scholarly journals Development, Sex Steroid Regulation, and Phenotypic Characterization of RFamide-Related Peptide (Rfrp) Gene Expression and RFamide Receptors in the Mouse Hypothalamus

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1827-1840 ◽  
Author(s):  
Matthew C. Poling ◽  
Joshua Kim ◽  
Sangeeta Dhamija ◽  
Alexander S. Kauffman
Keyword(s):  
In Situ Hybridization ◽  
Estrogen Receptor Α ◽  
Cell Number ◽  
Phenotypic Characterization ◽  
Related Peptide ◽  
Adult Mice ◽  
Gnrh Neurons ◽  
Double Label ◽  
Hormonal Milieu

Arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3, encoded by the Rfrp gene) is the mammalian ortholog of gonadotropin-inhibiting hormone and can inhibit GnRH neuronal activity and LH release. However, the development and regulation of the RFRP-3 system in both sexes is poorly understood. Using in situ hybridization, we examined changes in Rfrp-expressing neurons in mice of both sexes during development and under different adulthood hormonal milieus. We found no sex differences in Rfrp expression or cell number in adult mice. Interestingly, we identified two interspersed subpopulations of Rfrp cells (high Rfrp-expressing, HE; low Rfrp-expressing, LE), which have unique developmental and steroidal regulation characteristics. The number of LE cells robustly decreases during postnatal development, whereas HE cell number increases significantly before puberty. Using Bax knockout mice, we determined that the dramatic developmental decrease in LE Rfrp cells is not due primarily to BAX-dependent apoptosis. In adults, we found that estradiol and testosterone moderately repress Rfrp expression in both HE and LE cells, whereas the nonaromatizable androgen dihydrotestosterone has no effect. Using double-label in situ hybridization, we determined that approximately 25% of Rfrp neurons coexpress estrogen receptor-α in each sex, whereas Rfrp cells do not readily express androgen receptor in either sex, regardless of hormonal milieu. Lastly, when we looked at RFRP-3 receptors, we detected some coexpression of Gpr147 but no coexpression of Gpr74 in GnRH neurons of both intact and gonadectomized males and females. Thus, RFRP-3 may exert its effects on reproduction either directly, via Gpr147 in a subset of GnRH neurons, and/or indirectly, via upstream regulators of GnRH.

scholarly journals Hypothalamic Kiss1 but Not Kiss2 Neurons Are Involved in Estrogen Feedback in Medaka (Oryzias latipes)

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1751-1759 ◽  
Author(s):  
Yuta Mitani ◽  
Shinji Kanda ◽  
Yasuhisa Akazome ◽  
Buntaro Zempo ◽  
Yoshitaka Oka
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Estrogen Receptor Α ◽  
Strongly Correlated ◽  
Hypothalamic Nuclei ◽  
Model Animal ◽  
Double Label ◽  
Breeding Condition ◽  
Relative Potencies

Kiss2, a paralogous gene for kiss1, has recently been identified in several vertebrates. However, their relative potencies for the regulation of reproductive functions appear to differ among species. Here we used medaka as a model animal to examine the kiss1 and kiss2 expression dynamics by in situ hybridization under different conditions: breeding or nonbreeding and ovariectomized or sham operated. Medaka kiss1-expressing neurons and kiss2-expressing neurons were mainly localized in two hypothalamic nuclei, nucleus ventralis tuberis (NVT) and nucleus recessus lateralis (NRL), respectively. NRL kiss2 expression did not change according to differences in breeding condition, whereas NVT kiss1 expression was strongly correlated with breeding condition. In addition, ovariectomy did not change kiss2 expression but significantly decreased the kiss1 expression. Moreover, double-label in situ hybridization revealed that NVT Kiss1 neurons coexpress estrogen receptor-α, whereas NRL Kiss2 neurons do not. From these results, we conclude that the NVT Kiss1 neurons are positively regulated by ovarian estrogen via their coexpressed estrogen receptor-α and are directly involved in the central regulation of reproduction in medaka. In contrast, we argue that the NRL Kiss2 neurons in medaka may serve nonreproductive functions. These functional differences between Kiss1 and Kiss2 neurons are discussed from a phylogenetic viewpoint.

Gonadal expression of c-kit encoded at the W locus of the mouse

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1057-1069 ◽  
Author(s):  
K. Manova ◽  
K. Nocka ◽  
P. Besmer ◽  
R.F. Bachvarova
Keyword(s):  
In Situ Hybridization ◽  
Germ Cells ◽  
Leydig Cells ◽  
Interstitial Tissue ◽  
Type B ◽  
Age Dependence ◽  
Oocyte Growth ◽  
Adult Mice

Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.

scholarly journals Gastric Parietal Cells: Potent Endocrine Role in Secreting Estrogen as a Possible Regulator of Gastro-Hepatic Axis

Endocrinology ◽  
2002 ◽  
Vol 143 (8) ◽  
pp. 3162-3170 ◽  
Author(s):  
Takashi Ueyama ◽  
Nobuyuki Shirasawa ◽  
Mitsuteru Numazawa ◽  
Keiko Yamada ◽  
Momoko Shelangouski ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Gastric Mucosa ◽  
Estrogen Receptor Α ◽  
Parietal Cells ◽  
Rt Pcr ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Gastric Parietal Cells

