The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) – the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) – a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

The nuclear envelope (NE) is an endoplasmic reticulum (ER) subdomain that contains characteristic components dedicated to nuclear functions. These include nuclear pore complexes (NPCs) -the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) -a scaffold for NE and chromatin organization at the nuclear periphery. Since numerous human diseases associated with NE/NL proteins occur in mesenchyme-derived cells, a more comprehensive characterization of proteins concentrated at the NE in these cell types is warranted. Accordingly, we used proteomics to analyze NE and other subcellular fractions isolated from mesenchymal stem cells and from differentiated adipocytes and myocytes. We evaluated the proteomics datasets to calculate relative protein enrichment in the NE fraction, using a spectral abundance-based scoring system that accurately described most benchmark proteins. We then examined five high-scoring transmembrane proteins expressed in all three cell types that were not previously known to be enriched at the NE. Using quantitative immunofluorescence microscopy to track ectopically expressed proteins, we validated that all five of these components are substantially concentrated at the NE of multiple cell types. One (Itprip) is exposed to the outer nuclear membrane, a second (Smpd4) is enriched at the NPC, and the three others (Mfsd10, Tmx4, and Arl6ip6) are suggested to reside in the inner nuclear membrane. Considering their sequences and other features, these proteins provide new focal points for studying the functions and membrane dynamics of the NE. Our datasets should be useful for identifying additional NE-concentrated proteins, and for evaluating candidates that are identified in screening.

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, a species in which females and males exhibit both overlapping and distinct pair bond behaviors. We 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 coordinated behavior emerged — females consistently huddled more with their partner than males did, and partner huddle time became correlated between partners. When animals were allowed to freely interact with a partner or a novel in sequential free interaction tests, pairs spent more time interacting together than either animal did with a novel. Pair interaction 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 coordinated behavior emerge, and that these intra-pair behavioral changes are likely organized and driven by the female animal.

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.

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 pairbonded 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.