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Reproductive Biology and Endocrinology... Feb 2010Rats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous...
BACKGROUND
Rats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.
METHODS
To investigate some of the mechanisms involved in these reproductive abnormalities, hypothyroidism was induced in virgin rats by propilthyouracil (0.1 g/L in the drinking water) and we determined circulating hormones by radioimmunoassay and whole ovary expression of ovarian hormone receptors, growth factors and steroidogenic enzymes using semi-quantitative RT-PCR.The study was performed on days 6 to 9 of treatment, corresponding to diestrus I (at 20.00-22.00 h), diestrus II (at 20.00-22.00 h), proestrus and estrus (both at 8.00-10.00 h and 20.00-22.00 h) of the second estrous cycle after beginning propilthyouracil treatment. Another group of rats was mated on day 8 and the treatment continued through the entire pregnancy to evaluate reproductive performance.
RESULTS
Hypothyroidism increased circulating prolactin and estradiol on estrus 5 to 7-fold and 1.2 to 1.4-fold respectively. Growth hormone and insulin-like growth factor 1 diminished 60 and 20% respectively on proestrus morning. Hypothyroidism doubled the ovarian mRNA contents of estrogen receptor-beta on proestrus and estrus evenings, cyp19A1 aromatase mRNA on estrus evening and of growth hormone receptor on proestrus evening. Hypothyroidism did not influence ovulation rate or the number of corpora lutea at term, but a diminished number of implantation sites and pups per litter were observed (Hypothyroid: 11.7 +/- 0.8 vs.
CONTROL
13.9 +/- 0.7).
CONCLUSIONS
Short term hypothyroidism alters normal hormone profile in the cycling rat increasing the expression of estrogen receptor-beta and cyp19A1 aromatase on estrus, which in turn may stimulate estradiol and prolactin secretion, favouring corpus luteum survival and the subsequent instauration of pseudopregnancy.
Topics: Animals; Embryo Implantation; Estrous Cycle; Female; Gonadal Steroid Hormones; Growth Hormone; Hypothyroidism; Insulin-Like Growth Factor I; Male; Ovary; Ovulation; Pregnancy; Prolactin; Propylthiouracil; Rats; Rats, Wistar; Thyroid Hormones; Time Factors
PubMed: 20149258
DOI: 10.1186/1477-7827-8-14 -
Scientific Reports Feb 2020Embryo transfer has been used as one of the essential reproductive technologies for production of new strains and maintenance of genetic resources in animals. Mating...
Embryo transfer has been used as one of the essential reproductive technologies for production of new strains and maintenance of genetic resources in animals. Mating with vasectomised male rats is a requirement for inducing pseudopregnancy in female rats selected for embryo transfer. Although this procedure has been used routinely, large breeding space and high expenditure are required to maintain a sufficient number of females and vasectomised males. This study was performed to induce pseudopregnancy in females by artificial stimulation using sonic vibration instead of vasectomised males. The females continued to be in the dioestrus stage for at least 14 days after artificial stimulation was performed. Of fresh 2-cell embryos that transferred into the oviducts of females after artificial stimulation, 56% was implanted and 50% was developed to offspring. Approximately 46% of the frozen 2-cell embryos were implanted and 24% developed into offspring. Furthermore, 66% of the fresh pronuclear embryos were implanted and 60% developed into offspring. This study successfully induced pseudopregnancy in rat females by artificial stimulation using a sonic vibration. This method, 'Easy-ET', was useful for efficient production and maintenance of rat strains.
Topics: Animals; Breeding; Cryopreservation; Embryo Implantation; Embryo Transfer; Embryo, Mammalian; Estrus; Fallopian Tubes; Female; Male; Pregnancy; Pseudopregnancy; Rats; Sound; Vasectomy; Vibration
PubMed: 32066799
DOI: 10.1038/s41598-020-59611-1 -
Biology of Reproduction Feb 2014The in vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate (DEHP) on the reproductive function of peroxisome proliferator-activated receptor gamma...
In vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate on pseudopregnant rabbit corpora lutea: possible involvement of peroxisome proliferator-activated receptor gamma.
