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American Journal of Reproductive... Nov 2023Decidualization is critical to the establishment of mouse normal pregnancy. The fibroblast-like stromal cells in the process form polyploid multinucleated cells. Aurora...
RESEARCH QUESTION
Decidualization is critical to the establishment of mouse normal pregnancy. The fibroblast-like stromal cells in the process form polyploid multinucleated cells. Aurora kinase B (Aurora B) has previously been shown to regulate polyploidy in various cells. However, whether Aurora B regulates the formation of decidual cell polyploidization and its regulatory mechanisms remain poorly understood.
DESIGN
Establish decidualization model of mouse primary endometrial stromal cells in vitro. Construct pseudopregnancy mouse models and delayed-activation mouse models. Detect Aurora B and polyploidization related genes in mouse uteri treated by Aurora B specific inhibitor Barasertib and CPT.
RESULTS
In this study, we found that Aurora B was strongly expressed in endometrial stromal cells after implantation. Additionally, Aurora B was remarkably up regulated in the stromal cells of oil-induced deciduomoa and in vitro decidualization. As an Aurora B specific inhibitor, Barasertib significantly inhibits the mRNA expression of Prl8a2, a marker of mouse decidualization. Furthermore, the protein levels of p-Plk1, Survivin and p-Cdk1 were inhibited by Barasertib. CPT-induced DNA damage suppressed Aurkb (encodes Aurora B) expression, thus resulting in polyploidization.
CONCLUSION
Our data shows that Aurora B is expressed in decidual stromal cells of implantation sites and plays a key role for mouse decidualization. The protein of Plk1, Survivn, and Cdk1 may participate in formation of decidual cell polyploidization during mouse decidualization.
Topics: Animals; Female; Mice; Pregnancy; Aurora Kinase B; Decidua; Embryo Implantation; Polyploidy; Stromal Cells; Uterus
PubMed: 37881124
DOI: 10.1111/aji.13793 -
Toxicology May 2023Beta-cypermethrin (β-CYP) is a universally used pyrethroid pesticide with adverse effects on human health. β-CYP may impair endometrial remodeling in mice; however,...
Beta-cypermethrin (β-CYP) is a universally used pyrethroid pesticide with adverse effects on human health. β-CYP may impair endometrial remodeling in mice; however, the mechanism remains largely unknown. Endometrial remodeling plays a vital role in embryonic development and the maintenance of pregnancy. Therefore, we explored the mechanism by which peri-implantation β-CYP administration reduces uterine remodeling in pregnant mice. The C57BL/6 J pregnant mice were administered a dose of 20 mg/kg.bw. d β-CYP via oral gavage once daily from day 1 of gestation (GD1) to GD7. Molecular markers of endometrial remodeling, stromal cell proliferation, cell cycle regulation, and the PI3K/Akt/mTOR signaling pathway were evaluated in the decidual tissue of the uterus on GD7. An in vivo pseudopregnancy mouse model, a pregnant mouse model treated with an mTOR activator and an mTOR inhibitor and an in vitro decidualization model of mouse endometrial stromal cells were used to confirm β-CYP-induced defective endometrial remodeling and the key molecules expression of PI3K/Akt/mTOR signaling pathway. The results showed that β-CYP decreased the expression of the endometrial remodeling markers MMP9 and LIF in the uterine decidua. Peri-implantation β-CYP treatment markedly downregulated the expression of endometrial proliferation markers PCNA and Ki67 and decreased decidua thickness. Correspondingly, peri-implantation β-CYP exposure upregulated the expression of FOXO1, P57 and p-4E-BP1 in the decidua. Further experiments showed β-CYP significantly inhibited key molecules in the PI3K/Akt/mTOR pathway: PI3K, p-Akt/Akt, p-mTOR, and p-P70S6K in the uterine decidua. Additional experiments showed that aberrant endometrial remodeling induced by β-CYP was aggravated by rapamycin (an mTOR inhibitor) and partially reversed by MHY1485 (an mTOR agonist). In summary, our results indicated that a reduction in the PI3K/Akt/mTOR pathway may enhance defective endometrial remodeling by downregulating the proliferation and differentiation of endometrial stromal cells in early pregnant mice exposed to β-CYP. Our study elucidates the mechanism of defective endometrial remodeling induced by peri-implantation β-CYP exposure.
