The Luteinizing Hormone Receptor Knockout Mouse as a Tool to Probe the In Vivo Actions of Gonadotropic Hormones/Receptors in Females

Abstract

Mouse models with altered gonadotropin functions have provided invaluable insight into the functions of these hormones/receptors. Here we describe the repurposing of the infertile and hypogonadal luteinizing hormone receptor (LHR) knockout mouse model (LuRKO), to address outstanding questions in reproductive physiology. Using crossbreeding strategies and physiological and histological analyses, we first addressed the physiological relevance of forced LHR homomerization in female mice using BAC expression of 2 ligand-binding and signaling deficient mutant LHR, respectively, that have previously shown to undergo functional complementation and rescue the hypogonadal phenotype of male LuRKO mice. In female LuRKO mice, coexpression of signaling and binding deficient LHR mutants failed to rescue the hypogonadal and anovulatory phenotype. This was apparently due to the low-level expression of the 2 mutant LHR and potential lack of luteinizing hormone (LH)/LHR-dependent pleiotropic signaling that has previously been shown at high receptor densities to be essential for ovulation. Next, we utilized a mouse model overexpressing human chorionic gonadotropin (hCG) with increased circulating "LH/hCG"-like bioactivity to ~40 fold higher than WT females, to determine if high circulating hCG in the LuRKO background could reveal putative LHR-independent actions. No effects were found, thus, suggesting that LH/hCG mediate their gonadal and non-gonadal effects solely via LHR. Finally, targeted expression of a constitutively active follicle stimulating hormone receptor (FSHR) progressed antral follicles to preovulatory follicles and displayed phenotypic markers of enhanced estrogenic activity but failed to induce ovulation in LuRKO mice. This study highlights the critical importance and precise control of functional LHR and FSHR for mediating ovarian functions and of the potential repurposing of existing genetically modified mouse models in answering outstanding questions in reproductive physiology.

Keywords: G protein-coupled receptors; follicle-stimulating hormone; gonadotropin hormones; luteinizing hormone; reproduction.

Figure 1.

LHR functional complementation has no effect on the hypogonadal phenotype of female LuRKO animals due to low LHR^B–/LHR^S– expression. (A) Schematic detailing LHR functional complementation and forced homomerization using LHR^B– and LHR^S– mutant receptors. The effects of LHR^B– and LHR^S– BACs co-expressed in a LuRKO background on (B) day of vaginal opening. Statistical significance determined by 1-way ANOVA, a versus b = P < .001, WT versus LuRKO animals, with no significant difference between LuRKO, LHR^B–, LHR^S– and LHR^B–/LHR^S– groups (n = 4 for LuRKO, n = 5 for WT, LHR^S– and LHR^S–/LHR^B–, n = 6 for LHR^B–). (C) Representative ovarian histology in 3-month-old control WT, LuRKO alone, or a LuRKO background expressing LHR^B–/LHR^S– female littermates. *Presence of corpora lutea, arrows indicate failure of cumulus-oocyte-complex expansion. (D) Analysis of serum LH from WT, LuRKO, LURKO/LHR^B–, LuRKO/LHR^B– and LuRKO coexpressing LHR^B–/LHR^S–. Statistical significance was determined by 1-way ANOVA with Dunnett’s multiple comparisons to the control and between groups. Alphabetic denotation a versus b = P < .0001, WT versus LuRKO animals, with no significant difference between LuRKO, LHR^B–, LHR^S– and LHR^B–/LHR^S– groups (n = 6 animals per group). Relative transcript levels of (E) P450scc and (F) Lhr from ovarian extracts of female WT, LuRKO, LuRKO/LHR^B–, LuRKO/LHR^S– and LuRKO/LHR^B–/LHR^S– (n = 8 for LuRKO, LHR^B– and LHR^S–; n = 10 for WT; and n = 12 for LHR^B–/LHR^S–). (G) relative transcript levels of Lhr^B– and Lhr^S– in single female mice co-expressing Lhr^B–/Lhr^S–. Statistical significance in (E) was determined by 1-way ANOVA with Dunnett’s multiple comparisons to the control and between groups. Alphabetic denotation a versus b = P < .0001, WT versus LuRKO animals, with no significant difference between LuRKO, LHR^B–, LHR^S– and LHR^B–/LHR^S– groups.

Figure 2.

