Search

Acute estrogen deficiency in women can occur due to many conditions including hyperprolactinemia, chemotherapy, GnRH agonist treatment, and removal of hormone replacement therapy. Ovariectomized (OVX) rodent models, often combined with a high-fat diet (HFD), have been used to investigate the effects of decreased estrogen production on metabolism. Since evidence suggests that gut microbes may facilitate the protective effect of estrogen on metabolic dysregulation in an OVX + HFD model, we investigated whether the gut microbiome plays a role in the diet-independent weight gain that occurs after OVX in adult female mice. 16S rRNA gene sequence analysis demonstrated that OVX was not associated with changes in overall gut bacterial biodiversity but was correlated with a shift in beta diversity. Using differential abundance analysis, we observed a difference in the relative abundance of a few bacterial taxa, such as Turicibacter, 3 to 5 weeks after OVX, which was subsequent to the weight gain that occurred 2 weeks postsurgery. A cohousing study was performed to determine whether exposure to a healthy gut microbiome was protective against the development of the metabolic phenotype associated with OVX. Unlike mouse models of obesity, HFD maternal-induced metabolic dysregulation, or polycystic ovary syndrome, cohousing OVX mice with healthy mice did not improve the metabolic phenotype of OVX mice. Altogether, these results indicate that changes in the gut microbiome are unlikely to play a causal role in diet-independent, OVX-induced weight gain (since they occurred after the weight gain) and cohousing with healthy mice did not have a protective effect.

The preovulatory gonadotropin-releasing hormone (GnRH) and resulting luteinizing hormone (LH) surge is gated to the onset of the active period in mice by a circadian and steroid signal. The temporal signal that initiates this process is believed to arrive from the suprachiasmatic nucleus (SCN), the hypothalamic circadian pacemaker. The SCN may contribute to reproduction through two discrete projections: vasoactive inhibitory peptide (VIP) neurons in the SCN project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and arginine vasopressin (AVP) neurons in the SCN project to kisspeptin (Kiss1) neurons in the anteroventral periventricular (AVPV). Genomic deletion of the critical circadian clock component, Bmal1, results in infertility in females. We sought to determine whether Bmal1 was critical in the SCN VIP and AVP neurons by using cre/lox technology to create three lines of mutant mice: AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/-. We found no significant difference in LH levels following a kisspeptin challenge (2 mg/kg) between AVP-Bmal1-/- and Bmal1flox/flox (617.4 ± 216.9 vs 629.6 ± 110.6 pg/mL) or GnRH challenge (10 μg/kg) (1029.0 ± 246.2 vs 712.7 ± 142.6 pg/mL). Preliminary findings of an induced LH surge found AVP-Bmal1-/- and VIP-Bmal1-/- mice are able to mount a surge at the time of lights off (Bmal1flox/flox: 3 of 5; AVP-Bmal1-/-: 1 of 2, VIP-Bmal1:-/- 4 of 4; AVP-VIP-Bmal1-/- 0 of 2). Additionally, we were able to detect an endogenous surge at the time of lights off in both AVP-Bmal1-/-, VIP-Bmal1-/- mice, but not AVP-VIP-Bmal1-/-. However, AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/- females carried litters to term and show no differences in the time to first litter compared to Bmal1flox/flox control (AVP-Bmal1-/-: 48 ± 4.8 vs 44.3 ± 0.3 days; VIP-Bmal1-/-: 29.6 ± 7.7 vs 31.0 ± 4.2 days; AVP-VIP-Bmal1-/-;27.7 ± 7 vs 23 ± 0 days); number of litters in 100 days (AVP-Bmal1-/-: 3.7 ±0.6 vs 4 ± 0.6; VIP-Bmal1-/-: 3.0 ± 1.0 vs 3.0 ± 0.0; AVP-VIP-Bmal1-/- 3.5 ± 0.7 vs 3.5 ± 0.7 days); or pups per litter (AVP-Bmal1-/-: 7.2 ± 0.7 vs 7.3 ± 0.7; VIP-Bmal1-/-: 6.2 ± 1.6 vs 7.6 ± 2.9; AVP-VIP-Bmal1-/- 8.1 ± 0.9 vs 9 ± 1.4 pups). Overall, these findings suggest that Bmal1 expression in the SCN AVP or VIP neurons is not required for fertility in a 12:12 light:dark cycle.

