Systemic L-ornithine supplementation specifically increases ovarian putrescine levels during ovulation in mice

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of human chorionic gonadotropin (hCG). Aged mice have significantly reduced levels of this periovulatory ODC and putrescine rise. Putrescine supplementation, in vitro during oocyte maturation or in mouse drinking water during the periovulatory period, reduces egg aneuploidies and embryo resorption, improving fertility of aged mice. These studies suggest that periovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine supplementation is expected to increase widespread tissue putrescine levels, raising concerns of nonspecific and unwanted side effects. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that periovulatory supplementation of L-ornithine, the substrate of ODC, might be suitable for delivering putrescine specifically to the ovaries. In this study, we have demonstrated that systemic application of L-ornithine via oral gavage or subcutaneous injection increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in any other tissues. However, our attempts to improve fertility of aged mice through L-ornithine supplementation in mouse drinking water produced either no effects (1% L-ornithine) or negative impact on fertility (4% ornithine). Our results suggest that it might not be feasible to achieve fertility-enhancing ovarian putrescine levels via L-ornithine supplementation in drinking water without encountering undesired consequences of high dose of exogenous L-ornithine.

Temporal Changes In Cyclin D-CDK4/CDK6 And Cyclin E-CDK2 Pathways: Implications For The Mechanism of Deficient Decidualization In An Immune-Based Mouse Model of Unexplained Recurrent Spontaneous Abortion

Deficient endometrial decidualization has been associated with unexplained recurrent spontaneous abortion (URSA). However, the underlying mechanism is poorly understood. Here, we aimed to investigate the temporal cytokine changes and the involvement of the cyclin D-cyclin-dependent kinase (CDK)4/CDK6 and cyclin E-CDK2 pathways in the regulation of the G1 phase of the cell cycle during decidualization in a murine model of URSA. Serum and decidual tissues of URSA group and normal pregnant (NP) group mice were collected from gestation day 4 (GD4) to GD8. The embryo resorption and abortion rates were observed on GD8 and the decidual tissue status was assessed using hematoxylin and eosin staining. Cytokine levels in decidual tissues were analyzed using western blotting and reverse transcription polymerase chain reaction. We found that the embryo resorption rate was significantly increased in the URSA group compared to that in the NP group on GD8. The expression of the decidualization marker prolactin in the serum and decidual lysate of the URSA group was significantly decreased on GD6-8 compared to that of the NP group. Cyclin D, CDK4, CDK6, cyclin E, CDK2 and pRb levels in the URSA group mice were significantly lower compared to those in the NP group mice on GD6-8. Our results suggest that the hyperactivated cyclin D-CDK4/CDK6 and cyclin E CDK2 pathways inhibit the decidualization process on GD4, leading to deficient decidualization on GD8. Moreover, they clarify the role of cytokines in the cyclin D-CDK4/6 and cyclin E-CDK2 pathways during decidualization and provide new insight into URSA pathogenesis.

Human MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.

VCD induces premature ovarian insufficiency (POI) in rats by triggering the autophagy of granulosa cells through regulating the AKT/mTOR signal pathway

Purpose Investigating the pathological mechanism underlying POI modeling in animals with VCD. Methods QRT-PCR was used to detect the expression of miR-144 in the peripheral blood of POI patients, granulosa cells and the ovary tissues. MTT assay was used to evaluate the proliferation and the concentration of E2, FSH, LH, and AMH was determined by ELISA assay. The numbers of autophagosomes were detected by transmission electron microscope and autophagic flux assay. The expression of key proteins in AKT pathway and autophagy proteins was determined by Western blot. HE staining was used to check the state of follicles in the ovary tissues. Immunohistochemistry assay was used to evaluate the expression level of p-AKT, P62, and caspase-3 in the ovary tissues. Finally, the pregnancy rate and embryo resorption rate were calculated. Results MiR-144 was down-regulated in the peripheral blood of POI patients. Deceased cell viability, down-regulated miR-144, and increased autophagosomes were observed in VCD treated granulosa cells, which were reversed by the introduction of SC79 or miR-144. The increased concentration of FSH, and LH, decreased concentration of E2 and AMH, increased number of autophagosomes, up-regulated PTEN, and inactivated AKT/m-TOR signal pathway induced by VCD in both granulosa cells and ovary tissues were significantly reversed by SC79 or miR-144. Finally, the decreased follicles and pregnancy rate, as well as the increased embryo resorption rate induced by VCD were greatly reversed by SC79 or miR-144. Conclusion VCD induced POI in rats by triggering the autophagy of granulosa cells through regulating the AKT/mTOR signal pathway.

