RESUMEN
Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.
Asunto(s)
Cuerpo Lúteo/metabolismo , Citocinas/genética , Células de la Granulosa/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Oocitos/metabolismo , Células Tecales/metabolismo , Acrilamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Cuerpo Lúteo/citología , Cuerpo Lúteo/efectos de los fármacos , Cuerpo Lúteo/crecimiento & desarrollo , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Diestro/efectos de los fármacos , Diestro/genética , Inhibidores Enzimáticos/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Inmunohistoquímica , Ratones , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Piperidinas/farmacología , Proestro/efectos de los fármacos , Proestro/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tecales/citología , Células Tecales/efectos de los fármacosRESUMEN
The orofacial pain pathway projects to the parabrachial and amygdala, and sex steroids have been shown to affect neuronal activity in these regions. GABA positive cells in the amygdala are influenced by sex steroid metabolites to affect pain, and sex steroids have been shown to alter the expression of genes in the parabrachial, changing neuronal excitability. Mechanisms by which sex steroids affect amygdala and parabrachial signaling are unclear. The expression of genes in the parabrachial and amygdala in diestrus (low estradiol) and proestrus (high estradiol) female rats were evaluated in this study. First, varicella zoster virus was injected into the whisker pad of female rats to induce a pain response. Second, gene expression was quantitated using RNA-seq one week after injection. Genes that had the greatest change in expression and known to function in pain signaling were selected for the quantitation of protein content. Protein expression of four genes in the parabrachial and seven genes in the amygdala were quantitated by ELISA. In the parabrachial, neurexin 3 (Nrnx3) was elevated at proestrus. Nrnx3 has a role in AMPA receptor and GABA signaling. Neuronatin (Nnat) and protein phosphatase, Mg2+/Mn2+ dependent 1E (Ppm1e) were elevated in the parabrachial of diestrus animals both genes having a role in pain signaling. Epoxide hydroxylase (Ephx2) was elevated in the parabrachial at proestrus and the vitamin D receptor (Vdr) was elevated in the amygdala. Ephx2 antagonists and vitamin D have been used to treat neuropathic pain. In conclusion, sex steroids regulate genes in the parabrachial and amygdala that might result in the greater pain response observed during diestrus.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Diestro/genética , Regulación de la Expresión Génica , Herpesvirus Humano 3/fisiología , Inyecciones , Proestro/genética , Animales , Epóxido Hidrolasas/metabolismo , Femenino , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Calcitriol/metabolismoRESUMEN
Modifications of the endometrial transcriptome at day 7 of the estrus cycle are crucial to maintain gestation after transfer of in vitro-produced (IVP) embryos, although these changes are still largely unknown. The aim of this study was to identify genes, and their related biological mechanisms, important for pregnancy establishment based on the endometrial transcriptome of recipient lactating dairy cows that become pregnant in the subsequent estrus cycle, upon transfer of IVP embryos. Endometrial biopsies were taken from Holstein Friesian cows on day 6-8 of the estrus cycle followed by embryo transfer in the following cycle. Animals were classified retrospectively as pregnant (PR, n = 8) or nonpregnant (non-PR, n = 11) cows, according to pregnancy status at 26-47 days. Extracted mRNAs from endometrial samples were sequenced with an Illumina platform to determine differentially expressed genes (DEG) between the endometrial transcriptome from PR and non-PR cows. There were 111 DEG (false discovery rate < 0.05), which were mainly related to extracellular matrix interaction, histotroph metabolic composition, prostaglandin synthesis, transforming growth factor-ß signaling as well as inflammation and leukocyte activation. Comparison of these DEG with DEG identified in two public external data sets confirmed the more fertile endometrial molecular profile of PR cows. In conclusion, this study provides insights into the key early endometrial mechanisms for pregnancy establishment, after IVP embryo transfer in dairy cows.
