6 research outputs found
Increased expression of intra-ovarian adenylyl cyclase 3 following exposure to male-derived scent.
<p>Ovaries of 2 wild type (A, B) and 2 mutant (C, D) unisexually isolated mice, either unexposed (A, C) or exposed (B, D) to male-derived scent for 9 hours, were stained with a polyclonal antibody against adenylyl cyclase 3. Absence of estrous activity in mice not exposed to male-derived scent and presence of such activity in mice exposed to such scent were confirmed by vaginal cytology obtained just before the mice were euthanized. Stars are over the granulosa cell layers of ovarian follicles. CL: corpus luteum. Bars: 50 microns.</p
Cre-mediated recombination in olfactory tubercles of <i>Fshr-Cre</i> transgenic mice.
<p>Olfactory tubercles of R26R reporter mice either carrying (A) or not carrying (B) the <i>Fshr-Cre</i> transgene were stained for LacZ as reported earlier [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139013#pone.0139013.ref004" target="_blank">4</a>]. The blue color indicative of a positive LacZ colorimetric assay, present in olfactory bulbs from the transgenic line, indicates the presence of Cre-mediated recombination driven by the <i>Fshr-Cre</i> transgene in olfactory tubercles of mice harboring this transgene.</p
Olfactory receptor protein expression in mouse and human ovarian granulosa cells.
<p>Immunohistochemical stains of ovaries from wild type (A, C, E) and mutant (B, D, F) mice using either anti-Olfr68 (A-D) or anti-Olfr1508 (E-F). The high magnification images shown in C and D are from the same ovaries shown at lower magnification in A and B, respectively. All mice were littermates and were treated with 5 IU of PMSG 48 hours before being euthanized to mimic the conditions used for the mRNA profiling analyses. Bars: 100 microns. (G) Western blot analyses of protein extracts obtained from 3 different wild type mouse olfactory tubercles (samples 1–3), 3 different wild type mouse ovarian granulosa cells (samples 4–6), and 2 different normal donors of human luteinized granulosa cells undergoing <i>in vitro</i> fertilization procedures (samples 7–8). Cells from the donor from whom sample 7 was derived were also cultured <i>in vitro</i> for 8 days before being analyzed by western blotting (sample 7a). The blot was probed with an antibody against Olfr68, which shows 82% sequence homology to human OR52A5 and 73% homology to human OR52A1, both with molecular sizes of 36 KDa.</p
Increased expression of olfactory receptors in ovarian follicles lacking a functional Brca1.
<p>Two pairs of wild type (WT) and mutant (MT) mice, respectively 8 and 11 months old, were synchronized in the proestrus phase of their estrous cycle. Granulosa cells were isolated from frozen histological sections of their ovaries using laser capture microdissection. Expression profiling analyses were performed using total mRNA from each cell preparation. (A) Heat map illustrating the expression levels of genes showing differential down-regulation (blue) or up-regulation (yellow) of at least 2-fold in mutant mice from both age groups. The arrows indicate genes belonging to the olfactory receptor family. The heat map in (B) shows olfactory receptor genes with at least 2-fold differential expression in both age groups.</p
Correlation between gene expression levels determined from microarray <i>versus</i> RT-PCR analyses.
<p>Ten loci were randomly selected from the list of differentially expressed loci in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139013#pone.0139013.s002" target="_blank">S1 Table</a> and their relative expression levels were measured using quantitative RT-PCR. Relative expression values obtained from the expression profiling studies and from the RT-PCR data are shown in the table on the left and plotted in the graph shown on the right. The Pearson correlation coefficient was calculated to evaluate the correlation between expression array and real time RT-PCR data.</p
Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent
<div><p>We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type <i>Brca1</i> to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional <i>Brca1</i> gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that <i>Brca1</i><sup><i>-/-</i></sup> female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by <i>Brca1</i>-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.</p></div