Enhanced “LH-like” activity via hCG fails to rescue the hypogonadal phenotype of female LuRKO mice. The effects of hCG overexpression in the absence of LHR on key reproductive parameters as assessed by (A) day of vaginal opening checked daily from d21 weaning and (B) Uterine weights in WT, LuRKO, hCG beta (β) and LuRKO/hCG beta animals (LuRKO/β) females. Statistical analysis via 1-way ANOVA with Dunnett’s multiple comparisons to the control and between groups. N Differential letter denotation equaled statistical significance between experimental groups, with a vs b = P < .0001, a vs c = P < .001, and b vs c = P < .0001. n = 5 for WT, LuRKO, and hCG beta groups, and n = 4 for LuRKO/hCG beta mice. (C) Ovarian weights showing hypogonadal phenotype of LuRKO/hCG beta females, 1-way ANOVA with Dunnett’s multiple comparisons analysis was conducted comparing between all groups. Differential letter denotation equaled statistical significance between experimental groups, with a versus b = P < .05. n = 5 for WT, LuRKO, and hCG beta groups, and n = 4 for LuRKO/hCG beta mice. (D) histological analysis of ovarian sections, with representative ovarian sections taken from the central part of the ovary with multiple large antral follicles displayed. The ovaries of LuRKO/hCG beta mice were comparable to LuRKO animals, with follicles arrested at the large antral phase (enlarged image inset) lacking cumulus oocyte complex expansion, and absence of corpora lutea (*example corpus luteum in WT animals). For the hCG beta mice, a hemorrhagic cyst, typical for mice with hCG beta expression, was also observed (arrowhead). Scale bars = 200 µm, with inset 400 µm. WT included Lhr^+/– females. Representative from n = 4 for all experimental groups. (E) Relative expression of Cyp19a1. Statistical analysis via One-way ANOVA with Dunnett’s multiple comparisons analysis, with a versus b, P < .05. n = 4 for all experimental groups (F) Representative examples of estrous cycles of each experimental group. Staging was Met, metestrous; Di, diestrous; Pro, proestrous; Estr, estrous. Transition stages have been marked between the 2 stages. n = 4 for all experimental groups.

Figure 3.

Constitutive activation of FSHR in the absence of LHR partially restores ovarian function and mammary gland development. Key reproductive parameters were assessed via (A) day of vaginal opening and (B) uterine weights in WT, LuRKO /CAM FSHR, CAM FSHR, and LuRKO females. One-way ANOVA conducted using log conversion of uterine weights. a versus b = P < .01. For WT, LuRKO and LuRKO/CAM-FSHR groups, n = 3, for CAM-FSHR group n = 4. (C) Representative images of the mammary gland tissue, internal reproductive tracts and ovaries. Histological characterization of mammary gland wholemounts (upper panel) with a reference point lymph node depicted by yellow arrow toward which ductal elongation occurs demonstrating the rescue of elongation in LuRKO/CAM FSHR female mice, versus rudimentary development in LuRKO females. Macroscopic images (middle panel) showing LuRKO females have thin threadlike uteri, while uteri of LuRKO/CAM FSHR mice resemble that of WT littermates. CAM FSHR expression typically results in hemorrhagic cysts in WT and also in LuRKO background (arrowheads in inserts, the backgrounds has been brightened for clarity). Representative ovarian images (lower panel) from central ovarian cross-sections, with the most advanced follicles in the sample presented. The ovaries of CAM FSHR mice were usually larger than those of WT mice and contain multiple developing follicles and luteinizing follicles (*). The ovaries of LuRKO/CAM FSHR mice have more advanced follicles (an arrow in the inset) than LuRKO mice but lack corpora lutea. A hemorrhagic cyst is marked (arrowhead). Scale bars: the uppermost row 1 mm; middle row 10 mm and inserts 50 mm: lowermost row 500 µm and inserts 250 µm. WT included Lhr^+/– females and CAM FSHR in WT and Lhr^+/– backgrounds. (D) Representative examples of estrous cycles. Met, metestrous; Di, diestrous; Pro, proestrous; Estr, estrous. Transition stages have been marked between the 2 stages. CAM FSHR females demonstrated variable cycles from seminormal to acyclic, in line with our previous publication (18), of which 1 has been shown here. For smear analyses WT, n = 8; LuRKO, n = 4; CAM-FSHR, n = 13; LuRKO/CAM-FSHR, n = 6.