The naturally occurring luteinizing hormone surge is diminished in mice lacking estrogen receptor Beta in the ovary

Abstract

Female ESR2-null mice (betaERKO) display defects in ovarian function and are subfertile. Follicular maturation is impaired and explains smaller litters, but betaERKO also produce fewer litters, which may be partially due to inadequate ovulatory signals. To test this, the amplitude and timing of the naturally occurring luteinizing hormone (LH) surge was measured in individual intact betaERKO and wild-type (WT) mice. Vaginal cytology was evaluated daily, and blood samples were taken from mice in proestrus. The amplitude of the LH surge was severely blunted in betaERKO mice compared to WT, but pituitary LH levels revealed no differences. The betaERKO mice did not produce a preovulatory estradiol surge. To determine if the smaller LH surges and the reduced number of litters in betaERKO were due to the lack of ESR2 in the hypothalamic-pituitary axis or due to the absence of ESR2 in the ovary, ovaries were transplanted from WT into betaERKO mice and vice versa. The size of the LH surge was reduced only in mice lacking ESR2 within the ovary, and these mice had fewer litters. Fertility and size of the LH surge were rescued in betaERKO mice receiving a WT ovary. These data provide the first experimental evidence that the LH surge is impaired in betaERKO females and may contribute to their reduced fertility. ESR2 is not necessary within the pituitary and hypothalamus for the generation of a normal LH surge and for normal fertility, but ESR2 is essential within the ovary to provide proper signals.

Keywords: ESR2; HPO axis; estrogen receptor beta; fertility; naturally occurring luteinizing hormone surge; ovarian transplants; positive feedback.

FIG. 1

βERKO mice spend more time in E and MET but less time in DI compared to WT mice. Cyclicity in WT and βERKO mice as determined by daily vaginal smears. A) Vaginal smears were taken for 45–50 consecutive days and scored to be either PRO, E, MET, or DI. Representative profiles of one WT and one βERKO animal are shown. B) Percentage of time spent in each stage of the cycle (mean ± SEM). Significant P-values between WT and βERKO mice in each stage are shown. Genotypes were compared using two-sample t-tests; P-values are two sided. PRO, proestrus; MET, metestrus; DI, diestrus; E, estrus.

FIG. 2

Timing and shape of the LH surge are similar between WT and βERKO mice. Consecutive LH surge profiles are shown from one representative WT and one βERKO mouse. Each animal was sampled during proestrus of three different cycles during the 50-day experimental period. Each LH surge profile is defined by three LH values measured in blood samples taken at D − 2 h, D, and D + 2 h. An LH surge threshold was defined as >0.43 ng/ml (represented by the gray line).

FIG. 3

Maximum LH measured in βERKO mice is significantly decreased relative to WT mice. Maximum LH values of LH surges detected in blood samples from WT and βERKO mice in the evening of proestrus. The solid bars represents the mean ± SEM, while the individual markers represent the distribution of all values (WT, n = 51; βERKO, n = 24). The distribution of WT maximum LH into quartiles is indicated by the horizontal dotted lines, and the percentile distributions of maximum LH values for βERKO within those quartiles are lowest quartile (58.3%), second quartile (29.2%), third quartile (12.5%), and fourth quartile (0%). Quartile distributions were analyzed by chi-square analysis. *P < 0.01. Maximum LH was compared using Mann-Whitney test; P-values are two sided.

FIG. 4

LH pituitary content is not different between WT and βERKO mice. LH content in pituitaries collected from intact cycling mice (mean ± SEM) 5 h before dark on the day of proestrus. Pituitaries from βERKO mice contained similar amounts of LH as WT. P > 0.1 by t-tests; P-values are two sided. n = 8 per group.

FIG. 5

Systemic estradiol (E2) does not increase in βERKO mice during DI. E2 concentrations in peripheral blood samples collected during all stages of the estrous cycle at different times before dark (D) as indicated on the x-axis. ** P < 0.01 compared to WT by t-tests; P-values are two sided. Sample sizes: MET (WT, n = 9; βERKO, n = 6), DI D − 5 h (WT, n = 8; βERKO, n = 8), PRO D − 12 h (WT, n = 7; βERKO, n = 6), PRO D − 9 h (WT, n = 9; βERKO, n = 7), PRO D − 5 h (WT, n = 8; βERKO, n = 8), E D− 12 h (WT, n = 7; βERKO, n = 4).

FIG. 6

Estradiol (E2) supplementation does not rescue the LH surge in βERKO mice. Maximum serum LH concentrations (mean ± SEM) during the LH surge in animals injected with oil vehicle or estradiol (10 μg/kg BW) on the afternoon of DI. Sample sizes: WT, n = 6 per group; βERKO oil, n = 5; and βERKO E2, n = 4. P > 0.1, two-way ANOVA with Bonferroni posttest.

FIG. 7

The serum LH level increases to WT levels when a WT ovary is transplanted into a βERKO host. Maximum serum LH concentrations (mean ± SEM) during the LH surge in animals with ovarian transplants. *P < 0.03; one-way ANOVA with the Dunnett multiple comparison posttest. Sample sizes: WT no surgery, n = 6; βERKO no surgery, n = 13; WT in WT, n = 4; WT in βERKO, n = 5; and βERKO in WT, n = 5.