Oxytocin and arginine vasopressin receptor evolution: implications for adaptive novelties in placental mammals.

Oxytocin receptor (OXTR) and arginine vasopressin receptors (AVPR1a, AVPR1b, and AVPR2) are paralogous genes that emerged through duplication events; along the evolutionary timeline, owing to speciation, numerous orthologues emerged as well. In order to elucidate the evolutionary forces that shaped these four genes in placental mammals and to reveal specific aspects of their protein structures, 35 species were selected. Specifically, we investigated their molecular evolutionary history and intrinsic protein disorder content, and identified the presence of short linear interaction motifs. OXTR seems to be under evolutionary constraint in placental mammals, whereas AVPR1a, AVPR1b, and AVPR2 exhibit higher evolutionary rates, suggesting that they have been under relaxed or experienced positive selection. In addition, we describe here, for the first time, that the OXTR, AVPR1a, AVPR1b, and AVPR2 mammalian orthologues preserve their disorder content, while this condition varies among the paralogues. Finally, our results reveal the presence of short linear interaction motifs, indicating possible functional adaptations related to physiological and/or behavioral taxa-specific traits.

Embryo mortality in Isg15-/- mice is exacerbated by environmental stress.

The interferon-stimulated gene 15 (Isg15) encodes a ubiquitin-like protein that is induced in the endometrium by pregnancy in mice, humans, and ruminants. Because ISG15 is a component of the innate immune system, we hypothesized that development of the embryo, fetus, and postnatal pup may be impaired in mice lacking Isg15 (Isg15(-/-)) and that this development would be further impaired in response to environmental insults such as hypoxia. The number of implantation sites, resorption sites, dead embryos, and the changes in overall gross morphology of the uterus were evaluated in Isg15(-/-) mice on Days 7.5 and 12.5 postcoitum (dpc). Postnatal development also was monitored from birth to 12 wk of age. On 7.5 dpc, the number of implantation sites and serum progesterone concentrations were similar. However, embryo mortality increased (P < 0.05) in Isg15(-/-) dams by 12.5 dpc, resulting in smaller litter sizes (4.26 ± 0.21 embryos; n = 83 litters) compared to Isg15(+/+) females (7.78 ± 0.29 pups; n = 47 litters). Embryo mortality in Isg15(-/-) mice was further exacerbated to 70% when dams were stressed through housing under hypoxic conditions (PB = 445 mmHg; 6.5-12.5 dpc). Transmission electron microscopy revealed lesions in antimesometrial decidua as well as trophoblast cells adjacent to decidual cells on 7.5 dpc. ISG15 was localized to mesometrial decidua on 7.5 dpc. By 12.5 dpc, ISG15 was intensely localized to the labyrinth of the placenta. By 7.5 dpc, uterine natural killer cell migration into the mesometrial pole was diminished by 65% and was less prevalent in Isg15(-/-) compared to Isg15(+/+) deciduum. Postnatal growth rate of offspring that survived to birth from Isg15(-/-) and Isg15(+/+) dams was not different. Embryo mortality occurs in pregnant Isg15(-/-) mice, is exacerbated by environmental insults like maternal hypoxia, and might result from impaired early decidualization, vascular development, and formation of the labyrinth.

Endocrine delivery of interferon tau protects the corpus luteum from prostaglandin F2 alpha-induced luteolysis in ewes.