In rodents, the preovulatory LH surge is temporally gated, but the timing cue is unknown. Estrogen primes neurons in the anteroventral periventricular nucleus (AVPV) to secrete kisspeptin, which potently activates GnRH neurons to release GnRH, eliciting a surge of LH to induce ovulation. Deletion of the circadian clock gene Bmal1 results in infertility. Previous studies have found that Bmal1 knockout (KO) females do not display an LH surge at any time of day. We sought to determine whether neuroendocrine disruption contributes to the absence of the LH surge. Because Kiss1 expression in the AVPV is critical for regulating ovulation, we hypothesized that this population is disrupted in Bmal1 KO females. However, we found an appropriate rise in AVPV Kiss1 and Fos mRNA at the time of lights out in ovariectomized estrogen-treated animals, despite the absence of a measureable increase in LH. Furthermore, Bmal1 KO females have significantly increased LH response to kiss-10 administration, although the LH response to GnRH was unchanged. We then created Kiss1- and GnRH-specific Bmal1 KO mice to examine whether Bmal1 expression is necessary within either kisspeptin or GnRH neurons. We detected no significant differences in any measured reproductive parameter. Our results indicate that disruption of the hypothalamic regulation of fertility in the Bmal1 KO females is not dependent on endogenous clocks within either the GnRH or kisspeptin neurons.

Light provides the primary timing signal that enables fine-tuned behavioral and hormonal entrainment of circadian rhythms to the environment. Light is transmitted from the eye to the brain through the retinohypothalamic tract, where one target is the hypothalamic suprachiasmatic nucleus (SCN), which generates self-sustained circadian rhythms. The vasoactive intestinal polypeptide (VIP) expressing neurons of the SCN relay light information to peripheral cells and tissues through control of hormonal and nervous signals, allowing synchronization of molecular clocks located in individual cells throughout the body. Non-natural light cycles, ie shiftwork, and weakened SCN function through genetic manipulation, disrupt the body’s circadian rhythms, causing deregulated hormone release, metabolic disorders, and negative effects on reproductive systems such as irregular menstrual cycles and decreased sperm count. To further our understanding of how the SCN translates light information into neuroendocrine control of fertility, we conditionally deleted the SCN enriched transcription factor Ventral anterior homeobox 1 (Vax1) in post-developmental VIP neurons, generating Vax1-flox/flox:Vip-Cre+ (cKO) mice. To determine if the SCN timekeeping function was impacted in cKO mice, we single housed males and females with running wheels to examine activity during both 12-hour light/dark cycles and in constant darkness. Wheel-running behavior in constant darkness revealed a shortening of the endogenous free-running period (Tau) of the SCN. Aside from Tau, wheel running behaviors were comparable to controls. Weakened SCN output can negatively impact fertility. While on 12-hour light/dark cycles, we found a modest, but significant change in follicle stimulating hormone and estrogen in cKO females and a reduced sensitivity of GnRH neurons to kisspeptin in males. The changes in hormone release were associated with a slightly lengthened estrous cycle in cKO females and reduced sperm quality in cKO males. To identify the molecular origin of the shortened SCN period, we used immunohistochemistry and RNAscope to examine expression of Vip. We found that diestrus cKO females had a significant reduction in Vip expression at ZT16 and preliminary data suggest a reduction in the circadian clock gene Bmal1. Together, these studies identify a novel role of VAX1 in VIP neurons where VAX1 is required for VIP expression and circadian timekeeping. Loss of VAX1 in VIP neurons weakens SCN output, deregulating reproductive hormone release and modestly reducing reproductive function in both males and females.