Topics: Pregnancy; Female; Mice; Humans; Animals; Decidua; Proto-Oncogene Proteins c-akt; Pesticides; Phosphatidylinositol 3-Kinases; Mice, Inbred C57BL; Endometrium; Embryo Implantation; TOR Serine-Threonine Kinases; Pyrethrins; Stromal Cells
PubMed: 37011868
DOI: 10.1016/j.tox.2023.153497 -
Scientific Reports Oct 2022The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized...
The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized by distinct hormone concentration profiles. Given the difficulty of repeatedly sampling plasma steroid hormones from rodents, the primary method for classifying estrous stage is by identifying vaginal epithelial cell types. However, manual classification of epithelial cell samples is time-intensive and variable, even amongst expert investigators. Here, we use a deep learning approach to achieve classification accuracy at expert level. Due to the heterogeneity and breadth of our input dataset, our deep learning approach ("EstrousNet") is highly generalizable across rodent species, stains, and subjects. The EstrousNet algorithm exploits the temporal dimension of the hormonal cycle by fitting classifications to an archetypal cycle, highlighting possible misclassifications and flagging anestrus phases (e.g., pseudopregnancy). EstrousNet allows for rapid estrous cycle staging, improving the ability of investigators to consider endocrine state in their rodent studies.
Topics: Female; Animals; Rodentia; Deep Learning; Estrus; Estrous Cycle; Hormones
PubMed: 36271290
DOI: 10.1038/s41598-022-22392-w -
Endocrinology Sep 2023Gonadotrophin releasing hormone (GnRH) facilitates the migration of mast cells (MCs) into the involuting mammary gland. As GnRH is also expressed in the ovary, we...
Gonadotrophin releasing hormone (GnRH) facilitates the migration of mast cells (MCs) into the involuting mammary gland. As GnRH is also expressed in the ovary, we examined changes in ovarian MCs. MCs in the ovary were mainly in interstitial tissue and their number increased during the estrous cycle to produce 2 peaks, one at diestrus 2 (20:00 hours) and another at proestrus (17:00 hours). Laser microdissection demonstrated that GnRH mRNA is expressed throughout ovarian tissues (corpora lutea, follicles, and interstitial tissues). GnRH immunoreactivity was also ubiquitous, but MCs were the most strongly immunostained. Analysis of GnRH mRNA in the ovary showed it to fluctuate similarly to the variation in MC number during the estrous cycle, and MCs also expressed GnRH. Local administration of a GnRH agonist (GnRHa) into the hemilateral ovarian bursa increased MCs in the administered ovary. MC number and GnRH mRNA were significantly lowered in the pregnant ovary. Prolactin administration suppressed the normal peaks in MC number in the ovary at both diestrus and proestrus. By contrast, a dopamine agonist, administered when prolactin was elevated during pseudopregnancy, increased ovarian MC number. Furthermore, prolactin inhibited GnRHa-induced peritoneal MC migration in a Transwell assay. These data clearly demonstrate that ovarian MC number is regulated positively by local GnRH expression and negatively by prolactin. The suppressive effect of prolactin on GnRH and MCs would be part of its luteotrophic action.
Topics: Female; Pregnancy; Animals; Gonadotropin-Releasing Hormone; Ovary; Prolactin; Mast Cells; RNA, Messenger
PubMed: 37797313
DOI: 10.1210/endocr/bqad144 -
Animal Science Journal = Nihon Chikusan... 2023This study was conducted to examine the effects of different pseudopregnancy periods in nonpregnant sows on artificial lactation induction efficiency and milk...