Asunto(s)
Bovinos/genética , Diestro/genética , Transferencia de Embrión/veterinaria , Endometrio/metabolismo , Fertilidad/genética , Fertilización In Vitro/veterinaria , Transcriptoma , Animales , Biopsia , Bovinos/sangre , Transferencia de Embrión/métodos , Endometrio/patología , Femenino , Fertilización In Vitro/métodos , Regulación de la Expresión Génica , Lactancia , Embarazo , Progesterona/sangre , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , RNA-Seq , Estudios RetrospectivosRESUMEN
Frizzled 3 is an important receptor in the Wnt/ß-catenin pathway, a conserved signaling pathway that regulates gene expression and controls diverse developmental processes. However, the role of this protein during follicular development in the adult ovary is not known. The present study was designed to investigate the expression and localization of Frizzled 3 mRNA and protein during the estrous cycle in the mouse ovary through in situ hybridization (ISH), real-time quantitative polymerase chain reaction, immunohistochemistry and western blot. ISH results showed that in proestrus, high expression of Frizzled 3 was found in the granulosa and stroma with weak levels in the corpus luteum. In estrus and diestrus, the stroma had high Frizzled 3 expression, but levels were low in granulosa cells and corpus luteum. In the metestrus, moderate expression of Frizzled 3 was found in the stroma but low to no expression was found in luteal cells and follicles. The mRNA and protein levels of Frizzled 3 were found to be the highest in proestrus and diestrus compared to estrus and metestrus (P < 0.05), confirming the ISH results. During estrus and diestrus, high Frizzled 3 expression was observed in the stroma and moderate levels in granulosa cells, and during estrus and proestrus, low expression was seen in the oocyte cell membrane. The western blot results further confirmed this change during the estrous cycle. Together, these results indicate that Frizzled 3 is involved in regulating follicular development and oocyte maturation during the estrous cycle.
Asunto(s)
Cuerpo Lúteo/metabolismo , Receptores Frizzled/genética , Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Animales , Cuerpo Lúteo/crecimiento & desarrollo , Cuerpo Lúteo/ultraestructura , Diestro/genética , Estro/genética , Femenino , Receptores Frizzled/metabolismo , Hibridación in Situ , Ratones , Ratones Endogámicos ICR , Oocitos/crecimiento & desarrollo , Oocitos/ultraestructura , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/ultraestructura , Proestro/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de TiempoRESUMEN
Gonadotropes in the anterior pituitary gland are of particular importance within the hypothalamic-pituitary-gonadal axis because they provide a means of communication and thus a functional link between the brain and the gonads. Recent results indicate that female gonadotropes may be organized in the form of a network that shows plasticity and adapts to the altered endocrine conditions of different physiological states. However, little is known about functional changes on the molecular level within gonadotropes during these different conditions. In this study we capitalize on a binary genetic strategy in order to fluorescently label murine gonadotrope cells. Using this mouse model allows to produce an enriched gonadotrope population using fluorescence activated cell sorting to perform mRNA sequencing. By using this strategy, we analyze and compare the expression profile of murine gonadotropes in different genders and developmental and hormonal stages. We find that gonadotropes taken from juvenile males and females, from cycling females at diestrus and at proestrus, from lactating females, and from adult males each have unique gene expression patterns with approximately 100 to approximately 500 genes expressed only in one particular stage. We also demonstrate extensive gene-expression profile changes with up to approximately 2200 differentially expressed genes when comparing female and male development, juveniles and adults, and cycling females. Differentially expressed genes were significantly enriched in the GnRH signaling, calcium signaling, and MAPK signaling pathways by Kyoto Encyclopedia of Genes and Genomes analysis. Our data provide an unprecedented molecular view of the primary gonadotropes and reveal a high degree of molecular plasticity within the gonadotrope population.
