A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons

Emergent Intra-Pair Sex Differences and Organized Behavior in Pair Bonded Prairie Voles (Microtus ochrogaster)

In pair bonding animals, coordinated behavior between partners is required for the pair to accomplish shared goals such as raising young. Despite this, experimental designs rarely assess the behavior of both partners within a bonded pair. Thus, we lack an understanding of the interdependent behavioral dynamics between partners that likely facilitate relationship success. To identify intra-pair behavioral correlates of pair bonding, we used socially monogamous prairie voles (Microtus ochrogaster) and tested both partners using social choice and non-choice tests at short- and long-term pairing timepoints. Females developed a preference for their partner more rapidly than males, with preference driven by different behaviors in each sex. Further, as bonds matured, intra-pair behavioral sex differences and organized behavior emerged—females consistently huddled more with their partner than males did regardless of overall intra-pair affiliation levels. When animals were allowed to freely interact with a partner or a novel vole in sequential free interaction tests, pairs spent more time interacting together than either animal did with a novel vole, consistent with partner preference in the more commonly employed choice test. Total pair interaction in freely moving voles was correlated with female, but not male, behavior. Via a social operant paradigm, we found that pair-bonded females, but not males, are more motivated to access and huddle with their partner than a novel vole. Together, our data indicate that as pair bonds mature, sex differences and organized behavior emerge within pairs, and that these intra-pair behavioral changes are likely organized and driven by the female animal

A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons

The Contribution of the Circadian Gene Bmal1 to Female Fertility and the Generation of the Preovulatory Luteinizing Hormone Surge.

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

Prolonged partner separation erodes nucleus accumbens transcriptional signatures of pair bonding in male prairie voles

The loss of a spouse is often cited as the most traumatic event in a person’s life. However, for most people, the severity of grief and its maladaptive effects subside over time via an understudied adaptive process. Like humans, socially monogamous prairie voles (Microtus ochrogaster) form opposite-sex pair bonds, and upon partner separation, show stress phenotypes that diminish over time. We test the hypothesis that extended partner separation diminishes pair bond-associated behaviors and causes pair bond transcriptional signatures to erode. Opposite-sex or same-sex paired males were cohoused for 2 weeks and then either remained paired or were separated for 48 hours or 4 weeks before collecting fresh nucleus accumbens tissue for RNAseq. In a separate cohort, we assessed partner-directed affiliation at these time points. We found that these behaviors persist despite prolonged separation in both same-sex and opposite-sex paired voles. Opposite-sex pair bonding led to changes in accumbal transcription that were stably maintained while animals remained paired but eroded following prolonged partner separation. Eroded genes are associated with gliogenesis and myelination, suggesting a previously undescribed role for glia in pair bonding and loss. Further, we pioneered neuron-specific translating ribosomal affinity purification in voles. Neuronally enriched transcriptional changes revealed dopaminergic-, mitochondrial-, and steroid hormone signaling-associated gene clusters sensitive to acute pair bond disruption and loss adaptation. Our results suggest that partner separation erodes transcriptomic signatures of pair bonding despite core behavioral features of the bond remaining intact, revealing potential molecular processes priming a vole to be able to form a new bond

GnRH-E1 RNA is localized in the GT1-7 neuron nucleus.

<p>(A) Schematic diagram of the conserved regulatory elements upstream of the mouse <i>Gnrh1</i> TSS, which contains enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the <i>Gnrh1</i> gene with four exons (white boxes). Coordinates above the regulatory elements indicate positions with respect to the <i>Gnrh1</i> TSS. RT-PCR primers used in B-D are indicated by arrows, and expected PCR products are represented by a connecting line. Positions of PCR primers are aligned to the mouse conserved regulatory region diagrammed above. Nuclear and cytoplasmic extracts from GT1-7 neurons were analyzed for GnRH-E1 RNA (B), <i>Gnrh1</i> pre-mRNA (C), <i>Gnrh1</i> mRNA (D), and <i>H2afz</i> mRNA control (E) by RT-PCR. RT-PCR analysis was performed on random hexamer-primed cDNA, where cDNA synthesized with (+) and without (-) reverse transcriptase were analyzed in parallel. PCR loading controls are plasmid containing the -3568/-1128 bp segment upstream of the <i>Gnrh1</i> TSS and no-template control (NTC). The sizes of the PCR amplicons were marked by a 100 bp DNA ladder or a 1 kbp DNA ladder where indicated, that were resolved on the agarose gel in parallel.</p

Oligonucleotide sequences.

<p>Oligonucleotide sequences.</p

RT-qPCR analysis of rat and mouse GnRH-E1 RNA expression in cell lines.

