Differential expression of inhibin α and βA subunit genes in rat and mouse ovarian follicles during pregnancy

ABSTRACT Relative levels of rat ovarian α inhibin (αI) and βA inhibin (βAI) mRNAs were measured during pregnancy by dot-blot hybridization of ovarian poly(A+) RNA. Follicular patterns of αI and βAI expression in contralateral ovaries from the same rats were also studied by hybridization histochemistry. Oligodeoxynucleotide probes specific for porcine αI and βAI were synthesized, 32P end-labelled and used as hybridization probes on dot-blots of ovarian RNA and frozen sections of ovarian tissue from pregnant rats. During pregnancy, levels of αI and βAI mRNAs remained fairly constant from day 7 after mating until parturition and then fell within 16 h post partum. In all ovaries observed, expression of inhibin genes was located in granulosa cells of healthy antral follicles. In general, the strongest signals for αI and βAI mRNAs were obtained in large follicles, with weaker signals in smaller follicles. Follicular patterns of αI and βAI expression during pregnancy were often dissimilar when βI and βAI were compared over a range of follicles. Considerable βI mRNA was detectable in some follicles in which βAI was reduced or undetectable, despite strong signals for both αI and βAI in an adjacent follicle. Essentially, αI mRNA levels were relatively consistent between groups of follicles, whereas βAI levels varied considerably. βAI mRNA was never observed in a follicle in the absence of αI mRNA, indicating that activin production in any follicle occurs in the presence of αI mRNA. Similar patterns of expression were observed in ovaries from pregnant mice. We have shown that expression of αI and βAI inhibin genes is not regulated uniformly within follicles of pregnant rat and mouse ovaries.

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In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0120107 ◽

1994 ◽

Vol 12 (1) ◽

pp. 107-118 ◽

Cited By ~ 21

Author(s):

A Van Bael ◽

R Huygen ◽

B Himpens ◽

C Denef

Keyword(s):

Cell Cycle ◽

Cell Population ◽

Blot Hybridization ◽

Postnatal Period ◽

S Phase ◽

Female Rats ◽

Mrna Levels ◽

Dot Blot ◽

Total Cell Population

ABSTRACT We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

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The expression of transforming growth factor β in pregnant rat myometrium is hormone and stretch dependent

Reproduction ◽

10.1530/rep-07-0004 ◽

2007 ◽

Vol 134 (3) ◽

pp. 503-511 ◽

Cited By ~ 29

Author(s):

Oksana Shynlova ◽

Prudence Tsui ◽

Anna Dorogin ◽

B Lowell Langille ◽

Stephen J Lye

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Late Gestation ◽

Quiescent State ◽

Mrna Levels ◽

Pregnant Rats ◽

Pregnant Rat ◽

Threefold Increase ◽

Antagonist Ru486

From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor β (TGFβ) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFβs. The expression of TGFβ1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfβ1-3 genes were expressed differentially in pregnant myometrium. Tgfβ2 gene was not affected by pregnancy, whereas the Tgfβ1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfβ3 gene expression throughout pregnancy. Tgfβ3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfβ3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20–23) caused a significant decrease in the expression of Tgfβ3 gene. In addition, Tgfβ3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFβ family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.

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Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes

Biochemical Journal ◽

10.1042/bj2510935 ◽

1988 ◽

Vol 251 (3) ◽

pp. 935-938 ◽

Cited By ~ 6

Author(s):

A H al-Hakim ◽

R Hull

Keyword(s):

Nucleic Acid ◽

Blot Hybridization ◽

Nucleic Acid Hybridization ◽

Cross Linking ◽

Dot Blot ◽

Hybridization Probes ◽

Target Dna ◽

Amino Derivatives ◽

Biotin Molecule ◽

Long Chain

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.

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P-glycoprotein expression and localization in the rat uterus throughout gestation and labor

Reproduction ◽

10.1530/rep-16-0161 ◽

2016 ◽

pp. 195-204 ◽

Cited By ~ 4

Author(s):

Qi-Tao Huang ◽

Oksana Shynlova ◽

Mark Kibschull ◽

Mei Zhong ◽

Yan-Hong Yu ◽

...