Asunto(s)
Plasticidad de la Célula/genética , Ciclo Estral/genética , Gonadotrofos/metabolismo , Lactancia/genética , ARN Mensajero/genética , Maduración Sexual/genética , Transcriptoma , Animales , Señalización del Calcio/genética , Diestro/genética , Diestro/metabolismo , Ciclo Estral/metabolismo , Femenino , Citometría de Flujo , Hormona Liberadora de Gonadotropina/metabolismo , Lactancia/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Proestro/genética , Proestro/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/genéticaRESUMEN
The kisspeptin (Kp) neurons in the anteroventral periventricular nucleus (AVPV) are essential for the preovulatory LH surge, which is gated by circulating estradiol (E2) and the time of day. We investigated whether AVPV Kp neurons in intact female mice may be the site in which both E2 and daily signals are integrated and whether these neurons may host a circadian oscillator involved in the timed LH surge. In the afternoon of proestrous day, Kp immunoreactivity displayed a marked and transient decrease 2 hours before the LH surge. In contrast, Kp content was stable throughout the day of diestrus, when LH levels are constantly low. AVPV Kp neurons expressed the clock protein period 1 (PER1) with a daily rhythm that is phase delayed compared with the PER1 rhythm measured in the main clock of the suprachiasmatic nuclei (SCN). PER1 rhythm in the AVPV, but not in the SCN, exhibited a significant phase delay of 2.8 hours in diestrus as compared with proestrus. Isolated Kp-expressing AVPV explants from PER2::LUCIFERASE mice displayed sustained circadian oscillations of bioluminescence with a circadian period (23.2 h) significantly shorter than that of SCN explants (24.5 h). Furthermore, in AVPV explants incubated with E2 (10 nM to 1 µM), the circadian period was lengthened by 1 hour, whereas the SCN clock remained unaltered. In conclusion, these findings indicate that AVPV Kp neurons display an E2-dependent daily rhythm, which may possibly be driven by an intrinsic circadian clock acting in combination with the SCN timing signal.
Asunto(s)
Núcleo Hipotalámico Anterior/metabolismo , Relojes Circadianos/genética , Kisspeptinas/genética , Animales , Diestro/efectos de los fármacos , Diestro/genética , Diestro/metabolismo , Estradiol/metabolismo , Estradiol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proestro/efectos de los fármacos , Proestro/genética , Proestro/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMEN
The objective of this study was to compare expression of estrogen receptor alpha (ER-α), ß (ER-ß), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-α (ESR1), ER-ß (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-ß, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT method. Expression for ESR1 (P<0.05) tended to be higher during estrus than diestrus in the endometrium (P=0.1) and myometrium (P=0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P<0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P=0.1). Expression for PGR was greater (P<0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P=0.05) and tended to be higher during diestrus in relation to pregnancy (P=0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P>0.05). Immunolabeling in the endometrium appeared to be more intense for ER-α during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares.
Asunto(s)
Endometrio/metabolismo , Ciclo Estral , Caballos , Miometrio/metabolismo , Preñez , Receptores de Prostaglandina E/genética , Receptores de Esteroides/genética , Animales , Diestro/genética , Diestro/metabolismo , Ciclo Estral/genética , Ciclo Estral/metabolismo , Estro/genética , Estro/metabolismo , Femenino , Edad Gestacional , Hormonas Esteroides Gonadales/metabolismo , Caballos/genética , Caballos/metabolismo , Ovario/metabolismo , Embarazo , Preñez/genética , Preñez/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Esteroides/metabolismoRESUMEN
Progesterone is a neuroactive hormone with non-genomic effects on GABA(A) receptors (GABA(A)R). Changes in the expression of GABA(A)R subunits are related to depressive-like behaviors in rats. Moreover, sex differences and depressive behaviors have been associated with prefrontal brain asymmetry in rodents and humans. Thus, our objective was to investigate the effect of progesterone on the GABA(A)R α1 and γ2 subunits mRNA expression in the right and left prefrontal cortex of diestrus female and male rats exposed to the forced swimming test (FST). Male and female rats (n = 8/group) were randomly selected to receive a daily dose of progesterone (0·4 mg·kg⻹) or vehicle, during two complete female estrous cycles (8-10 days). On the experiment day, male rats or diestrus female rats were euthanized 30 min after the FST. Our results showed that progesterone significantly increased the α1 subunit mRNA in both hemispheres of male and female rats. Moreover, there was an inverse correlation between depressive-like behaviors and GABA(A)R α1 subunit mRNA expression in the right hemisphere in female rats. Progesterone decreased the GABA(A)R γ2 mRNA expression only in the left hemisphere of male rats. Therefore, we conclude that the GABA(A) system displays an asymmetric distribution according to sex and that progesterone, at lower doses, presents an antidepressant effect after increasing the GABA(A) R α1 subunit expression in the right prefrontal cortex of female rats.
