Loss of cannabinoid receptor CB1 induces preterm birth

Abstract

Background: Preterm birth accounting approximate 10% of pregnancies in women is a tremendous social, clinical and economic burden. However, its underlying causes remain largely unknown. Emerging evidence suggests that endocannabinoid signaling via cannabinoid receptor CB1 play critical roles in multiple early pregnancy events in both animals and humans. Since our previous studies demonstrated that loss of CB1 defers the normal implantation window in mice, we surmised that CB1 deficiency would influence parturition events.

Methods and findings: Exploiting mouse models with targeted deletion of Cnr1, Cnr2 and Ptgs1 encoding CB1, CB2 and cyclooxygenase-1, respectively, we examined consequences of CB1 or CB2 silencing on the onset of parturition. We observed that genetic or pharmacological inactivation of CB1, but not CB2, induced preterm labor in mice. Radioimmunoassay analysis of circulating levels of ovarian steroid hormones revealed that premature birth resulting from CB1 inactivation is correlated with altered progesterone/estrogen ratios prior to parturition. More strikingly, the phenotypic defects of prolonged pregnancy length and parturition failure in mice missing Ptgs1 were corrected by introducing CB1 deficiency into Ptgs1 null mice. In addition, loss of CB1 resulted in aberrant secretions of corticotrophin-releasing hormone and corticosterone during late gestation. The pathophysiological significance of this altered corticotrophin-releasing hormone-driven endocrine activity in the absence of CB1 was evident from our subsequent findings that a selective corticotrophin-releasing hormone antagonist was able to restore the normal parturition timing in Cnr1 deficient mice. In contrast, wild-type females receiving excessive levels of corticosterone induced preterm birth.

Conclusions: CB1 deficiency altering normal progesterone and estrogen levels induces preterm birth in mice. This defect is independent of prostaglandins produced by cyclooxygenase-1. Moreover, CB1 inactivation resulted in aberrant corticotrophin-releasing hormone and corticosterone activities prior to parturition, suggesting that CB1 regulates labor by interacting with the corticotrophin-releasing hormone-driven endocrine axis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Genetic or pharmacological silencing of CB1 induces preterm labor.

(A & B) Mice missing Cnr1, but not Cnr2, show early onset of labor with reduced fetal weights at birth. (C & D) Preterm labor occurs in wild-type (WT) pregnant mice receiving a CB1-selective antagonist SR141716 (SR1), but not a CB2-selective antagonist SR144528 (SR2), on days 14–18 with little effects on fetal weights. Numbers within bars indicate the number of mice examined in panels A and C. The average fetal weights (mg) at birth are shown in panels B and D. The bars with different letters are significantly different (P<0.01).

Figure 2. Expression of CB1 transcript and protein in the wild-type (WT) brain (A) and ovary (B) on day 18 of pregnancy.

Bar, 100 µm. 3V, third ventricle; Arc, arcuate nucleus of the hypothalamus; Cx, cortex; Hip, hippocampus; Subl, subincertal nucleus; VMH, ventromedial hypothalamic nucleus; CL, corpus luteum; f, follicle; o, oocyte.

Figure 3. CB1 deficiency alters normal progesterone (P4) and estradiol-17β (E2) levels prior to parturition in mice.

Serum P4 and E2 levels were analyzed by radioimmunoassay. While CB1 deficiency induced an early drop in serum P4 levels on day 19 (A), circulating E2 levels were elevated on days 16–18 (B), resulting in a remarkable decrease in P4/E2 ratio prior to labor in Cnr1−/− females (C) (n = 6–10, *P<0.05).

Figure 4. Western blot analysis of cytochrome P450 aromatase (P450Arom), 17β-hydroxysteroid dehydrogenase 7 (17β-HSD7) and 20α-HSD in wild-type (WT) and Cnr1−/− ovaries during late gestation.

Figure 5. Loss of CB1 overrides delayed parturition that occurs in Ptgs1−/− mice.

(A & B) The onset of parturition and survival rate of newborn pups in mice missing both Cnr1 and Ptgs1 are substantially improved in contrast to delayed parturition in Ptgs1−/− mice. (C & D) Parturition defects were largely restored in pregnant Ptgs1−/− mice receiving a CB1-selective antagonist SR141716 (SR1), but not a CB2-selective antagonist SR144528 (SR2), on days 14–18. Numbers within the bars indicate number of mice examined in panels A and C. The bars with different letters are significantly different (P<0.01).

Figure 6. Aberrant levels of CRH and corticosterone from CB1 deficiency contribute to preterm birth in mice.

(A & B) Circulating levels of CRH and corticosterone in pregnant WT and Cnr1−/− mice during late gestation (n = 4–5, *P<0.05). (C) Western blot analysis of CRH in WT and Cnr1−/− ovaries during late gestation, showing an early induction of ovarian CRH expression in mutant females.

Figure 7. Pharmacological silencing of CRH activities by Antalarmin hydrochloride (AH) on days 15–17 restores normal labor in Cnr1−/− mice with little effects on fetal birth weights (A & B), while enhanced corticosterone (CTS) activity on days 14–18 induces preterm birth with impaired fetal growth in wild-type (WT) mice (C & D).

Numbers within bars indicate the number of mice examined in panels A and C. The average fetal weights (mg) are shown in panels B and D. The bars with different letters are significantly different (P