Keyword(s):

Cellular Localization ◽

Post Partum ◽

Late Gestation ◽

Mrna Levels ◽

Microvascular Endothelium ◽

Rat Uterus ◽

Pregnant Rats ◽

Transporter Protein ◽

P Glycoprotein

Uterine tissues contain the efflux transporter P-glycoprotein (P-gp, encoded by Abcb1a/1b gene), but little is known about how it changes through gestation. Our aim was to investigate the expression profile and cellular localization of P-gp in the pregnant, laboring and post-partum (PP) rat uterus. We propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial Abcb1a/1b/P-gp. Samples from bilaterally and unilaterally pregnant rats were collected throughout gestation, during labor, and PP (n=4–6/gestational day). RNA and protein were isolated and subjected to quantitative PCR and immunoblotting; P-gp transcript and protein were localized by in situ hybridization and immunohistochemistry. Expression of Abcb1a/1b gene and membrane P-gp protein in uterine tissue (1) increased throughout gestation, peaked at term (GD19-21) and dropped during labor (GD23L); and (2) was upregulated only in gravid but not in empty horn of unilaterally pregnant rats. (3) The drop of Abcb1a/1b mRNA on GD23 was prevented by artificial maintenance of elevated progesterone (P4) levels in late gestation; (4) injection of the P4 receptor antagonist RU486 on GD19 caused a significant decrease in Abcb1 mRNA levels. (5) In situ hybridization and immunohistochemistry indicated that Abcb1/P-gp is absent from myometrium throughout gestation; (6) was expressed exclusively by uterine microvascular endothelium (at early gestation) and luminal epithelium (at mid and late gestation), but was undetectable during labor. In conclusion, ABC transporter protein P-gp in pregnant uterus is hormonally and mechanically regulated. However, its substrate(s) and precise function in these tissues during pregnancy remains to be determined.

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Variation in the amounts of hepatic copper, zinc and metallothionein mRNA during development in the rat

Biochemical Journal ◽

10.1042/bj2380023 ◽

1986 ◽

Vol 238 (1) ◽

pp. 23-27 ◽

Cited By ~ 20

Author(s):

J F B Mercer ◽

A Grimes

Keyword(s):

Placental Transfer ◽

Blot Hybridization ◽

Neonatal Rat ◽

Dot Blot ◽

Pregnant Rats ◽

Hepatic Copper ◽

Zinc Concentrations ◽

Copper Zinc ◽

Hepatic Metallothionein ◽

Ornithine Transcarbamoylase

Amounts of hepatic metallothionein mRNA were assessed in RNA from foetal and neonatal rat livers by using dot-blot hybridization. Metallothionein mRNA began to increase about day 15 of gestation and reached a foetal maximum of 5-fold higher than adult values between 18 and 21 days of gestation. The amounts fell significantly for the first 3 days after parturition, and rose again to 6-fold above adult values 6 days after birth. By 15 days after birth the metallothionein mRNA had declined to adult amounts. In comparison, amounts of ornithine transcarbamoylase mRNA did not vary greatly during development. Hepatic zinc concentrations increased from day 14 of gestation to a maximum just before birth, and remained above adult values until 30 days after birth. From 14 days of gestation to 8 days after birth, hepatic copper concentrations were about 4-fold higher than in the adult, but a substantial increase (to about 9-fold higher than in the adult) occurs between 10 and 15 days after birth. CdCl2 administered to pregnant rats on day 18 of gestation was shown to block placental transfer of zinc, and we found decreased foetal hepatic zinc concentration after the CdCl2 treatment, but this failed to cause a significant decrease in metallothionein mRNA, suggesting that zinc may not be the primary inducer of hepatic metallothionein mRNA during foetal life.

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LH down-regulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis

Journal of Endocrinology ◽

10.1677/joe.0.1620409 ◽

1999 ◽

Vol 162 (3) ◽

pp. 409-415 ◽

Cited By ~ 12

Author(s):

MC Botte ◽

Y Lerrant ◽

A Lozach ◽

A Berault ◽

R Counis ◽

...