Asunto(s)
Expresión Génica/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Progesterona/farmacología , Receptores de GABA-A/genética , Análisis de Varianza , Animales , Diestro/genética , Femenino , Masculino , Corteza Prefrontal/metabolismo , Progestinas/farmacología , Subunidades de Proteína/genética , Distribución Aleatoria , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Natación , Factores de TiempoRESUMEN
The aim of this investigation was to evaluate overall DNA damage induced by experimental paradoxical sleep deprivation (PSD) in estrous-cycling and ovariectomized female rats to examine possible hormonal involvement during DNA damage. Intact rats in different phases of the estrous cycle (proestrus, estrus, and diestrus) or ovariectomized female Wistar rats were subjected to PSD by the single platform technique for 96 h or were maintained for the equivalent period as controls in home-cages. After this period, peripheral blood and tissues (brain, liver, and heart) were collected to evaluate genetic damage using the single cell gel (comet) assay. The results showed that PSD caused extensive genotoxic effects in brain cells, as evident by increased DNA migration rates in rats exposed to PSD for 96 h when compared to negative control. This was observed for all phases of the estrous cycle indistinctly. In ovariectomized rats, PSD also led to DNA damage in brain cells. No significant statistically differences were detected in peripheral blood, the liver or heart for all groups analyzed. In conclusion, our data are consistent with the notion that genetic damage in the form of DNA breakage in brain cells induced by sleep deprivation overrides the effects related to endogenous female sex hormones.
Asunto(s)
Daño del ADN , Hormonas Esteroides Gonadales/metabolismo , Privación de Sueño/genética , Privación de Sueño/metabolismo , Sueño REM , Animales , Sangre/metabolismo , Encéfalo/metabolismo , Corticosterona/metabolismo , Diestro/genética , Diestro/fisiología , Estro/genética , Estro/fisiología , Femenino , Hígado/metabolismo , Miocardio/metabolismo , Ovariectomía , Proestro/genética , Proestro/fisiología , Progesterona/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Testosterona/metabolismoRESUMEN
Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRalpha, mPRbeta and mPRgamma) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRgamma. The mPRalpha mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRbeta mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRgamma and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRalpha and mPRbeta in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period.
Asunto(s)
Hipotálamo/metabolismo , Proteínas de la Membrana/metabolismo , Proestro/metabolismo , Receptores de Progesterona/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Ritmo Circadiano , Diestro/genética , Diestro/metabolismo , Estro/genética , Estro/metabolismo , Femenino , Expresión Génica , Espacio Intracelular/genética , Espacio Intracelular/metabolismo , Proteínas de la Membrana/genética , Fotoperiodo , Proestro/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Progesterona/genética , Factores de TiempoRESUMEN
Studies in people and animal models suggest that depression is influenced by natural fluctuations in the levels of 17beta-oestradiol (E(2)), as well as administration of E(2)-based therapies, such as selective oestrogen receptor modulators (SERMs). Elucidating the effects and mechanisms of E(2) is important to improve future E(2)-based therapeutics. An important question is whether effects of E(2) or SERMs for mood regulation act at the alpha or beta isoform of the oestrogen receptor (ER) because some of the unwanted trophic effects of E(2)-based therapies may involve actions at ERalpha, rather than ERbeta. In the present study, whether there are sex differences in depression-like behaviour of adult mice (experiment 1), and the effects of natural fluctuations in E(2) (experiment 2), or administration of E(2) or a SERM that has higher affinity for ERbeta than for ERalpha (diarylpropionitrile; DPN) to ovariectomised (experiment 3) wildtype and ERbeta knockout (betaERKO) mice were investigated. Results of this study supported our hypotheses that: there would be sex differences favouring males for depression-like behaviour and endogenous increases in, or exogenous administration of, E(2) or administration of an ERbeta SERM would decrease depression-like behaviour in wildtype, but not betaERKO, mice. In experiment 1, adult male mice spent less time immobile in the forced swim test (i.e., showed less depression-like behaviour) compared with female mice. In experiment 2, pro-oestrous (higher circulating E(2) levels), compared with dioestrous (lower circulating E(2) levels), mice had reduced immobility in the forced swim test; this effect was not observed in betaERKO mice. In experiment 3, administration of E(2) or DPN to ovariectomised wildtype, but not betaERKO, mice decreased immobility compared with vehicle administration, these data suggest that ERbeta may be required for some of the anti-depressant-like effects of E(2).
