Granulosa Cell Endothelin-2 Expression is Fundamental for Ovulatory Follicle Rupture

Ovulation is dependent upon numerous factors mediating follicular growth, vascularization, and ultimately oocyte release via follicle rupture. Endothelin-2 (EDN2) is a potent vasoconstrictor that is transiently produced prior to follicle rupture by granulosa cells of periovulatory follicles and induces ovarian contraction. To determine the role of Edn2 expression, surgical transplant and novel conditional knockout mice were super-ovulated and analyzed. Conditional knockout mice utilized a new iCre driven by the Esr2 promoter to selectively remove Edn2. Follicle rupture and fertility were significantly impaired in the absence of ovarian Edn2 expression. When ovaries of Edn2KO mice were transplanted in wild type recipients, significantly more corpora lutea containing un-ovulated oocytes were present after hormonal stimulation (1.0 vs. 5.4, p = 0.010). Following selective ablation of Edn2 in granulosa cells, Esr2-Edn2KO dams had reduced oocytes ovulated (3.8 vs. 16.4 oocytes/ovary) and smaller litters (4.29 ± l.02 vs. 8.50 pups/dam). However, the number of pregnancies per pairing was not different and the reproductive axis remained intact. Esr2-Edn2KO ovaries had a higher percentage of antral follicles and fewer corpora lutea; follicles progressed to the antral stage but many were unable to rupture. Conditional loss of endothelin receptor A in granulosa cells also decreased ovulation but did not affect fecundity. These data demonstrate that EDN2-induced intraovarian contraction is a critical trigger of normal ovulation and subsequent fecundity

Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans

DNA methylation likely plays a role in the regulation of human stress reactivity. Here we show that in a genome-wide analysis of blood DNA methylation in 85 healthy individuals, a locus in the Kit ligand gene (KITLG; cg27512205) showed the strongest association with cortisol stress reactivity (P=5.8 � 10?6). Replication was obtained in two independent samples using either blood (N=45, P=0.001) or buccal cells (N=255, P=0.004). KITLG methylation strongly mediates the relationship between childhood trauma and cortisol stress reactivity in the discovery sample (32% mediation). Its genomic location, a CpG island shore within an H3K27ac enhancer mark, and the correlation between methylation in the blood and prefrontal cortex provide further evidence that KITLG methylation is functionally relevant for the programming of stress reactivity in the human brain. Our results extend preclinical evidence for epigenetic regulation of stress reactivity to humans and provide leads to enhance our understanding of the neurobiological pathways underlying stress vulnerability

Loss of Fertility in the Absence of Progesterone Receptor Expression in Kisspeptin Neurons of Female Mice

<div><p>Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the <i>Pgr</i> gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the <i>Pgr</i> deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.</p></div

PGR does not regulate <i>Kiss1</i> gene expression.

<p><i>Kiss1</i> mRNA expression level in 6-month-old WT (n = 9) and Kiss1-PgrKO (n = 4) mice.</p

Quantification of kisspeptin positive immunofluorescent cells in the AVPV of WT and Kiss1-PgrKO mice.

<p>A. Immunofluorescence of kisspeptin in the AVPV of 5-month-old WT and Kiss1-PgrKO; arrows indicate kisspeptin positive cells (bar = 25 μ). B. Quantification of the raw cell number of kisspeptin positive cells from the AVPV of 2-3- and 5-month-old WT and Kiss1-PgrKO mice; the raw cell number was calculated from 3 sections per animal. Data represent means ± SD.</p

Establishment of a murine graft-versus-myeloma model using allogeneic stem cell transplantation

Background: Multiple myeloma (MM) is a malignant plasma cell disorder with poor long-term survival and high recurrence rates. Despite evidence of graft-versus-myeloma (GvM) effects, the use of allogeneic hematopoietic stem cell transplantation (allo-SCT) has remained controversial in MM. In the current study, we investigated the anti-myeloma effects of allo-SCT from B10.D2 mice into MHC-matched myeloma-bearing Balb/cJ mice (previously injected with the MOPC315.BM myeloma cell line), based on a chronic graft-versus-host disease (GvHD) murine model. Methods and results: Balb/cJ mice were injected intravenously with luciferase-transfected MOPC315.BM cells, and received 30 days later an allogeneic (B10.D2 donor) or autologous (Balb/cJ donor) transplantation by intravenous administration of bone marrow cells and splenocytes. We observed a graft-versus-myeloma effect in 94% of the allogeneic transplanted mice, as luciferase signal completely disappeared after transplantation, whereas all the autologous transplanted mice showed myeloma evolution. Lower serum paraprotein levels and myeloma infiltration in bone marrow and spleen in the allogeneic setting confirmed the observed GvM effect, while allogeneic mice also displayed chronic GvHD symptoms. In vivo and in vitro data suggest the involvement of effector memory CD4 and CD8 T cells in the GvM effect. The essential role of CD8 T cells was demonstrated in vivo where CD8 T-cell depletion of the graft resulted in reduced GvM effects. Finally, TCR V spectratyping analysis identified V families within CD4 and CD8 T cells which were associated with both GvM effects and GVHD, whereas other V families within CD4 T cells were associated exclusively with either GvM or GvHD responses. Conclusions: We successfully established an immunocompetent murine model of graft-versus-myeloma. This is the first immunocompetent murine model which is based on a MM model closely resembling human MM disease (bone marrow tropism, ...) and using allo-SCT after the disease establishment, as a curative treatmen

PGR and kisspeptin are co-localized with ERα in kisspeptin neurons but are not expressed in AVPV kisspeptin neurons of ERαKO mice.

<p>A. Double immunohistochemistry of PGR and kisspeptin in WT and ERαKO mice. Black arrows indicate dark brown nuclear PGR staining. Black arrowheads indicate brown red cytoplasmic kisspeptin staining. Open arrowhead indicates hematoxylin-stained nucleus of a kisspeptin positive and PGR negative neuron. Bar = 25 μm. B. Co-localization of PGR, ERα, and kisspeptin in kisspeptin neuron in the hypothalamic AVPV nucleus from a 2-month-old female C57BL/6 mouse in the estrus stage. From left to right: PGR (blue), kisspeptin (red), ERα (green), and merged image. Yellow arrows indicate nuclear PGR, ERα, and merged PGR/ERα. Green arrows indicate cytoplasmic kisspeptin. White arrows indicate ERα positive nucleus.</p

Immunohistochemical labeling of PGR and kisspeptin in neurons from the AVPV, and cells from the ovary and uterus of 2-3-month-old WT (left), Kiss1-PgrKO (middle), and global-PgrKO (right).

<p>Black arrows indicate PGR positive nuclei, black arrowheads indicate cytoplasmic kisspeptin, open arrowheads indicate nuclei that do not express PGR. Bar in AVPV = 20 μm; bar in uterus and ovary = 25 μm.</p