PMC

Med12^fl/fl Amhr2-Cre uterine anatomy and histology. (A) Gross morphology of 12-week-old female mouse reproductive tract. The Med12^fl/fl (control mice) display normal reproductive tract. In comparison to the controls, Med12^fl/fl Amhr2-Cre reproductive tract is atrophic. Scale bar = 1cm. (B) Twelve-week-old uterine weight is significantly decreased in Med12^fl/fl Amhr2-Cre as compared to Med12^fl/fl and Med12^fl/+ Amhr2-Cre group (n = 7). Data are presented as mean ± SEM. ^**P < 0.01. (C–H) Hematoxylin and eosin staining of uteri from 12-week-old Med12^fl/fl, Med12^fl/+ Amhr2-Cre and Med12^fl/fl Amhr2-Cre mice synchronized with PMSG and hCG. Note the atrophic uteri of Med12^fl/fl Amhr2-Cre female. EM, endometrium; MY, myometrium. Scale bars = 100 μm.

Med12^fl/fl Amhr2-Cre female mice are infertile. (A) Breeding crosses used to generate Med12^fl/fl Amhr2-Cre and Med12^fl/y Amhr2-Cre mice for experimental studies. (B) Schematic representation of floxed Med12 allele. LoxP sites bracket exons 1–7 of the Med12 gene. In Amhr2-Cre transgenic mice, Amhr2 promoter drives Cre expression in uterine mesenchyme, oviduct, and granulosa cells of ovaries. (C) Fertility data of control (Med12^fl/fl) (n = 7), Med12^fl/+ Amhr2-cre (n = 13), and Med12^fl/fl Amhr2-cre (n = 7) female mice. The mice were bred with wild-type stud males for 6 months. (D) Mating success was evaluated by vaginal plug detection (n = 8). Data are presented as mean ± SEM. ^***P < 0.001 (C, D).

Estrous cycle is disrupted in Med12^fl/fl Amhr2-Cre mice. (A) Schematic representation of the estrous cycles in Med12^fl/fl and Med12^fl/fl Amhr2-Cre females. (B) Ages at first vaginal cornification and onset of first cyclicity are plotted against the genotype and shows a delay in Med12^fl/fl Amhr2-Cre versus Med12^fl/fl (control) mice (n = 5). (C) Number of estrous cycles counted over a 6-week period in Med12^fl/fl and Med12^fl/fl Amhr2-Cre females (n = 6) shows a significant decrease in Med12^fl/fl Amhr2-Cre females. (D) Days spent at each stage of the estrous cycle over a 6-week period plotted against each stage (n = 5). Stages are defined as P, proestrus; E, estrus; M, metestrus; D, diestrus. (E) Estradiol concentration was measured in the serum of 3-week-old Med12^fl/fl and Med12^fl/fl Amhr2-Cre females collected in mice without (–) and with PMSG treatment (+) after 48 h (n = 5). Data are represented as mean ± SEM. ^*P < 0.05 (B, E), ^**P < 0.01 (C, D), ^***P < 0.001 (E).

Oocyte-specific deletion of Med12 causes infertility without affecting folliculogenesis. (A) Breeding strategy used to assess the effects of Med12 deficiency on folliculogenesis with Gdf9-Cre that is active in oocytes of primordial (smallest) ovarian follicles, and Zp3-Cre that is active in oocytes of primary follicles. Gdf9-Cre introduced from the male side never produced Gdf9-Cre positive pups likely due to leaky Gdf9-Cre expression from the paternal side that inactivated Med12 floxed allele from the maternal side and caused embryonic lethality (D). (B, C) Periodic acid-Schiff staining of 12-week-old Med12^fl/fl and Med12^fl/fl Zp3-Cre ovaries generated in crosses described in (A). Note the presence of normal follicles at all stages as well as corpora luteua in both Med12^fl/fl and Med12^fl/fl Zp3-Cre mice. Med12^fl/fl Zp3-Cre mice are infertile. (D–G) Fertility results from various breeding strategies depicted in (A) are shown. Note lack of mortality when Zp3-Cre is transmitted from the male side (G) as opposed to Gdf9-Cre (D) (D, n = 7; E, n = 5; F, n = 4; G, n = 3). ^****P < 0.0001. Scale bars = 100 μm.

