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. 2016 Dec;157(12):4914-4929.
doi: 10.1210/en.2016-1620. Epub 2016 Oct 5.

Bmal1 Is Required for Normal Reproductive Behaviors in Male Mice

Erica L Schoeller  1 , Daniel D Clark  1 , Sandeepa Dey  1 , Nathan V Cao  1 , Sheila J Semaan  1 , Ling W Chao  1 , Alexander S Kauffman  1 , Lisa Stowers  1 , Pamela L Mellon  1
Affiliations

Bmal1 Is Required for Normal Reproductive Behaviors in Male Mice

Erica L Schoeller et al. Endocrinology. 2016 Dec.

Abstract

Circadian rhythms synchronize physiological processes with the light-dark cycle and are regulated by a hierarchical system initiated in the suprachiasmatic nucleus, a hypothalamic region that receives direct photic input. The suprachiasmatic nucleus then entrains additional oscillators in the periphery. Circadian rhythms are maintained by a molecular transcriptional feedback loop, of which brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a key member. Disruption of circadian rhythms by deletion of the BMAL1 gene (Bmal1 knockout [KO]) induces a variety of disease states, including infertility in males, due to unidentified mechanisms. We find that, despite normal sperm function, Bmal1 KO males fail to mate with receptive females, indicating a behavioral defect. Mating is dependent on pheromone detection, as are several other behaviors. We determined that Bmal1 KO males also fail to display aggression and avoidance of predator scent, despite intact main olfactory function. Moreover, the vomeronasal organ, a specialized pheromone-responsive organ, was also functionally intact, as determined by calcium imaging in response to urine pheromone stimulus. However, neural circuit tracing using c-FOS activation revealed that, although Bmal1 KO males displayed appropriate activation in the olfactory bulb and accessory olfactory bulb, the bed nucleus of the stria terminalis and the medial preoptic area (areas responsible for integration of copulatory behaviors) failed to activate highly in response to the female scent. This indicates that neural signaling in select behavioral centers is impaired in the absence of BMAL1, likely underlying Bmal1 KO male copulatory defects, demonstrating the importance of the BMAL1 protein in the maintenance of neural circuits that drive pheromone-mediated mating behaviors.