Asunto(s)
Depresión/tratamiento farmacológico , Depresión/genética , Estradiol/uso terapéutico , Receptor beta de Estrógeno/genética , Nitrilos/uso terapéutico , Proestro/genética , Propionatos/uso terapéutico , Animales , Diestro/genética , Evaluación Preclínica de Medicamentos , Receptor beta de Estrógeno/agonistas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Caracteres SexualesRESUMEN
As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100 per cent identical to a rat DNA sequence (Accession No. NW_047400)downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.
Asunto(s)
Femenino , Ratas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trompas Uterinas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Perfilación de la Expresión Génica , ADN Complementario/genética , Secuencia de Bases , Northern Blotting , Ciclo Estral/genética , Ciclo Estral/metabolismo , Diestro/genética , Diestro/metabolismo , Expresión Génica/genética , Datos de Secuencia Molecular , Ovariectomía , Embarazo , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Útero/metabolismoRESUMEN
As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100% identical to a rat DNA sequence (Accession No. NW_047400) downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.
Asunto(s)
Trompas Uterinas/metabolismo , Perfilación de la Expresión Génica , ARN Mensajero/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Northern Blotting , ADN Complementario/genética , Diestro/genética , Diestro/metabolismo , Ciclo Estral/genética , Ciclo Estral/metabolismo , Femenino , Expresión Génica/genética , Datos de Secuencia Molecular , Ovariectomía , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción/metabolismo , Útero/metabolismoRESUMEN
Previous studies have revealed that in several animal models, N-methyl-D,L-Aspartate (NMA) stimulates LH secretion by acting at a suprapituitary site. In addition, NMDA receptor antagonists appear to block GnRH neuronal activation on the afternoon of proestrous as evidenced by the lack of c-Fos expression in the neurons and by the absence of an ovulatory LH surge. However, administration of NMA does not induce c-Fos or c-Jun expression in GnRH neurons. To better understand the effects of NMDA receptor activation on GnRH neuronal function, we examined whether GnRH neurons express the NMDA receptor in male rats, and in female rats during diestrus and proestrus, by performing double label in situ hybridization. An 35S-labeled cRNA probe for the NMDA receptor subunit (NMDAR1) was used to quantify NMDAR1 mRNA and a digoxigenin-labeled cRNA probe for GnRH was used to identify GnRH neurons. The data were quantified and expressed as grains/average cell area. In male and female rats, less than 5% of GnRH neurons expressed grain levels twice the minimum detectable level and were considered double-labeled. However, many non-GnRH neurons in the same areas as GnRH neurons expressed high levels of NMDAR1 mRNA. These results suggest that the effects of NMA on GnRH secretion are unlikely to be mediated solely by the activation of NMDA receptors on GnRH neurons. Given the widespread expression of NMDAR1 mRNA in the hypothalamus, it is possible that the stimulatory effects of NMA on GnRH neurons are indirect through activation of other neurons.