Unexpected Plasma Gonadal Steroid and Prolactin Levels Across the Mouse Estrous Cycle

Abstract

Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (±SEM) concentration of 11 ± 3 ng/mL and occurred coincident with the peak in progesterone levels (22 ± 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 ± 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 ± 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 ± 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.

Keywords: estradiol; luteinizing hormone; progesterone; prolactin.

Figure 1.

Vaginal cytology of estrous stage smears. A, estrus is represented by cornified epithelial cells; B, metestrus is represented by the presence of cornified epithelial cells and leukocytes; C, early diestrus is represented by a dense appearance of leukocytes; D, late diestrus is represented by a scattered appearance of leukocytes and a small number of nucleated epithelial cells; and E, proestrus is represented by nucleated epithelial cells and often a small number of cornified epithelial cells. Scale bar represents 20 μm.

Figure 2.

Levels of gonadal and productive hormones across the mouse estrous cycle measured using liquid chromatography–mass spectrometry. Estradiol (E2) levels on diestrus 10 Am are statistically significant compared to all other time points except metestrus 10 Am and proestrus 6 Pm. Progesterone (P4) on proestrus 6 Pm is statistically significant compared to all other time points. Luteinizing hormone (LH) on proestrus 6 Pm is statistically significant compared to all other data points. Prolactin (Prl) on proestrus 2 Am is statistically significant compared to all other time points except proestrus 6 Pm. N = 4-11/time point (see text). Gray shaded area represents lights off. Mean ± SEM. Analysis of variance with post hoc Tukey; *P less than .05.

Figure 3.

Levels of A, estrone, and B, androstenedione across the mouse estrous cycle measured using liquid chromatography–mass spectrometry. The gray dotted line represents the LOD/√2. N = 4-11/group. Mean ± SEM. LOD, limit of detection.