This study was conducted to examine the effects of different pseudopregnancy periods in nonpregnant sows on artificial lactation induction efficiency and milk composition. Sixteen pseudopregnant sows (n = 4 per group) were treated with 5 mg of estradiol dipropionate at 28 (Group D38), 35 (Group D45), 42 (Group D52), and 49 (Group D59) days after the end of estrus, followed by prostaglandin F as 0.175-mg cloprostenol twice at 12 h intervals 10 days later. The overall success rate of lactation induction was 81.3%. The lactation rates were significantly higher in Groups D38, D45, and D59 (100.0%) than in Group D52 (25.0%). The milk immunoglobulin (Ig) G concentration was significantly higher in Group D38 than in Group D59. However, IgA levels and milk compositions (protein, ash, and lactose) did not differ among the groups. Lactation induction was successful between 38 and 59 days of pseudopregnancy. Apart from IgG, pseudopregnancy length did not affect milk components from 38 to 59 days of pseudopregnancy.
Topics: Pregnancy; Swine; Animals; Female; Pseudopregnancy; Estrus; Prostaglandins F; Milk; Lactation
PubMed: 36752079
DOI: 10.1111/asj.13815 -
Molecular Human Reproduction Mar 2020The study investigated the effect of normal and supraphysiological (resulting from gonadotropin-dependent ovarian stimulation) levels of estradiol (E2) and progesterone...
Uterine aquaporin expression is dynamically regulated by estradiol and progesterone and ovarian stimulation disrupts embryo implantation without affecting luminal closure.
The study investigated the effect of normal and supraphysiological (resulting from gonadotropin-dependent ovarian stimulation) levels of estradiol (E2) and progesterone (P4) on mouse uterine aquaporin gene/protein (Aqp/AQP) expression on Day 1 (D1) and D4 of pregnancy. The study also examined the effect of ovarian stimulation on uterine luminal closure and uterine receptivity on D4 of pregnancy and embryo implantation on D5 and D7 of pregnancy. These analyses revealed that the expression of Aqp3, Aqp4, Aqp5 and Aqp8 is induced by E2 while the expression of Aqp1 and Aqp11 is induced by P4. Additionally, P4 inhibits E2 induction of Aqp3 and Aqp4 expression while E2 inhibits Aqp1 and Aqp11 expression. Aqp9, however, is constitutively expressed. Ovarian stimulation disrupts Aqp3, Aqp5 and Aqp8 expression on D4 and AQP1, AQP3 and AQP5 spatial expression on both D1 and D4, strikingly so in the myometrium. Interestingly, while ovarian stimulation has no overt effect on luminal closure and uterine receptivity, it reduces implantation events, likely through a disruption in myometrial activity and embryo development. The wider implication of this study is that ovarian stimulation, which results in supraphysiological levels of E2 and P4 and changes (depending on the degree of stimulation) in the E2:P4 ratio, triggers abnormal expression of uterine AQP during pregnancy, and this is associated with implantation failure. These findings lead us to recognize that abnormal expression would also occur under any pathological state (such as endometriosis) that is associated with changes in the normal E2:P4 ratio. Thus, infertility among these patients might in part be linked to abnormal uterine AQP expression.
Topics: Animals; Aquaporins; Embryo Implantation; Embryo Transfer; Estradiol; Female; Gene Expression Regulation, Developmental; Mice; Mice, Inbred C57BL; Mifepristone; Ovulation Induction; Pregnancy; Progesterone; Pseudopregnancy; Uterus; Water
PubMed: 31977023
DOI: 10.1093/molehr/gaaa007 -
Lab Animal Feb 2021For the production and rederivation of mouse strains, pseudopregnant female mice are used for embryo transfer and serve as surrogate mothers to support embryo...