Paracrine release of ovine interferon tau (oIFNT) from the conceptus alters release of endometrial prostaglandin F2 alpha (PGF) and prevents luteolysis. Endocrine release of oIFNT into the uterine vein occurs by Day 15 of pregnancy and may impart resistance of the corpus luteum (CL) to PGF. It was hypothesized that infusion of recombinant oIFNT (roIFNT) into the uterine or jugular veins on Day 10 of the estrous cycle would protect the CL against exogenous PGF-induced luteolysis. Osmotic pumps were surgically installed in 24 ewes to deliver bovine serum albumin (BSA; n = 12) or roIFNT (200 µg/day; n = 12) for 24 h into the uterine vein. Six ewes in each treatment group received a single injection of PGF (4 mg/58 kg body weight) 12 h after pump installation. In a second experiment, BSA or roIFNT was delivered at 20 or 200 µg/day into the uterine vein or 200 µg/day into the jugular vein for 72 h in 30 ewes. One half of these ewes received an injection of PGF 24 h after pump installation. Concentrations of progesterone in serum declined in BSA-treated ewes injected with PGF, but were sustained in all ewes infused with 20 µg/day of roIFNT into the uterine vein and 200 µg of roIFNT into the jugular vein followed 24 h later with injection of PGF. All concentrations of roIFNT and modes of delivery (uterine or jugular vein) increased luteal concentrations of IFN-stimulated gene (i.e., ISG15) mRNA. Infusion of 200 µg of IFNT over 24 h induced greater mRNA concentrations for cell survival genes, such as BCL2-like 1 (BCL2L1 or Bcl-xL), serine/threonine kinase (AKT), and X-linked inhibitor of apoptosis (XIAP) and decreased prostaglandin F receptor (PTGFR) mRNA concentrations, when compared to controls. It is concluded that endocrine delivery of roIFNT, regardless of route (uterine or jugular vein), effectively protects CL from the luteolytic actions of PGF by mechanisms that involve ISGs and stabilization of cell survival genes.

Acid sphingomyelinase involvement in tumor necrosis factor alpha-regulated vascular and steroid disruption during luteolysis in vivo.

TNF is well known for its role in inflammation, including direct effects on the vasculature. TNF also is implicated in the regulation of reproduction by its actions to affect ovarian steroidogenic cells and to induce apoptosis of corpus luteum (CL)-derived endothelial cells in vitro. We hypothesized that the disruption of TNF signaling would postpone the regression of the highly vascularized CL in vivo, and this effect could be replicated in mutant mouse models lacking TNF receptor (TNFRI(-/-)) and/or a critical enzyme of TNF signaling, acid sphingomyelinase (ASMase(-/-)). In the current study, the treatment of pseudopregnant mice with the luteolytic mediator prostaglandin F2-alpha (PGF) significantly increased TNF in the ovaries when compared with saline-treated controls. Treatment with PGF also reduced serum progesterone (P4) concentrations and caused involution of the CL. However, pretreatment of pseudopregnant mice with Etanercept (ETA), a TNF-neutralizing antibody, inhibited the PGF-induced decrease in P4 and delayed luteal regression. A similar outcome was evident in pseudopregnant TNFRI(-/-) animals. Treatment of luteal microvascular endothelial cells (MVECs) with TNF provoked a significant increase in ASMase activity when compared with the corresponding controls. Furthermore, TNF-induced MVEC death was inhibited in the ASMase(-/-) mice. The ASMase(-/-) mice displayed no obvious evidence of luteal regression 24 h after treatment with PGF and were resistant to the PGF-induced decrease in P4. Together these data provide evidence that TNF plays an active role in luteolysis. Further studies are required to determine the deleterious effects of anti-inflammatory agents on basic ovarian processes.

Uterine vein infusion of interferon tau (IFNT) extends luteal life span in ewes.

Interferon tau (IFNT) from the ovine conceptus has paracrine actions on the endometrium that alter release of prostaglandin F(2alpha) (PGF) and protect the corpus luteum (CL). Antiviral activity in uterine vein blood and expression of interferon-stimulated genes (ISGs) in CL is greater in pregnant than in nonpregnant ewes. We hypothesized that IFNT contributes to antiviral activity in uterine vein blood and has endocrine actions on the CL. Preadsorption of IFNT with antiserum against recombinant ovine (ro) IFNT revealed that antiviral activity in uterine vein blood from pregnant ewes was mediated by IFNT. Endocrine actions of IFNT were examined after infusing either roIFNT or bovine serum albumin (BSA; 200 microg/24 h; mini-osmotic pump) into the uterine vein of nonpregnant ewes from Day 10 to Day 11 postestrus. The abundance of ISG15 mRNA and protein was greater in CL (P < 0.05) from ewes receiving 24-h roIFNT infusion compared to that from ewes receiving 24-h BSA infusion. Injection of PGF at 12 h following insertion of mini-osmotic pumps resulted in a decline in serum progesterone concentrations 6 through 12 h later in BSA-infused ewes; however, in roIFNT-infused ewes, a similar decline in progesterone concentrations at 6 h was followed by recovery to control values at 12 h. Ewes then received infusions (200 microg/day) of either roIFNT or BSA for 7 days beginning on Day 10 of the estrous cycle. All BSA-infused ewes returned to estrus by Day 19, whereas 80% of roIFNT-infused ewes maintained luteal-phase concentrations of progesterone through Day 32. In conclusion, IFNT is released from the uterus into the uterine vein and acts through an endocrine mechanism to induce ISGs in the CL and delay luteolysis.

