSD17B7, and FOXL2 expression in 24-wk-old mice. At least three animals of each genotype were analyzed with each antibody. Insets represent nonimmune serum controls. CL, corpus luteum. Bars = 100 μm.
FIG. 7
FIG. 7
Superovulation cannot rescue the anovulatory phenotype. WT and KiLHRD582G mice at 3 wk of age were primed with eCG and then treated with an ovulatory dose of hCG for 24 h. A) Number of oocytes ovulated per WT (n = 3) and KiLHRD582G mouse (n = 5). Representative photomicrographs of H&E stained ovarian sections from superovulated WT (B) and KiLHRD582G mice (C). Data are expressed as mean ± SEM. CL, corpus luteum. Arrows indicate nonovulated oocytes. Bars = 100 μm.
FIG. 8
FIG. 8
Normal glucose tolerance in KiLHRD582G mice. A) Body weights from 2 to 24 wk of age (n = 9∼18). B) Glucose tolerance test of WT andKiLHRD582G mice at 16 wk of age (n = 7–9). Glucose was measured in tail blood samples at 0, 15, 30, 45, 60, and 120 min after glucose was administered intraperitoneally. C) The area under the curve (AUC) was calculated by trapezoid analysis. D) Plasma insulin was measured from blood samples drawn at 0 and 15 min after glucose administration (n = 7). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT.

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