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Figures

Figure 1.
Figure 1.
Mating and aggressive behavior in Bmal1 KO males. A, Male mating assay: days until appearance of copulatory plug after introduction of an estrous WT female (n = 4, Student's t test). B, Serum T measurement in WT and Bmal1 KO males (n = 10–12, Mann-Whitney test). C, Mating behavior: total number of mounting attempts in 30 minutes during a mating trial with a receptive WT female (n = 9 intact, 3–4 GDX + T, one way ANOVA followed by Tukey post hoc). D, Aggression (resident): aggressive behavior during introduction of a WT male into the test subject's (resident's) home cage (n = 5–6 intact, 3–4 GDX + T). *, P < .05; **, P < .01.
Figure 2.
Figure 2.
Fear behavior in Bmal1 KO male mice. A and B, Fear behavior: time spent in marked area near (area 1) predator scent (fox urine) or away (area 2) from predator scent. Fox urine placement is marked by a red box (n = 6, one way ANOVA). C, Serum corticosterone was measured by an ELISA after exposure to predator scent for 10 minutes (n = 5 for scent exposed groups, n = 2 for baseline groups, one way ANOVA followed by Tukey post hoc). *, P < .05; **, P < .01; ***, P < .001.
Figure 3.
Figure 3.
Hypothalamic and pituitary function in Bmal1 KO males. A, Male serum FSH (n = 6, Student's t test). B, Male serum LH (n = 6, Student's t test). C, Pituitary gene expression: mRNA expression of FSHβ, LHβ, and GnRH receptor mRNAs in the pituitary of WT and Bmal1 KO males (n = 5, Student's t test). D, Hypothalamic gene expression (n = 4–6, Student's t test). E, GnRH challenge: LH response to exogenous injection of GnRH (n = 5–6, two way ANOVA followed by Sidak post hoc). F, Kisspeptin challenge: LH response to injection of kisspeptin peptide (kiss-10) (n = 4, two way ANOVA followed by Sidak post hoc). G, AVPV kisspeptin expression: mRNA expression of kisspeptin in the AVPV, a marker of brain masculinization (n = 3, one way ANOVA followed by Tukey pot hoc). H, Preputial separation: postnatal day when preputial separation occurs in male mice (marker of puberty) (n = 8–9, Student's t test). *, P < .05; **, P < .01; ***, P < .001; ****, P < .0001.
Figure 4.
Figure 4.
Olfactory function in Bmal1 KO males. A, Buried food test: latency to discovery of buried food pellet (n = 7, Student's t test). B, Chemoinvestigation: time spent performing anogenital sniffing upon introduction of receptive female (n = 3–9, one way ANOVA). C and D, Territorial marking: urine marks in response to a spot of receptive female estrous urine scent on Whatman paper (n = 6–8, Student's t test). E, VNO function: isolated vomeronasal neuron calcium flux response to whole urine from a WT male (n = 3, Student's t test). F, VNO function: isolated vomeronasal neuron response to high molecular weight (HMW) fraction of urine from a WT male in culture (n = 3 Student's t test, P < .05). **, P < .01; ***, P < .001.
Figure 5.
Figure 5.
c-FOS expression in the granule cell layer of the OB and AOB in WT vs Bmal1 KO males after exposure to estrous female scent. A, WT control OB. B, WT + estrous female scent OB. C, Bmal1 KO control OB. D, Bmal1 KO + estrous female scent OB. E, Dotted outline indicates quantified portion. Images were obtained from the Allen Mouse Brain Atlas (http://mouse.brain-map.org). F, Quantification of c-FOS-positive cells in defined area. G, WT control AOB. H, WT + estrous female scent AOB. I, Bmal1 KO control AOB. J, Bmal1 KO + estrous female scent AOB. K, Dotted outline indicates quantified portion. Images obtained from the Allen Institute web site. L, Quantification of c-FOS-positive cells in defined area (n = 3 animals per treatment group). *, P < .5, **, P < .01, ***, P < .001, by ANOVA followed by Tukey post hoc. Scale bar, 100 μm. Image reproduced with permission from the Allen Institute.
Figure 6.
Figure 6.
Expression of c-FOS in the medial amygdala in WT vs Bmal1 KO males after exposure to estrous female scent. A, WT control. B, WT + estrous female scent. C, Bmal1 KO control. D, Bmal1 KO + estrous female scent. E, Dotted outline indicates quantified portion. Images were obtained from the Allen Mouse Brain Atlas (http://mouse.brain-map.org). F, Quantification of c-FOS-positive cells in defined area (n = 4 animals per treatment group). *, P < .5, **, P < .01, ***, P < .001, by ANOVA followed by Tukey post hoc. Scale bar, 100 μm. Image reproduced with permission from the Allen Institute.
Figure 7.
Figure 7.
Expression of c-FOS in the BNST in WT vs Bmal1 KO males after exposure to estrous female scent. A, WT control. B, WT + estrous female scent. C, Bmal1 KO control. D, Bmal1 KO + estrous female scent. E, Dotted outline indicates quantified portion. Images obtained from the Allen Mouse Brain Atlas (http://mouse.brain-map.org). F, Quantification of c-FOS-positive cells in defined area (n = 4 animals per treatment group). *, P < .5, **, P < .01, ***, P < .001, by ANOVA followed by Tukey post hoc. Scale bar, 100 μm. Image reproduced with permission from the Allen Institute.
Figure 8.
Figure 8.
Expression of c-FOS in the MPO in WT vs Bmal1 KO males after exposure to estrous female scent. A, WT control. B, WT + estrOus female scent. C, Bmal1 KO control. D, Bmal1 KO + estrous female scent. E, Dotted outline indicates quantified portion. Images obtained from the Allen Mouse Brain Atlas (http://mouse.brain-map.org). F, Quantification of c-FOS-positive cells in defined area (n = 4 animals per treatment group). *, P < .5, **, P < .01, ***, P < .001, by ANOVA followed by Tukey post hoc. Scale bar, 100 μm. Image reproduced with permission from the Allen Institute.
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