For the production and rederivation of mouse strains, pseudopregnant female mice are used for embryo transfer and serve as surrogate mothers to support embryo development to term. Vasectomized males are commonly used to render pseudopregnancy in females, generated by surgical procedures associated with considerable pain and discomfort. Genetically modified mouse strains with a sterility phenotype provide a non-surgical replacement and represent an important application of the 3Rs (Replacement, Reduction, Refinement). However, the maintenance of such genetically modified mouse strains requires extensive breeding and genotyping procedures, which are regulated procedures under national legislation. As an alternative, we have explored the use of sterile male hybrids that result when two wild-type mouse subspecies, Mus musculus musculus and Mus musculus domesticus, interbreed. We find the male STUSB6F1 hybrid, resulting from the mating of female STUS/Fore with male C57BL/6J, ideally suited and demonstrate that its performance for the production of oviduct and uterine transfer recipients is indistinguishable when compared to surgically vasectomized mice. The use of these sterile hybrids avoids the necessity for surgical procedures or the breeding of sterile genetically modified lines and can be generated by the simple mating of two wild-type laboratory strains-a non-regulated procedure. Furthermore, in contrast with the breeding of genetically sterile mice, all male offspring are sterile and suitable for the generation of pseudopregnancy, allowing their efficient production with minimal breeding pairs.
Topics: Animals; Female; Infertility; Male; Mice; Mice, Inbred C57BL; Phenotype; Pregnancy; Pseudopregnancy; Vasectomy
PubMed: 33398200
DOI: 10.1038/s41684-020-00692-w -
Reproductive Sciences (Thousand Oaks,... Mar 2022Cadherins play an essential role in the attachment of the blastocyst to the endometrium, a process known as endometrial receptivity. Loss of E-cadherin expression is...
Cadherins play an essential role in the attachment of the blastocyst to the endometrium, a process known as endometrial receptivity. Loss of E-cadherin expression is essential during the process, while the expression level of the other cadherin, N-cadherin, has been reported to be altered in cases of infertility. Both E-cadherin and N-cadherin can be regulated by members of the PARP family. Specifically, PARP-2, which is under the epigenetic control of miR-149, has been observed to promote E-cadherin expression in other human cells. We investigated the roles of E-cadherin and N-cadherin in endometrial receptivity using mouse models for normal endometrial receptivity, pseudopregnancy, and LPS-induced endometrial receptivity failure. E-cadherin and phosphorylated E-cadherin were predominantly expressed during pre-receptive stages as well as in the implantation site of the receptive stage, which were observed reduced during the later stages of implantation in both implantation and non-implantation regions, while N-cadherin was detected only at pre-receptive stages. E-cadherin and N-cadherin were also seen in the uterus during pseudopregnancy, showing a downregulation trend during receptive and post-receptive stages. LPS-induced failed endometrial receptivity showed upregulation of E-cadherin and downregulation of N-cadherin. The E-cadherin expression promoter, GSK-3, was lost and its suppressor, SLUG was upregulated during normal course of endometrial receptivity in mouse model, while GSK-3 was increased during LPS-induced failed embryo implantation. In an in vitro model of embryo implantation, E-cadherin expression is promoted by PARP-2 and regulated by miR-149 epigenetically in human endometrium epithelial cells. In conclusion, E-cadherin is predominantly expressed during pre-receptive stage and promoted by PARP-2, which is regulated by miR-149 in the endometrial epithelial cells.
Topics: Animals; Cadherins; Embryo Implantation; Endometrium; Female; Mice; MicroRNAs; Poly(ADP-ribose) Polymerases; Pregnancy; Signal Transduction
PubMed: 34460092
DOI: 10.1007/s43032-021-00710-3 -
Reproduction (Cambridge, England) May 2020We previously demonstrated that 5'-AMP-activated protein kinase (AMPK) is essential for normal reproductive functions in female mice. Conditional ablation of Prkaa1 and...