The in vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate (DEHP) on the reproductive function of peroxisome proliferator-activated receptor gamma (PPARG) were studied in rabbit corpora lutea (CL) at early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. The rabbits were in vivo treated with DEHP for 15 days before induction of pseudopregnancy. Immunohistochemistry provided evidence for the presence of PPARG, prostaglandin endoperoxide synthase 1 (PTGS1), PTGS2, prostaglandin E2-9-ketoreductase (PGE2-9-K), and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in all the luteal cells during pseudopregnancy. DEHP decreased progesterone plasma levels and CL production in all the luteal stages and PPARG protein and gene expressions in early and mid-CL. DEHP in vivo treatment reduced PTGS2 protein expression at the late stage and that of PGE2-9-K at all the stages, whereas PTGS1 and 3beta-HSD were not affected. In in vitro cultured CL, DEHP alone, the PPARG antagonist T0070907 alone, or DEHP plus T0070907 diminished progesterone production and 3beta-HSD activity and increased PGF2alpha and PTGS2 in early and mid-CL, whereas DEHP plus the PPARG agonist 15d-PGJ2 did not affect these hormones and enzymes. All the in vitro treatments did not affect PGE2 secretion as well as PTGS1 and PGE2-9-K enzymatic activities in all the luteal stages. These results provided evidence that DEHP favors functional luteolysis of pseudopregnant rabbit CL, with a mechanism that seems to involve PPARG expression down-regulation, an increase of PTGS2 activity and prostaglandin F2alpha secretion, 3beta-HSD down-regulation, and decrease in progesterone.
Topics: Animals; Cells, Cultured; Corpus Luteum; Cyclooxygenase 2; Diethylhexyl Phthalate; Environmental Pollutants; Female; Ovulation; PPAR gamma; Plasticizers; Pseudopregnancy; Rabbits; Time Factors; Toxicity Tests, Acute; Toxicity Tests, Chronic
PubMed: 24403546
DOI: 10.1095/biolreprod.113.109223 -
Reproductive Sciences (Thousand Oaks,... May 2015Endoplasmic reticulum stress (ERS), which is a novel pathway of regulating cellular apoptosis and the function of ERS during corpus luteum (CL) regression, is explored....
Endoplasmic reticulum stress (ERS), which is a novel pathway of regulating cellular apoptosis and the function of ERS during corpus luteum (CL) regression, is explored. Early-luteal stage (day 2), mid-luteal stage (day 7), and late-luteal stage (day 14 and 20) were induced, and the apoptosis of luteal cells was detected by a terminal 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay. The apoptotic cells were increased with the regression of CL, especially during the late-luteal stage. The ERS markers glucose-regulated protein 78 (Grp78), CCAAT/enhancer-binding protein homologous protein (CHOP), X-box binding protein 1 (XBP1), activating transcription factor 6α (ATF6α), eukaryotic initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α), caspase 12, and apoptosis marker caspase 3 were analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry, in agreement with the results of the TUNEL assay; the expression levels of CHOP, caspase 12, and caspase 3 were increased during the process of CL regression. Luteal cells were isolated and cultured in vitro, and the apoptosis of luteal cells was induced by prostaglandin F2α. The ERS was attenuated by the ERS inhibitor tauroursodeoxycholic acid, and the apoptotic rate was analyzed by flow cytometry. The ERS markers Grp78, CHOP, XBP1s, ATF6α, eIF2α, IRE1α, caspase 12, and apoptotic execute marker caspase 3 were analyzed by real-time PCR and immunofluorescence, and the results suggested that the expression of CHOP, caspase 12, and caspase 3 were increased, and there was increased apoptosis of luteal cells. But the expression of IRE1α/XBP1s and eIF2α was not detected. Taken together, the ERS is involved in the CL regression of rats through the CHOP and caspase 12 pathway.
Topics: Activating Transcription Factor 6; Animals; Apoptosis; Caspase 12; Caspase 3; Cells, Cultured; Corpus Luteum; DNA-Binding Proteins; Dinoprost; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Eukaryotic Initiation Factor-2; Female; Gene Expression Regulation; Heat-Shock Proteins; Immunohistochemistry; In Situ Nick-End Labeling; Luteal Cells; Luteal Phase; Luteolysis; Mice; Multienzyme Complexes; Protein Serine-Threonine Kinases; Pseudopregnancy; Rats; Real-Time Polymerase Chain Reaction; Regulatory Factor X Transcription Factors; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor CHOP; Transcription Factors; X-Box Binding Protein 1
PubMed: 25332219
DOI: 10.1177/1933719114553445 -
Molecular Pathology : MP Oct 2001To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta1 (TGF-beta1) in the mouse uterus.
AIMS
To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta1 (TGF-beta1) in the mouse uterus.