Cytokeratin 18 expression inhibits cytokine-induced death of cervical cancer cells.

OBJECTIVES: In cervical cancer, increased cytokeratin 18 (CK18) filament expression is associated with disease progression. However, it may also provide resistance to cytokine-induced apoptosis. The present study tested whether CK18 expression influences susceptibility to cytokine-induced apoptosis. METHODS: The cervical cancer cell lines C-4II (high CK18 expression), ME-180 (low CK18 expression), and 2 subtypes of HeLa cells containing or lacking CK18 expression (CK18+ and CK18- cells, respectively) were exposed to vehicle (control), Fas ligand (FasL) (50 ng/mL), or tumor necrosis factor α (TNF-α; 10 ng/mL) without/with cycloheximide (CHX; 2.5 µg/mL) to test the hypothesis that diminished CK18 expression increases susceptibility to cytokine-induced apoptosis. RESULTS: Flow cytometric analysis of cell death via TUNEL staining revealed that cytokine-induced apoptosis was 2-fold greater in ME-180 cells than C-4II cells in response to FasL+CHX or TNF-α+CHX (P < 0.05). Similarly, there was a higher incidence of FasL-induced apoptosis in CK18- HeLa cells (23% and 91% apoptotic for FasL and FasL+CHX, respectively) than CK18+ HeLa cells (1% and 11%, respectively; P < 0.05). Surprisingly, TNF-α had no effect on either CK18+ or CK18- HeLa cells (P > 0.05). Caspase 3 activity was greater in CK18- HeLa cells than in CK18+ HeLa cells at 8 and 18 hours after FasL treatment (P < 0.05), an effect abrogated by the caspase 8 inhibitor IETD-fmk (P < 0.05). CONCLUSIONS: Cervical cancer cells with diminished CK18 expression are more susceptible to cytokine-induced apoptosis, particularly in response to FasL treatment. These observations suggest that relative CK18 expression is an important factor when considering therapeutic strategies to enhance immune cell-mediated death of cervical cancer cells.

The involvement of proline-rich 15 in early conceptus development in sheep.

The ruminant conceptus undergoes a period of elongation that is required for maternal recognition of pregnancy, prior to attaching to the endometrium. The purpose of these studies was to investigate the role of proline-rich 15 (PRR15) in the sheep conceptus by examining mRNA expression, protein localization, and the effect of PRR15 mRNA degradation. Conceptuses were collected on Days 11, 13, 15, 16, 17, 21, and 30 after mating. Quantitative RT-PCR showed expression of PRR15 mRNA corresponded with the process of trophoblast elongation, with peak expression occurring on Days 15 and 16. A recombinant ovine PRR15 was generated and used to create polyclonal antibodies against PRR15. Immunohistochemistry of a Day 15 conceptus indicated that PRR15 was localized predominantly in the nucleus of the trophectoderm and extraembryonic primitive endoderm. To test whether PRR15 was required during early conceptus development, RNA interference was employed. Blastocysts collected on Day 8 after mating were infected with a lentivirus expressing a short-hairpin RNA (shRNA) that targeted PRR15 mRNA for degradation, an shRNA containing a three-nucleotide mismatch to PRR15 mRNA, or a lentivirus expressing no shRNA. After infection, blastocysts were transferred into recipient ewes and collected back on Day 15 of gestation. Although the majority of the control and mismatched shRNA-treated conceptuses elongated and survived to Day 15, none of the embryos treated with the lentivirus expressing shRNA against PRR15 mRNA elongated, and most died. In conclusion, expression of PRR15 mRNA occurred during a narrow window of conceptus development, and degradation of PRR15 mRNA led to conceptus demise or abnormal development.