We previously demonstrated that 5'-AMP-activated protein kinase (AMPK) is essential for normal reproductive functions in female mice. Conditional ablation of Prkaa1 and Prkaa2, genes that encode the α1 and α2 catalytic domains of AMPK, resulted in early reproductive senescence, faulty artificial decidualization, uterine inflammation and fibrotic postparturient endometrial regeneration. We also noted a delay in the timing of embryo implantation in Prkaa1/2d/d female mice, suggesting a role for AMPK in establishing uterine receptivity. As outlined in new studies here, conditional uterine ablation of Prkaa1/2 led to an increase in ESR1 in the uteri of Prkaa1/2d/d mice, resulting in prolonged epithelial cell proliferation and retention of E2-induced gene expression (e.g. Msx1, Muc1, Ltf) through the implantation window. Within the stromal compartment, stromal cell proliferation was reduced by five-fold in Prkaa1/2d/d mice, and this was accompanied by a significant decrease in cell cycle regulatory genes and aberrant expression of decidualization marker genes such as Hand2, Bmp2, Fst and Inhbb. This phenotype is consistent with our prior study, demonstrating a failure of the Prkaa1/2d/d uterus to undergo decidualization. Despite these uterine defects, ovarian function seemed to be normal following ablation of Prkaa1/2 from peri-ovulatory follicles in which ovulation, luteinization and serum progesterone levels were not different on day 5 of pregnancy or pseudopregnancy between Prkaa1/2fl/fl and Prkaa1/2d/d mice. These cumulative findings demonstrate that AMPK activity plays a prominent role in mediating several steroid hormone-dependent events such as epithelial cell proliferation, uterine receptivity and decidualization as pregnancy is established.
Topics: AMP-Activated Protein Kinases; Animals; Cell Proliferation; Embryo Implantation; Epithelial Cells; Estradiol; Estrogen Receptor alpha; Female; Mice; Mice, Knockout; Stromal Cells; Uterus
PubMed: 32191914
DOI: 10.1530/REP-19-0402 -
Theriogenology Sep 2019To investigate subtle pregnancy-associated changes in the lab opossum, Monodelphis domestica, an induced ovulator, we compared pregnant with non-pregnant and...
To investigate subtle pregnancy-associated changes in the lab opossum, Monodelphis domestica, an induced ovulator, we compared pregnant with non-pregnant and pseudopregnant animals with regard to serum P levels and progesterone receptor (PR) expression. Using video-verified, time-mated lab opossums as sources of biological material, we compared ovaries, uteri and sera obtained on odd-numbered days of the 14.5-day pregnancy in this animal. Females that mated successfully but did not produce embryos were classified as pseudopregnant. P levels differed significantly between pregnant (N = 21) and either non-pregnant (N = 3) or pseudopregnant (N = 3) opossums, but not between the non-pregnant and pseudopregnant groups. A significant decline in serum P occurred between pregnancy days 3 and 5, coinciding with an elevated probability of pregnancy failure between days 5 and 9. PR was detected in the nuclei of uterine-gland epithelial cells on pregnancy days 5 and 7 as well as variably in the corpora lutea (CL) of animals on pregnancy days 3-11. PR expression in the CL suggests that P may be autostimulatory in lab opossums and that certain levels of this steroid are required during normal pregnancy. The significant day-3 drop in P may explain why pregnancy failure in this polyovular metatherian is likeliest to occur between days 5 and 9, an interval during which the extended period of blastocyst morphogenesis and expansion occurs. Taken together, these results suggest that P may have unrecognized signaling roles not only in pregnancy but perhaps embryonic development as well in the lab opossum.
Topics: Abortion, Veterinary; Animals; Female; Litter Size; Opossums; Pregnancy; Pregnancy, Animal; Progesterone; Pseudopregnancy
PubMed: 31254723
DOI: 10.1016/j.theriogenology.2019.06.026