METHODS
In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) and TGF-beta1 in uteri from sexually mature female mice that had either been (1) mated with sterile males to induce pseudopregnancy or (2) ovariectomised (OVX) and administered estradiol-17beta (E2) or progesterone (P4), either alone or in combination. Uteri collected on days 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 of pseudopregnancy or at one, three, six, 12, or 24 hours after steroid administration were fixed, sectioned, and incubated with specific riboprobes or antibodies to permit detection and localisation of mRNA or protein for CTGF and TGF-beta1.
RESULTS
On days 0.5-2.5 of pseudopregnancy, CCN2 (CTGF) and TGF-beta1 were principally colocalised to uterine epithelial cells, with much smaller amounts in the stroma. On days 3.5-4.5, there was a reduction of CCN2 (CTGF) and TGF-beta1 in the epithelium but an increase in stromal and endothelial cells, corresponding to a period of extracellular matrix remodelling and neovascularisation within the endometrium. In OVX mice, epithelial cells were weakly positive for both CCN2 (CTGF) and TGF-beta1 in the absence of steroid hormones. Epithelial CTGF mRNA production were strongly but transiently stimulated in OVX mice cells by E2. These effects were antagonised by P4, which itself transiently stimulated epithelial CCN2 (CTGF) production, although less robustly than E2. CTGF and TGF-beta1 protein amounts were high in epithelial cells throughout steroid treatment and were increased in the stroma, where they were relatively long lived. Stromal CCN2 (CTGF) and TGF-beta1 were lower after co-administration of E2 and P4 than in response to each hormone individually. Although ccn2 (ctgf) is a TGF-beta1 inducible gene in other systems, and both growth factors were often co-localised in uterine tissues in these studies, several treatment regimens resulted in high amounts of TGF-beta1 protein in stromal cells without the concomitant production of ccn2 (ctgf) mRNA.
CONCLUSIONS
Maternal factors are principal cues for CCN2 (CTGF) and TGF-beta1 production in the uterus because (1) their expression during pseudopregnancy is comparable to that seen in pregnancy and (2) they are regulated by ovarian steroids. TGF-beta dependent and independent mechanisms of ccn2 (ctgf) gene transcription exist in the uterus that are variably regulated by steroid hormones. Collectively, the data support a role for CCN2 (CTGF) in mediating the effects of steroid hormones and TGF-beta on endometrial function.
Topics: Animals; Connective Tissue Growth Factor; Estradiol; Extracellular Matrix; Female; Growth Substances; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Mice; Neovascularization, Physiologic; Progesterone; Pseudopregnancy; RNA, Messenger; Transforming Growth Factor beta; Uterus
PubMed: 11577177
DOI: 10.1136/mp.54.5.338 -
Methods in Molecular Biology (Clifton,... 2009Introduction of foreign DNA into the mouse germ line is considered a major technical advancement in the fields of developmental biology and genetics. This technology now...
Introduction of foreign DNA into the mouse germ line is considered a major technical advancement in the fields of developmental biology and genetics. This technology now referred to as transgenic mouse technology has revolutionized virtually all fields of biology and provided new genetic approaches to model many human diseases in a whole animal context. Several hundreds of transgenic lines with expression of foreign genes specifically targeted to desired organelles/cells/tissues have been characterized. Further, the ability to spatio-temporally inactivate or activate gene expression in vivo using the "Cre-lox" technology has recently emerged as a powerful approach to understand various developmental processes including those relevant to molecular endocrinology. In this chapter, we will discuss the principles of transgenic mouse technology, and describe detailed methodology standardized at our institute.
Topics: Animals; Female; Genes, Reporter; Mice; Mice, Transgenic; Microinjections; Polymerase Chain Reaction; Pseudopregnancy; Transgenes
PubMed: 19763515
DOI: 10.1007/978-1-60327-378-7_22 -
Scientific Reports Jan 2022Psuedopregnancy for embryo transfer (ET) is usually induced in rats by mating with vasectomized males. Previously, we successfully induced pseudopregnancy using sonic...