CXCR3-dependent immune pathology in mice following infection with Toxoplasma gondii during early pregnancy

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular parasite, Toxoplasma gondii. The symptoms of congenital toxoplasmosis range from embryonic death and resorption to subclinical infection, but the mechanism of disease onset remains unclear. The C-X-C motif chemokine receptor 3 (CXCR3) is highly expressed in Th1-associated immune cells and plays an important role in the trafficking and activation of immune cells. However, the roles of CXCR3 in T. gondii-induced fetal loss and the molecular mechanism of embryo resorption remain poorly understood. In this study, we investigated the role of CXCR3 in fetal wastage caused by T. gondii infection using CXCR3-deficient (CXCR3−/−) mice. CXCR3−/− and wild-type pregnant mice were inoculated intraperitoneally with T. gondii tachyzoites on day 3.5 of gestation (Gd3.5). Pregnancy rates decreased as the pregnancy progressed in both infected groups; however, infected CXCR3−/− mice showed a significant fetal loss at Gd13.5 compared with Gd7.5. All embryos of the infected groups showed necrosis, and embryo resorption was significantly increased in infected CXCR3−/− compared with wild-type mice at Gd13.5. The parasite load of fetoplacental tissues was significantly increased in CXCR3−/− mice at Gd10.5. Moreover, mRNA expression levels of inducible nitric oxide synthase were significantly increased in fetoplacental tissues from infected wild-type mice compared to infected CXCR3−/− mice following the infection. These results suggested that CXCR3-dependent immune responses provide anti-Toxoplasma activity and play an essential role in reducing embryo resorption and fetal loss caused by T. gondii infection during early pregnancy.

Spontaneous embryo resorption in the mouse is triggered by embryonic apoptosis followed by rapid removal via maternal sterile purulent inflammation

Background In normal mammalian development a high percentage of implantations is lost by spontaneous resorption. This is a major problem in assisted reproduction and blastocyst transfer. Which embryo will be resorbed is unpredictable. Resorption is very fast, so that with conventional methods only final haemorrhagic stages are encountered. Here we describe the histology and immunohistochemistry of 23 spontaneous embryo resorptions between days 7 and 13 of murine development, which were identified by high-resolution ultrasound (US) in a previous study. Results In the early resorptions detected at day 7, the embryo proper was replaced by maternal haemorrhage and a suppurate focus of maternal neutrophils. In the decidua maternal macrophages transformed to foam cells and formed a second focus of tissue dissolution. In the late resorptions detected at day 9, the embryo underwent apoptosis without involvement of maternal cells. The apoptotic embryonic cells expressed caspase 3 and embryonic blood cells developed a macrophage like phenotype. Subsequently, the wall of the embryonic vesicle ruptured and the apoptotic embryo was aborted into the uterine lumen. Abortion was initiated by degeneration of the embryonic lacunar trophoblast and dissolution of the maternal decidua capsularis via sterile inflammation and accompanied by maternal haemorrhage, invasion of the apoptotic embryo by maternal neutrophils, and contraction rings of the uterine muscle layers. Conclusions We conclude that spontaneous resorption starts with endogenous apoptosis of the embryo without maternal contribution. After break down of the foetal-maternal border, the apoptotic embryo is invaded by maternal neutrophils, aborted into the uterine lumen, and rapidly resorbed. We assume that the innate maternal unspecific inflammation is elicited by disintegrating apoptotic embryonic cells. Graphical abstract