Keyword(s):

Blot Hybridization ◽

Fold Increase ◽

Inhibitory Effect ◽

Gonadotropin Releasing Hormone ◽

Mrna Levels ◽

Dot Blot ◽

Releasing Hormone ◽

Rat Testis ◽

Gnrh Analogs ◽

Serum Lh

The demonstration of an inhibitory effect of gonadotropin-releasing hormone (GnRH) agonists upon steroidogenesis in hypophysectomized rats and the presence of mRNA coding for GnRH and GnRH receptors (GnRH-R) in rat gonads suggests that GnRH can act locally in the gonads. To assess this hypothesis, we investigated the effects of GnRH analogs, gonadotropins and testosterone on the levels of both GnRH and GnRH-R mRNA in the rat testis. Using dot blot hybridization, we measured the mRNA levels 2 to 120 h after the administration of the GnRH agonist, triptorelin. We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection (about 38% of control). However, the kinetics for testis GnRH-R mRNA were different from those previously found for pituitary GnRH-R mRNA under the same conditions. Initially, the concentrations of serum LH and FSH peaked, then declined, probably due to the desensitization of the gonadotrope cells. In contrast, the GnRH antagonist, antarelix, after 8 h induced a 2.5-fold increase in GnRH-R mRNA, but not in GnRH mRNA, while gonadotropins levels were reduced. Human recombinant FSH had no significant effect on either GnRH or GnRH-R mRNA levels. Inversely, GnRH-R mRNA levels markedly decreased by 21% of that of control 24 h after hCG injection. Finally, 24 h after testosterone injection, a significant increase in GnRH-R mRNA levels (2.3 fold vs control) was found, but a reduction in the concentration of serum LH, probably by negative feedback on the pituitary, was observed. In contrast, GnRH mRNA levels were not significantly altered following testosterone treatment. Since LH receptors, GnRH-R and testosterone synthesis are colocalized in Leydig cells, our data suggest that LH could inhibit the GnRH-R gene expression or decrease the GnRH-R mRNA stability in the testis. However, this does not exclude the possibility that GnRH analogs could also affect the GnRH-R mRNA levels via direct binding to testicular GnRH-R. In contrast, the regulation of GnRH mRNA levels appeared to be independent of gonadotropins. Taken together, our results suggest a regulation of GnRH and GnRH-R mRNA specific for the testis.

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Distinct trans-placental effects of maternal immune activation by TLR3 and TLR7 agonists: implications for schizophrenia risk

10.1101/2021.09.20.460754 ◽

2021 ◽

Author(s):

Jaedeok Kwon ◽

Maria Suessmilch ◽

Alison McColl ◽

Jonathan Cavanagh ◽

Brian J. Morris

Keyword(s):

Disease Risk ◽

Later Life ◽

Maternal Blood ◽

Maternal Plasma ◽

Poly I:C ◽

Psychiatric Disease ◽

Mrna Levels ◽

Pregnant Rats ◽

Pregnant Mice ◽

Virus Exposure

AbstractExposure to infection in utero predisposes towards psychiatric diseases such as autism, depression and schizophrenia in later life. The mechanisms involved are typically studied by administering mimetics of double-stranded (ds) RNA viral or bacterial infection to pregnant rats or mice. The effect of single-stranded (ss) virus mimetics has been largely ignored, despite evidence linking prenatal ss virus exposure specifically with psychiatric disease. Understanding the effects of gestational ss virus exposure has become even more important with the current SARS-CoV-2 pandemic. In this study, in pregnant mice, we compare directly the effects, on the maternal blood, placenta and the embryonic brain, of maternal administration of ds-virus mimetic poly I:C (to activate toll-like receptor 3, TLR3) and ss-virus mimetic resiquimod (to activate TLR7/8). We find that, 4h after the administration, both poly I:C and resiquimod elevated the levels of IL-6, TNFα, and chemokines including CCL2 and CCL5, in maternal plasma. Both agents also increased placental mRNA levels of IL-6 and IL-10, but only resiquimod increased placental TNFα mRNA. In foetal brain, poly I:C produced no detectable immune-response-related increases, whereas pronounced increases in cytokine (e.g. Il-6, Tnfα) and chemokine (e.g. Ccl2, Ccl5) expression were observed with maternal resiquimod administration. The data show substantial differences between the effect of maternal exposure to a TLR7/8 activator as compared to a TLR3 activator. There are significant implications for future modelling of diseases where maternal ss virus exposure contributes to environmental disease risk in offspring.

