Embryonic exposures can increase the risk of congenital cardiac birth defects and adult disease. The present study identifies the predominant pathways modulated by an acute embryonic mouse exposure during gastrulation to lithium or homocysteine that induces cardiac defects. High dose periconceptional folate supplementation normalized development. Microarray bioinformatic analysis of gene expression demonstrated that primarily lipid metabolism is altered after the acute exposures. The lipid-related modulation demonstrated a gender bias with male embryos showing greater number of lipid-related Gene Ontology biological processes altered than in female embryos. RT-PCR analysis demonstrated significant change of the fatty acid oxidation gene Acadm with homocysteine exposure primarily in male embryos than in female. The perturbations resulting from the exposures resulted in growth-restricted placentas with disorganized cellular lipid droplet distribution indicating lipids have a critical role in cardiac-placental abnormal development. High folate supplementation protected normal heart-placental function, gene expression and lipid localization.

A comprehensive analysis of transposable element (TE) expression in mammalian full-grown oocytes reveals that LTR class III retrotransposons make an unexpectedly high contribution to the maternal mRNA pool, which persists in cleavage stage embryos. The most abundant transcripts in the mouse oocyte are from the mouse transcript (MT) retrotransposon family, and expression of this and other TE families is developmentally regulated. Furthermore, TEs act as alternative promoters and first exons for a subset of host genes, regulating their expression in full-grown oocytes and cleavage stage embryos. To our knowledge, this is the first example of TEs initiating synchronous, developmentally regulated expression of multiple genes in mammals. We propose that differential TE expression triggers sequential reprogramming of the embryonic genome during the oocyte to embryo transition and in preimplantation embryos.

The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a G(s) G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, G(s) is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-G(s) signaling, we examined the effect of LH on the localization of Galpha(s). G(s) activation in response to stimulation of an exogenously expressed beta(2)-adrenergic receptor causes Galpha(s) to move from the oocyte plasma membrane into the cytoplasm, whereas G(s) inactivation in response to inhibition of the beta(2)-adrenergic receptor causes Galpha(s) to move back to the plasma membrane. However, LH does not cause a change in Galpha(s) localization, indicating that LH does not act by terminating receptor-G(s) signaling.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

The rapid expansion of transcriptomics and affordability of next-generation sequencing (NGS) technologies generate rocketing amounts of gene expression data across biology and medicine, including cancer research. Concomitantly, many bioinformatics tools were developed to streamline gene expression and quantification. We tested the concordance of NGS RNA sequencing (RNA-seq) analysis outcomes between two predominant programs for read alignment, HISAT2, and STAR, and two most popular programs for quantifying gene expression in NGS experiments, edgeR and DESeq2, using RNA-seq data from breast cancer progression series, which include histologically confirmed normal, early neoplasia, ductal carcinoma in situ and infiltrating ductal carcinoma samples microdissected from formalin fixed, paraffin embedded (FFPE) breast tissue blocks. We identified significant differences in aligners' performance: HISAT2 was prone to misalign reads to retrogene genomic loci, STAR generated more precise alignments, especially for early neoplasia samples. edgeR and DESeq2 produced similar lists of differentially expressed genes, with edgeR producing more conservative, though shorter, lists of genes. Gene Ontology (GO) enrichment analysis revealed no skewness in significant GO terms identified among differentially expressed genes by edgeR versus DESeq2. As transcriptomics of FFPE samples becomes a vanguard of precision medicine, choice of bioinformatics tools becomes critical for clinical research. Our results indicate that STAR and edgeR are well-suited tools for differential gene expression analysis from FFPE samples.

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 minutes of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 hours after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 hours after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

More adolescents are coming out as transgender each year and are put on puberty blockers to suppress natal puberty, which is then followed by cross-hormone treatment to achieve puberty of the desired gender. Studies to examine the effects of puberty suppression and virilizing therapy on future reproductive potential among transgender males are lacking. This study used a translational murine in vitro fertilization model to examine the effects of female puberty suppression with depot leuprolide acetate (LA), followed by virilizing therapy with testosterone cypionate (T), on embryologic and pregnancy outcomes. LA effectively inhibited puberty when mice were treated beginning at 3 weeks of age. LA treatment was associated with higher mouse weight but lower ovarian weight. LA-treated mice ovulated developmentally competent eggs in response to gonadotropin administration, albeit at a higher dose than controls. Ovaries from mice treated with LA and T produced oocytes that had morphologically normal meiotic spindles after in vitro maturation and responded to gonadotropin stimulation. Eggs from mice treated with LA and T were fertilizable and produced developmentally competent embryos that led to births of fertile pups. These results suggest that fertility may not be impaired after puberty suppression and cross-hormone therapy for transgender males.