Critical role of mTOR, PPARγ and PPARδ signaling in regulating early pregnancy decidual function, embryo viability and feto-placental growth

STUDY QUESTION What are the consequences of inhibiting mTOR, the mechanistic target of rapamycin (mTOR), and the peroxisome proliferator activated receptor gamma (PPARγ) and PPARδ pathways in the early post-implantation period on decidual function, embryo viability and feto-placental growth in the rat? SUMMARY ANSWER mTOR inhibition from Days 7 to 9 of pregnancy in rats caused decidual PPARγ and PPARδ upregulation on Day 9 of pregnancy and resulted in embryo resorption by Day 14 of pregnancy. PPARγ and PPARδ inhibition differentially affected decidual mTOR signaling and levels of target proteins relevant to lipid histotrophic nutrition and led to reduced feto-placental weights on Day 14 of pregnancy. WHAT IS KNOWN ALREADY Although mTOR, PPARγ and PPARδ are nutrient sensors important during implantation, the role of these signaling pathways in decidual function and how they interact in the early post-implantation period are unknown. Perilipin 2 (PLIN2) and fatty acid binding protein 4 (FABP4), two adipogenic proteins involved in lipid histotrophic nutrition, are targets of mTOR and PPAR signaling pathways in a variety of tissues. STUDY DESIGN, SIZE, DURATION Rapamycin (mTOR inhibitor, 0.75 mg/kg, sc), T0070907 (PPARγ inhibitor, 0.001 mg/kg, sc), GSK0660 (PPARδ inhibitor, 0.1 mg/kg, sc) or vehicle was injected daily to pregnant rats from Days 7 to 9 of pregnancy and the studies were performed on Day 9 of pregnancy (n = 7 per group) or Day 14 of pregnancy (n = 7 per group). PARTICIPANTS/MATERIALS, SETTING, METHODS On Day 9 of pregnancy, rat decidua were collected and prepared for western blot and immunohistochemical studies. On Day 14 of pregnancy, the resorption rate, number of viable fetuses, crown–rump length and placental and decidual weights were determined. MAIN RESULTS AND THE ROLE OF CHANCE Inhibition of mTOR in the early post-implantation period led to a reduction in FABP4 protein levels, an increase in PLIN2 levels and an upregulation of PPARγ and PPARδ in 9-day-pregnant rat decidua. Most embryos were viable on Day 9 of pregnancy but had resorbed by Day 14 of pregnancy. This denotes a key function of mTOR in the post-implantation period and suggests that activation of PPAR signaling was insufficient to compensate for impaired nutritional/survival signaling induced by mTOR inhibition. Inhibition of PPARγ signaling resulted in decreased decidual PLIN2 and FABP4 protein expression as well as in inhibition of decidual mTOR signaling in Day 9 of pregnancy. This treatment also reduced feto-placental growth on Day 14 of pregnancy, revealing the relevance of PPARγ signaling in sustaining post-implantation growth. Moreover, following inhibition of PPARδ, PLIN2 levels were decreased and mTOR complex 1 and 2 signaling was altered in decidua on Day 9 of pregnancy. On Day 14 of pregnancy, PPARδ inhibition caused reduced feto-placental weight, increased decidual weight and increased resorption rate, suggesting a key role of PPARδ in sustaining post-implantation development. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION This is an in vivo animal study and the relevance of the results for humans remains to be established. WIDER IMPLICATIONS OF THE FINDINGS The early post-implantation period is a critical window of development and changes in the intrauterine environment may cause embryo resorption and lead to placental and fetal growth restriction. mTOR, PPARγ and PPARδ signaling are decidual nutrient sensors with extensive cross-talk that regulates adipogenic proteins involved in histotrophic nutrition and important for embryo viability and early placental and fetal development and growth. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided by the Agencia Nacional de Promoción Científica y Tecnológica de Argentina (PICT 2014-411 and PICT 2015-0130), and by the International Cooperation (Grants CONICET-NIH-2014 and CONICET-NIH-2017) to A.J. and T.J. The authors have no conflicts of interest.