Psuedopregnancy for embryo transfer (ET) is usually induced in rats by mating with vasectomized males. Previously, we successfully induced pseudopregnancy using sonic vibration instead (Easy-ET method). The transferred embryos developed normally. Conventionally, stimulation is performed 7 × 30 s with 5 min intervals at the day before ET. However, this protocol is time-consuming because it imitates natural mating behavior. Here, we investigated pseudopregnancy induction with shorter stimulation times. Stimulation was performed 2 × 30 s, with 30 s intervals at the proestrus stage at the day before ET. Of the transferred pronuclear or two-cell embryos, 43% or 62% developed normally, respectively. Furthermore, 67% or 68% of transferred pronuclear or two-cell embryos in rats at estrus stage stimulated on the day of ET developed normally, respectively. Pseudopregnancy was successfully induced with shorter stimulation. Furthermore, this protocol may be used to perform a single-day stimulation and ET operation at the estrus stage.
Topics: Animals; Embryo Transfer; Female; Male; Pregnancy; Pseudopregnancy; Rats; Sound
PubMed: 35075219
DOI: 10.1038/s41598-022-05293-w -
British Medical Journal Jun 1974
Topics: Endometriosis; Female; Fertility; Humans; Laparoscopy; Progestins; Pseudopregnancy
PubMed: 4277447
DOI: 10.1136/bmj.2.5921.682-a -
Biochemical and Biophysical Research... Nov 2013Progesterone (P4) and progesterone receptor (PR) have important functions in uterine environment. In previous studies, using high density DNA microarray analysis, we...
Progesterone (P4) and progesterone receptor (PR) have important functions in uterine environment. In previous studies, using high density DNA microarray analysis, we identified low density lipoprotein receptor-related protein 2 (Lrp2) is one of the genes upregulated by P4 and PR. In present studies, we examined the expression of Lrp2 through real-time PCR, in situ hybridization and immunohistochemistry by P4-PR response. Lrp2 mRNA transcript was significantly increased after P4 treatment in the luminal and glandular epithelium of the wild-type mice. However, Lrp2 expression was not observed in the progesterone receptor knock out (PRKO) mice treated with P4. The expression of Lrp2 expression is not regulated by estrogen. During early pregnancy, the expression of Lrp2 was detected at 2.5 dpc and then significantly increased at 3.5 dpc in luminal and glandular epithelium. These results suggest that Lrp2 is a novel target gene by P4 and PR.
Topics: Animals; Estradiol; Female; Gene Expression Regulation; Low Density Lipoprotein Receptor-Related Protein-2; Mice; Mice, Inbred C57BL; Mice, Knockout; Pregnancy; Progesterone; Pseudopregnancy; Uterus
PubMed: 24140060
DOI: 10.1016/j.bbrc.2013.10.037 -
Biology of Reproduction Oct 2013All mammalian uteri have luminal (LE) and glandular epithelia (GE) in their endometrium. The LE mediates uterine receptivity and blastocyst attachment for implantation,... (Comparative Study)
Comparative Study
All mammalian uteri have luminal (LE) and glandular epithelia (GE) in their endometrium. The LE mediates uterine receptivity and blastocyst attachment for implantation, and the GE synthesize and secrete or transport bioactive substances involved in blastocyst implantation, uterine receptivity, and stromal cell decidualization. However, the mechanisms governing uterine epithelial development after birth and their function in the adult are not fully understood. Here, comprehensive microarray analysis was conducted on LE and GE isolated by laser capture microdissection from uteri on Postnatal Day 10 (PD 10) and day of pseudopregnancy (DOPP) 2.5 and 3.5. This data was integrated with analysis of uteri from gland-containing control and aglandular progesterone-induced uterine gland knockout mice from PD 10 and DOPP 3.5. Many genes were expressed in both epithelia, but there was greater expression of genes in the LE than in the GE. In the neonate, GE-expressed genes were enriched for morphogenesis, development, migration, and retinoic acid signaling. In the adult, LE-expressed genes were enriched for metabolic processes and steroid biosynthesis, whereas retinoid signaling, tight junction, extracellular matrix, and regulation of kinase activity were enriched in the GE. The transcriptome differences in the epithelia support the idea that each cell type has a distinct and complementary function in the uterus. The candidate genes and regulatory networks identified here provide a framework to discover new mechanisms regulating development of epithelia in the postnatal uterus and their functions in early pregnancy.
Topics: Aging; Animals; Animals, Newborn; Decidua; Endometrium; Female; Fertility Agents, Female; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Knockout Techniques; Laser Capture Microdissection; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Oligonucleotide Array Sequence Analysis; Progesterone; Pseudopregnancy; Transcription, Genetic; Transcriptome; Uterus
PubMed: 23946541
DOI: 10.1095/biolreprod.113.111971