Expression of interferon (IFN)-stimulated genes in extrauterine tissues during early pregnancy in sheep is the consequence of endocrine IFN-tau release from the uterine vein.

The ruminant conceptus synthesizes and secretes interferon (IFN)-tau, which presumably acts via an intrauterine paracrine mechanism to signal maternal recognition of pregnancy. The aims of this study were to determine whether IFN-stimulated genes (ISG) such as ISG15 and OAS-1 are differentially expressed in blood cells circulating in the uterus of ewes; whether extrauterine components of the reproductive tract such as the corpus luteum (CL) also express mRNA for these ISG, and whether antiviral activity is greater in uterine vein than in uterine artery during early pregnancy. The concentrations of mRNA for both ISG were significantly greater (P < 0.0001) in endometrium and jugular blood of 15-d pregnant ewes than in nonpregnant ewes. ISG15 and OAS-1 mRNA concentrations were also greater (P < 0.05) in CL from 15-d pregnant ewes than in nonpregnant ewes. Immunohistochemistry revealed intense staining for ISG15 in large luteal cells on d 15 of pregnancy. Blood cells from uterine artery and vein of 15-d pregnant ewes had similar ISG15 and OAS-1 mRNA concentrations, suggesting that these cells were not conditioned by IFN-tau within the uterus. By using an antiviral assay, uterine venous blood was found to contain 500- to 1000-fold higher concentrations of bioactive IFN-tau than in uterine arterial blood on d 15 of pregnancy. It is concluded that uterine vein releases IFN-tau, which induces ISG in extrauterine tissues such as the CL during the time of maternal recognition of pregnancy.

Ubp43 gene expression is required for normal Isg15 expression and fetal development.

BACKGROUND: Isg15 covalently modifies murine endometrial proteins in response to early pregnancy. Isg15 can also be severed from targeted proteins by a specific protease called Ubp43 (Usp18). Mice lacking Ubp43 (null) form increased conjugated Isg15 in response to interferon. The Isg15 system has not been examined in chorioallantoic placenta (CP) or mesometrial (MM) components of implantation sites beyond 9.5 days post coitum (dpc). It was hypothesized that deletion of Ubp43 would cause disregulation of Isg15 in implantation sites, and that this would affect pregnancy rates. METHODS: Heterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc). RESULTS: Free and conjugated Isg15 were greater on 12.5 versus 17.5 dpc in MM. Free and conjugated Isg15 were also present in CP, but did not differ due to genotype on 12.5 dpc. However, null CP had greater free and conjugated Isg15 when compared to het/wt on 17.5 dpc. Null progeny died in utero with fetal genotype ratios (wt:het:null) of 2:5:1 on 12.5 and 2:2:1 on 17.5 dpc. Implantation sites were disrupted within the junctional zone and spongiotrophoblast, contained less vasculature based on lectin B4 staining and contained greater Isg15 mRNA and VEGF protein in Ubp43 null when compared to wt placenta. CONCLUSION: It is concluded that Isg15 and its conjugates are present in implantation sites during mid to late gestation and that deletion of Ubp43 causes an increase in free and conjugated Isg15 at the feto-maternal interface. Also, under mixed genetic background, deletion of Ubp43 results in fetal death.

Mutant mouse models and their contribution to our knowledge of corpus luteum development, function and regression.