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A profound decrease in maternal arginine uptake provokes endothelial nitration in the pregnant rat

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01051.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. H1156-H1163 ◽

Cited By ~ 13

Author(s):

Ran Reshef ◽

Doron Schwartz ◽

Merav Ingbir ◽

Alexander Shtabsky ◽

Tamara Chernichovski ◽

...

Keyword(s):

Northern Blot Analysis ◽

Late Pregnancy ◽

Endothelial Damage ◽

Mrna Levels ◽

Protein Nitration ◽

Pregnant Rats ◽

Pregnant Rat ◽

Cationic Amino Acid ◽

Kinase C ◽

Arginine Uptake

While a specific role for nitric oxide (NO) in inducing the hemodynamic alterations of pregnancy is somewhat controversial, it is widely accepted that excess NO is generated during pregnancy. l-Arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate l-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as the specific arginine transporter for endothelial NO synthase. The present study was designed to test the hypothesis that, during pregnancy, when arginine consumption by the fetus is significantly increased, compensatory changes in maternal arginine uptake affect the endothelium. Uptake of radiolabeled arginine (l-[3H]arginine) by freshly harvested maternal aortic rings from pregnant rats decreased by 65 and 30% in mid- and late pregnancy, respectively, compared with those obtained from virgin animals. This decrease was associated with a significant increase in endothelial protein nitration (the footprint of peroxynitrite generation), as shown by both Western blotting and immunohistochemistry utilizing anti-nitrotyrosine antibodies, reflecting endothelial damage. Northern blot analysis revealed that steady-state aortic CAT-1 mRNA levels did not change throughout pregnancy, whereas CAT-1 protein abundance was significantly increased, peaking at mid-pregnancy. Protein content of protein kinase C (PKC)-α, which was previously shown to decrease CAT-1 activity, increased significantly in the pregnant animals and was associated with a significant increase in CAT-1 phosphorylation. Intraperitoneal injection of α-tocopherol, a PKC-α inhibitor, prevented the decrease in arginine transport and attenuated protein nitration. In conclusion, aortic arginine uptake is reduced during pregnancy, through posttranslational modulation of CAT-1 protein, presumably via upregulation of PKC-α. The aforementioned findings are associated with an increase in protein nitration and, therefore, in selected individuals, may lead to the development of certain forms of endothelial dysfunction, like preeclampsia.

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Development and characteristics of an oestrogen sulphotransferase in placenta and uterus of the pregnant mouse. Comparison between mouse and rat

Biochemical Journal ◽

10.1042/bj2160451 ◽

1983 ◽

Vol 216 (2) ◽

pp. 451-457 ◽

Cited By ~ 20

Author(s):

R Hobkirk ◽

C A Cardy ◽

F Saidi ◽

T G Kennedy ◽

L R Girard

Keyword(s):

Post Partum ◽

Pregnant Mouse ◽

Late Gestation ◽

Tissue Concentrations ◽

Endometrial Stromal Cells ◽

Pregnant Rat ◽

Decidua Basalis ◽

Absolute Requirement ◽

Decidual Cells ◽

Pregnant Mice

The mouse placenta possesses a soluble oestrogen sulphotransferase activity which increases markedly from at least 12 days of gestation until term. At about 16 days of gestation, a similar activity is found in the uterus. This activity also increases until term and disappears rapidly post partum. The uterine enzyme activity appears to require the presence of the foetal unit for its onset, since unoccupied horns, whether their endometrial stromal cells are differentiated to decidual cells or not, are essentially devoid of it. Uterine cytosols from non-pregnant mice are also inactive in this respect. In late gestation, the uterine sulphotransferase is confined to the decidua basalis, the areas to which the placentas are attached. The sulphotransferase(s) of placenta and uterus has an absolute requirement for 3′-phosphoadenosine 5′-phosphosulphate, and possesses little activity in the absence of exogenous thiol groups. Stimulation is also seen in the presence of Mn2+, Mg2+ or Ca2+. Oestrone and oestradiol, and to a lesser degree oestriol, are substrates for the enzyme(s), whereas testosterone, cortisol and dehydroepiandrosterone are not. Oestrone and oestradiol at higher concentrations (1.0-1.5 microM) completely inhibit the enzyme(s). These enzymes could play a role in altering tissue concentrations of active oestrogens during gestation in the mouse. Oestrogen sulphotransferase activity is low or absent in reproductive tissues of the pregnant rat.

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