Impaired steroidogenesis and implantation failure in Bmal1-/- mice

Christine K Ratajczak, Katie L Boehle, Louis J Muglia, Christine K Ratajczak, Katie L Boehle, Louis J Muglia

Abstract

Evidence in humans and rodents suggests that normal circadian rhythmicity is important for supporting reproductive function. A molecular clock underlies circadian rhythmicity. Impaired fertility is observed in some genetically altered mice with deficiencies in genes of the molecular clock, suggesting a critical role for these genes in reproduction. Here we systematically characterize the reproductive phenotype of females deficient in the clock gene Bmal1. Bmal1(-/-) females are infertile. They exhibit progression through the estrous cycle, although these cycles are prolonged. Normal follicular development occurs in Bmal1(-/-) females, and healthy embryos of the expected developmental stage are found in the reproductive tract of Bmal1(-/-) females 3.5 d after mating to wild-type males. However, serum progesterone levels are significantly lower in Bmal1(-/-) vs. Bmal1(+/+/-) females on d 3.5 of gestation. Low progesterone levels in Bmal1(-/-) females are accompanied by decreased expression of steroidogenic acute regulatory protein in corpora lutea of Bmal1(-/-) vs. Bmal1(+/+/-) females. Whereas implantation of embryos is not observed in untreated or vehicle-treated Bmal1(-/-) females, exogenous administration of progesterone to Bmal1(-/-) females is able to reinstitute implantation. These data suggest that implantation failure due to impaired steroidogenesis causes infertility of Bmal1(-/-) females.

Figures

F ig . 1.
Fig. 1.
Prolonged estrous cycles in Bmal1−/− females. Estrous cycle was assessed by histological analysis of vaginal smears. Progression through the stages of the estrous cycle in representative Bmal1+/+ (A) and Bmal1−/− (B) females is shown. Mean cycle length (C) and proportion of time spent in each estrous stage (D) (n = 5 for each group) is also shown. Values are means ± sem. *, P < 0.001.
F ig . 2.
Fig. 2.
Normal follicular development in Bmal1−/− females. Follicles were counted on sections of hematoxylin and eosin-stained ovary. A, Normal preantral follicles (indicated by arrows), antral follicles (indicated by arrows), and corpora lutea inBmal1+/± and Bmal1−/− ovaries. Black bars, 50 μm. B, Abundance of follicles in different stages of development. Values are means ± sem (n = 4,Bmal1+/±; n = 3, Bmal1−/−). PF, Primordial and primary follicles; PrF, preantral follicles; APrF, atretic preantral follicles; AnF, antral follicles; AAnF, atretic antral follicles; CL, corpora lutea.
F ig . 3.
Fig. 3.
Healthy preimplantation embryos but low serum progesterone levels inBmal1−/− females at d 3.5 of gestation. Oocytes and embryos were flushed from the reproductive tract at d 3.5 of gestation and analyzed under a light microscope. Serum progesterone and estradiol at d 3.5 of gestation were assayed by solid-phase 125I-RIA. A, Average number of oocytes/embryos isolated from each gravid female. Values are means ± sem. Numbers in bars indicate the number of females with oocytes or embryos/total number of females analyzed. B, Percentage of embryos isolated identified as healthy, unfertilized, and degenerating. C, Percentage of healthy embryos isolated in the blastocyst or morula stage. D, Serum progesterone at ZT 4 on d 3.5 (n = 14,Bmal1+/±; n = 15, Bmal1−/−). Values are means ± sem. *, P < 0.0001. E, Circadian serum progesterone throughout d 3.5 (n = 3, Bmal1+/±; n = 3–4,Bmal1−/− per time point). Values are means ± sem (P < 0.0001 with respect to genotype). F, Serum estradiol (n = 12, Bmal1+/±; n = 15,Bmal1−/−). Values are means ± sem.
F ig . 4.
Fig. 4.
Bmal1 and StAR are expressed in corpora lutea of d 3.5 Bmal1+/± females. Ovary sections were subjected toin situ hybridization with radiolabeled antisenseBmal1 or StAR probes and then hematoxylin and eosin stained for histological analysis. A, Autoradiograph from in situ hybridization for Bmal1 at ZT 4 d 3.5. B, Hematoxylin and eosin staining of the same section. Arrows point to corpora lutea. Black bar, 200 μm. C and E, Representative autoradiographs from in situ hybridization for StAR at ZT 4 d 3.5. D and F, Hematoxylin and eosin staining of the same sections. Arrowspoint to corpora lutea. Black bars, 200 μm (n = 3,Bmal1+/±; n = 3, Bmal1−/−). G, Circadian StAR expression throughout d 3.5. Values are means ± sem (n = 3, Bmal1+/±; n = 3–7,Bmal1−/− per time point). +, No corpora lutea observed in any mice examined at this time point (P < 0.0001 with respect to genotype).
F ig . 5.
Fig. 5.
Implantation sites found in progesterone-supplementedBmal1−/− females are small but histologically normal. Paraffin sections of implantation sites were hematoxylin and eosin stained. Diameter of whole implantation sites was measured under a light microscope. Representative implantation sites from untreated Bmal1+/± females (A), vehicle-treated Bmal1+/± females (B), progesterone-treatedBmal1+/± females (C), and progesterone-treatedBmal1−/− females (D) are shown. Black bar, 1 mm. Em, Embryo; Pl, placenta; FM, fetal membranes; Ut, uterus. E, Implantation site diameter (n = 3 implantation sites from each of three to four females per group). *, P < 0.001 compared with each of the other groups. P4, Progesterone.

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