<p>(A) A schematic diagram of conserved regulatory elements upstream of the mouse <i>Gnrh1</i> transcription start site (TSS, curved arrow) and <i>Gnrh1</i> gene coding region. The <i>Gnrh1</i> gene regulatory region contains <i>Gnrh1</i> enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the <i>Gnrh1</i> gene consists of four exons (white boxes). PCR primers used in Fig 2C are indicated by arrows, and the predicted PCR product mouse GnRH-E1 RNA is represented by a connecting line. (B) Schematic diagram of transgene embedded in GT1-7 neurons carrying the 3’ portion of the rat GnRH-E2, GnRH-E1, GnRH-P, and the <i>Gnrh1</i> TSS, driving the SV40 T-antigen oncogene. PCR primers used in Fig 2C are indicated by arrows, and the predicted PCR product rat GnRH-E1 RNA from the transgene is represented by a connecting line. (C) RT-qPCR analysis of endogenous mouse GnRH-E1 RNA (black) and GnRH-E1 RNA expressed from the rat <i>Gnrh1</i> promoter transgene (white) in GT1-7, GN11, and NIH3T3 cells. Relative GnRH-E1 RNA expression is normalized to peptidylprolyl isomerase A (<i>Ppia</i>) mRNA control. Data are displayed as means ± SD. Asterisk indicates statistical significance by Student’s T-test on the comparison between mouse and rat GnRH-E1 RNA, where p<0.05.</p

GnRH-E1 RNA, <i>Gnrh1</i> mRNA, and <i>Gnrh1</i> pre-mRNA stability following actinomycin D treatment of GT1-7 neurons.

<p>GT1-7 neurons were treated with either DMSO control vehicle (black bars) or 1 μg/mL actinomycin D (white bars). Total RNA was harvest at 2, 4, 8, and 24 hours after treatment. RT-qPCR analysis was performed to determine changes in endogenous mouse GnRH-E1 RNA (A), transgene-derived rat GnRH-E1 RNA (B), <i>Gnrh1</i> pre-mRNA (C) and <i>Gnrh1</i> mRNA (D) expression. Relative expression is normalized to <i>H2afz</i> mRNA control. Data are displayed as the fold change from untreated cells that were harvested at the time of treatment, and as the mean ± SD. Statistical significance was determined by two-way ANOVA, followed by <i>post hoc</i> Tukey-Kramer HSD, where asterisks indicate statistical significance at p<0.05.</p

The effect of mouse GnRH-E1 RNA over-expression on <i>Gnrh1</i> gene transcriptional activity in GN11 cells.

<p>(A) A schematic diagram of the expression plasmid carrying the -3560 bp/-1128 bp segment from upstream of the mouse <i>Gnrh1</i> TSS, which contains the full-length mouse GnRH-E1 RNA that is integrated in the forward orientation. Expression of the sense GnRH-E1 RNA is driven by the CMV promoter (CMVp) and terminated by a plasmid-specific 3’ polyadenylation element (BGH-PolyA). B-C) GN11 neurons were transiently co-transfected with either empty pcDNA2.1 vector (white bars) or expression plasmid carrying the forward mouse GnRH-E1 RNA (black bars) for over-expression and the luciferase reporter plasmids as indicated. (B) GN11 cells were co-transfected with luciferase reporter plasmids containing -5000 bp, -4199 bp, -3175 bp, or -2168 bp of the rat <i>Gnrh1</i> regulatory region. Each luciferase reporter plasmid contains the indicated <i>Gnrh1</i> enhancers (E1, E2 and E3) and <i>Gnrh1</i> promoter (P). (C) GN11 cells were co-transfected with luciferase plasmids carrying the rat <i>Gnrh1</i> enhancer 1 and promoter (GnRH-E1/GnRH-P), RSV enhancer and the rat <i>Gnrh1</i> promoter (RSVe/GnRH-P), the rat <i>Gnrh1</i> enhancer 1 and RSV promoter (GnRH-E1/RSVp), or the RSV enhancer and promoter (RSVe/RSVp) control (inset). For reporter plasmids carrying GnRH-E1, the rat <i>Gnrh1</i> enhancer is integrated in the reverse orientation in the plasmid. (D) A schematic diagram of the antisense GnRH-E1 RNA expression plasmid carrying the full-length mouse GnRH-E1 RNA that is integrated in the reverse orientation, driven by CMVp and terminated by BGH-PolyA. (E) The antisense GnRH-E1 RNA expression plasmid (black bars) or empty pcDNA2.1 expression plasmid (white bars) were transiently co-transfected with a reporter plasmid carrying the -5 kb rat <i>Gnrh1</i> gene regulatory region, GnRH-E1/GnRH-P, GnRH-E1/RSVp, or RSVe/RSVp control (inset). Since the RSVe/RSVp is a very strong reporter, relative luciferase/β-galactosidase values for the RSVe/RSVp reporter are graphed separately for clarity in C and D. Luciferase/β-galactosidase values were normalized to pGL3. Data are displayed as the mean ± SD. Asterisks indicate statistical significance determined using Student’s T-test comparison between pcDNA2.1-transfected and GnRH-E1 RNA-transfected cells, where p<0.05.</p