Med12^fl/fl Amhr2-Cre ovarian response to PMSG stimulation and gene expression. (A–H) Med12^fl/fl and Med12^fl/fl Amhr2-Cre mice were treated with PMSG for 48 h, and ovaries were sectioned and stained by periodic acid-Schiff. Representative histology is shown in panels (A–H), comparing unstimulated and PMSG stimulated 3-week-old ovaries. Significantly more pre-ovulatory follicles were present in the Med12^fl/fl control group after PMSG treatment as compared to Med12^fl/fl Amhr2-Cre group. AnF, antral follicles; Gr, granulosa cells; PreOv, pre-ovulatory follicles. Scale bars = 100 μm. (I) Quantification of pre-ovulatory follicles from 3-week-old Med12^fl/fl and Med12^fl/fl Amhr2-Cre females treated with or without PMSG for 48 h (n = 4). (J) Granulosa cells were isolated from PMSG-treated Med12^fl/fl and Med12^fl/fl Amhr2-cre ovaries (n = 3), and RNA was extracted for cDNA conversion and real-time quantitative polymerase chain reaction (RT-qPCR). Data were normalized to Gapdh expression and were given as the mean relative quantity (compared with control), with error bars representing the standard error of the mean. Student t-test was used to calculate P values. The only significant difference was noted in the expression of Med12, as expected, as well as upregulation of Lhcgr, and downregulation of Nr5a2, Esr1, and Esr2. Pooled data represent mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Exogeneous estradiol and progesterone cannot fully rescue the uterine hypoplasia in Med12^fl/fl Amhr2-Cre mice. (A, B) Serum estradiol (A) and progesterone (B) concentrations were measured in ovariectomized Med12^fl/fl and Med12^fl/fl Amhr2-Cre mice after the implantation of placebo, estradiol, or estradiol combined with progesterone pellets for 4 weeks (n = 4). (C) Uterine weights in ovariectomized Med12^fl/fl and Med12^fl/fl Amhr2-Cre mice implanted with placebo, estradiol, or estradiol combined with progesterone pellets for 4 weeks (n = 5). (D) Gross morphology of uteri from 8-week-old ovariectomized Med12^fl/fl and Med12^fl/fl Amhr2-Cre mice implanted with placebo and estradiol combined with progesterone pellets for 4 weeks. Note significant increase in uterine size after exposure to combined estradiol and progesterone pellets. (E) Hematoxylin & eosin staining of uteri from 8-week-old ovariectomized Med12^fl/fl (control) and Med12^fl/fl Amhr2-Cre mice implanted with, placebo, estradiol, or estradiol combined with progesterone pellets for 4 weeks. Zoomed panels are shown below and to the left of the displayed histology section. Placebo treatment had little effect on ovariectomized control and Med12^fl/fl Amhr2-Cre uteri, with atrophic layers of endometrium and myometrium. After the implantation of estradiol or combined estradiol/progesterone implants, control uteri recovered well-developed endometrial and myometrial compartments (dotted yellow line). Unlike controls, Med12^fl/fl Amhr2-Cre uteri did not show well-developed endometrial and myometrial layers when exposed to exogenous estradiol or combined estradiol/progesterone implants (dotted yellow line). Moreover, Med12^fl/fl Amhr2-Cre uteri exposed to estradiol and progesterone implants displayed abnormal histology consisting of intramyometrium glandular structures (dotted yellow line). (F–H) Real-time quantitative polymerase chain reaction (RT-qPCR) on RNA isolated from uteri exposed to placebo, estradiol only, or estradiol and progesterone pellets. Results are shown for Gper1, Notch1, and Smo transcripts (n = 3). Pooled data represent mean ± SEM. ^*P < 0.05 (A, F, G, H); ^**P < 0.01 (A, B, F, G); ^***P < 0.001 (G); ^****P < 0.0001 (C, F, H). MY, myometrium; EM, endometrium. Scale bars = 100 μm.