The corpus luteum is a unique organ, which is transitory in nature. The development, maintenance and regression of the corpus luteum are regulated by endocrine, paracrine and autocrine signaling events. Defining the specific mediators of luteal development, maintenance and regression has been difficult and often perplexing due to the complexity that stems from the variety of cell types that make up the luteal tissue. Moreover, some regulators may serve dual functions as a luteotropic and luteolytic agent depending on the temporal and spatial environment in which they are expressed. As a result, some confusion is present in the interpretation of in vitro and in vivo studies. More recently investigators have utilized mutant mouse models to define the functional significance of specific gene products. The goal of this mini-review is to identify and discuss mutant mouse models that have luteal anomalies, which may provide some clues as to the significance of specific regulators of corpus luteum function.

Deletion of the Isg15 gene results in up-regulation of decidual cell survival genes and down-regulation of adhesion genes: implication for regulation by IL-1beta.

The ubiquitin homolog interferon stimulated gene 15 (ISG15) is up-regulated in the endometrium in response to pregnancy in primates, ruminants, pigs, and mice. ISG15 covalently attaches to intracellular proteins (isgylation) and regulates numerous intracellular responses. We hypothesized that ISG15 depletion (Isg15(-/-)) alters decidual tissue gene expression and that IL-1beta induces ISG15 expression and isgylation in cultured murine decidual explants and human uterine fibroblasts (HuFs). After studying the reproductive phenotype, contrary to earlier reports, up to 50% of the fetuses die between 7.5 and 12.5 d post coitum (dpc) in Isg15(-/-) mothers when mated to Isg15(-/-) fathers. Using microarray analysis, over 500 genes are differentially regulated in 7.5 dpc deciduas from Isg15(-/-) compared with Isg15(+/+) mice. The gene for interferon-inducible protein 202b, which functions in cell-survival mechanisms, was up-regulated (mRNA and protein) in deciduas from Isg15(-/-) mice. Culture of Isg15(+/+) mouse decidual explants (7.5 dpc) with IL-1beta decreased Isg15 mRNA but increased free and conjugated ISG15. In predecidual HuF cells, IL-1beta treatment increased ISG15 mRNA and isgylation. Additionally, IL-1beta up-regulated expression of enzymes (HERC5, UBCH8) that coordinate the covalent conjugation of ISG15 to target proteins, as well as the gene that encodes the deisglyation enzyme UBP43 in HuF cells. In conclusion, deletion of Isg15 gene results in 50% fetal loss after 7.5 dpc, which can be explained through differential decidual gene expression that is functionally tied to cell survival and adhesion pathways. This fetal death also might relate to impaired IL-1beta signaling, because ISG15 and isgylation are induced by IL-1beta in human and murine endometrial stromal cells.

Maternal and embryonic control of uterine sphingolipid-metabolizing enzymes during murine embryo implantation.

During early gestation in invasively implanting species, the uterine stromal compartment undergoes dramatic remodeling, defined by the differentiation of stromal fibroblast cells into decidual cells. Lipid signaling molecules from a number of pathways are well-established functional components of this decidualization reaction. Because of a correlation in the events that transpire in the uterus during early implantation with known functions of bioactive sphingolipid metabolites established from studies in other organ systems, we hypothesized that uterine sphingolipid metabolism would change during implantation. By a combination of Northern blot, Western blot, and immunohistochemical analyses, we establish that enzymes at each of the major catalytic steps in the sphingolipid cascade become transcriptionally up-regulated in the uterus during decidualization. Each of the enzymes analyzed was up-regulated from Days of Pregnancy (DOP) 4.5-7.5. When comparing embryo-induced decidualization (decidual) with mechanically induced decidualization (deciduomal), sphingomyelin phosphodiesterase 1 (Smpd1) mRNA and sphingosine kinase 1 (SPHK1) protein were shown to be dually regulated in the endometrium by both maternal and embryonic factors. As measured by the diacyl glycerol kinase assay, ceramide levels rose in parallel with Smpd1 gene expression, suggesting that elevated transcription of sphingolipid enzymes results in heightened catalytic activity of the pathway. Altogether, these findings place sphingolipids on a growing list of lipid signaling molecules that become increasingly present at the maternal-embryonic interface.

Cooperative expression of monocyte chemoattractant protein 1 within the bovine corpus luteum: evidence of immune cell-endothelial cell interactions in a coculture system.

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.