The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gα_q/11-coupled receptor, and mice bearing a global deletion ofKiss1r(Kiss1r^−/−) or a GnRH neuron-specific deletion ofKiss1r(Kiss1r^d/d) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via β-arrestin, and in mice lacking β-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gα_q/11in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gα_q/11-independent GnRH secretion would be sufficient to maintain fertility. To test this,Gnaq(encodes Gα_q) was selectively inactivated in the GnRH neurons of globalGna11(encodes Gα₁₁)-null mice by crossingGnrh-CreandGnaq^fl/fl;Gna11^−/−mice. ExperimentalGnaq^fl/fl;Gna11^−/−;Gnrh-Cre(Gnaq^d/d) and controlGnaq^fl/fl;Gna11^−/−(Gnaq^fl/fl) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected inGnaq^d/dmice than in previously characterizedKiss1r^−/−orKiss1r^d/dmice. Additionally,Gnaq^d/dmales were subfertile, and althoughGnaq^d/dfemales did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels inGnaq^d/dmice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gα_q/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis inGnaq^d/dmice.SIGNIFICANCE STATEMENTThe gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gα_q/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gα_q/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gα_q/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gα_q/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.

Expression of kisspeptin (protein) and Kiss1r (mRNA) was recently documented in the mouse uterus on D4 of pregnancy (the day of embryo implantation) suggesting that the uterine-based kisspeptin (KP)/kisspeptin receptor (KISS1R) signaling system regulates embryo implantation. Despite this important suggestion, it was never demonstrated that the uterus actually exhibits a functional KP/KISS1R signaling system on D4 of pregnancy. Thus, the goal of this study was to determine whether a functional KP/KISS1R signaling system exists in the mouse uterus on D4 of pregnancy. Since kisspeptin/KISS1R signaling triggers the phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2, through immunohistochemical analyses, we determined whether exogenously administered kisspeptin could trigger p38 and ERK1/2 phosphorylation in the uterus on D4 of pregnancy. The results clearly demonstrated that kisspeptin could and that its effects were mediated via KISS1R. Additionally, the robust kisspeptin-triggered response was observed in the pregnant uterus only. Finally, it was demonstrated that on D4 of pregnancy the Kiss1 null uterus expresses functional KISS1R molecules capable of mediating the effects of kisspeptin. These results lead us to conclude that on D4 of pregnancy, the mouse uterus expresses a functional KP/KISS1R signaling system strengthening the possibility that this signaling system regulates embryo implantation. These findings strengthen the rationale for determining whether such a functional system exists in the uterus of the human female and if so, what role it might play in human pregnancy.

Obesity is associated with altered fatty acid profiles, reduced fertility, and assisted reproductive technology (ART) success. The effects of palmitic acid (PA), oleic acid (OA), and their combination on mouse preimplantation development, endoplasmic reticulum (ER) stress pathway gene expression, lipid droplet formation, and mitochondrial reactive oxygen species (ROS) were characterized. Two-cell stage mouse embryos collected from superovulated and mated CD1 females were placed into culture with KSOMaa medium, or PA alone or in combination with OA for 46 h. PA significantly reduced blastocyst development in a concentration-dependent manner, which was prevented by co-treatment with OA. PA and OA levels in mouse reproductive tracts were assessed by liquid chromatography coupled to mass spectrometry (LC-MS). LC-MS indicated higher concentrations of PA in the mouse oviduct than the uterus. Transcript analysis revealed that PA alone groups had increased ER stress pathway (ATF3, CHOP, and XBP1 splicing) mRNAs, which was alleviated by OA co-treatment. OA co-treatment significantly increased lipid droplet accumulation and significantly decreased mitochondrial ROS from PA treatment alone. PA treatment for only 24 h significantly reduced its impact on blastocyst development from the 2-cell stage. Thus, PA affects ER stress pathway gene expression, lipid droplet accumulation, and mitochondrial ROS in treated preimplantation embryos. These mechanisms may serve to offset free fatty acid exposure effects on preimplantation development, but their protective ability may be overwhelmed by elevated PA.

What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0–2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR. Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. Not applicable. Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA’s detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.