Abstract Estrogen, if it is produced in the gastrointestinal tract, may overflow into the systemic circulation in the case of increased portal-systemic shunting. This idea is in accord with a significant step-up in serum estradiol (E2) concentration in the portal vein of rats, compared with that in the artery. Gene expression of aromatase, estrogen synthetase, was demonstrated by RT-PCR in the gastric mucosa of male and female adult rats, equivalent to that in the ovary. Aromatase activity and production of E2 in the gastric mucosa were demonstrated by 3H2O assay and gas chromatography-mass spectrometry, and they were inhibited by aromatase inhibitor, 4-hydroxyandrostenedione. Conversion of 14C-androstenedione to 14C-E2 through 14C-testosterone in cultured gastric mucosa was also demonstrated. Parietal cells exhibited strong signals for aromatase mRNA and immunoreactive protein by in situ hybridization histochemistry and immunohistochemistry. Estrogen receptor α mRNA and immunoreactive protein were demonstrated in hepatocytes by RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Total gastrectomy reduced portal venous E2 concentration, without changing systemic E2 concentration, together with down-regulation of estrogen receptor α mRNA level in the liver. These findings indicate that gastric parietal cells play a potent endocrine role in secreting estrogen that may function as a regulator of the gastro-hepatic axis.

scholarly journals RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of Gαo

2000 ◽  
Vol 350 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Sabine TRAVER ◽  
Carole BIDOT ◽  
Nathalie SPASSKY ◽  
Tania BALTAUSS ◽  
Marie-France DE TAND ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
G Protein ◽  
Rgs Proteins ◽  
Gtpase Activity ◽  
Α Subunit ◽  
Adult Mice ◽  
G Protein Signalling ◽  
Marked Preference

In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of the recently discovered family of RGS (‘regulators of G-protein signalling’) proteins that stimulate the GTPase activity of the GTP-binding α subunit of heterotrimeric G-proteins (Gα). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 is distinguished from other RGS proteins by its marked preference for Gαo over other Gα subunits: RGS14 binds preferentially to Gαo in isolated brain membranes, and also interacts preferentially with Gαo (as compared with Gαi1) to stimulate its GTPase activity. In adult mice, RGS14 expression is restricted to spleen and brain. In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression. Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Gαo, as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Gαo and Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the Gi/o family and those involving the Ras-related GTPase Rap2.

scholarly journals Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse

2010 ◽  
Vol 298 (1) ◽  
pp. E80-E88 ◽  
Author(s):  
Heather M. Dungan Lemko ◽  
Roxana Naderi ◽  
Valeriya Adjan ◽  
Lothar H. Jennes ◽  
Victor M. Navarro ◽  
...  
Keyword(s):  
Basal Forebrain ◽  
Neural Circuits ◽  
Direct Role ◽  
Lh Surge ◽  
Periventricular Nucleus ◽  
Gnrh Neurons ◽  
Gnrh Release ◽  
Double Label ◽  
Lh Secretion

In female mammals, increased ovarian estradiol (E2) secretion triggers GnRH release from neurons in the basal forebrain, which drives LH secretion from the pituitary and subsequently induces ovulation. However, the neural circuits that activate this preovulatory GnRH/LH surge remain unidentified. Neurotensin is expressed in neurons of the anteroventral periventricular nucleus (AVPV), a region thought to be critical for generating the preovulatory GnRH/LH surge. E2 induces neurotensin ( Nts) gene expression in this region, and blockade of neurotensin signaling reduces the LH surge in the rat. We postulated that neurotensin signaling plays a similar role in generating the E2-induced GnRH/LH surge in mice. We used in situ hybridization (ISH) to determine whether E2 induces Nts expression in the mouse and found evidence to support this proposition. Next, we determined that the neurotensin receptor (Ntsr2) is present in many GnRH-expressing neurons. Since the kisspeptin gene ( Kiss1) is expressed in the AVPV and is responsive to E2, we predicted that some neurons in this region express both Kiss1 and Nts; however, by double-label ISH, we observed no coexpression of the two mRNAs. We also postulated that Nts mRNA expression would increase in parallel with the E2-induced LH surge and that the central (icv) administration of neurotensin would stimulate LH secretion and activation of GnRH neurons but found no evidence to support either of these hypotheses. Together, these findings suggest that, although neurotensin neurons in the AVPV are targets for regulation by E2, neurotensin does not appear to play a direct role in generating the GnRH/LH surge in the mouse.

scholarly journals Formaldehyde-Inactivated Whole-Virus Vaccine Protects a Murine Model of Enterovirus 71 Encephalomyelitis against Disease

2009 ◽  
Vol 84 (1) ◽  
pp. 661-665 ◽  
Author(s):  
Kien Chai Ong ◽  
Shamala Devi ◽  
Mary Jane Cardosa ◽  
Kum Thong Wong
Keyword(s):  
In Situ Hybridization ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Neurological Complications ◽  
Viral Culture ◽  
Adult Mice ◽  
Therapeutic Drugs ◽  
Immunohistochemistry Analysis ◽  
Old Mice

ABSTRACT Enterovirus 71 (EV71) causes childhood hand, foot, and mouth disease and neurological complications, and no vaccines or therapeutic drugs are currently available. Formaldehyde-inactivated whole-virus vaccines derived from EV71 clinical isolates and a mouse-adapted virus (MAV) were tested in a mouse model of EV71 encephalomyelitis. After only two immunizations, given to mice at 1 and 7 days of age, the MAV vaccine protected mice at 14 days of age from disease. Tissues from immunized mice were negative for virus by viral culture, reverse transcriptase PCR, immunohistochemistry analysis, and in situ hybridization. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4, and C1 to C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease.

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