Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) regulate CD4+ T cells to induce Type 2 helper T cell (Th2) bias at the maternal-fetal interface.

STUDY QUESTION: Are the immune regulatory molecules programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) involved in regulating CD4+ T cell function during pregnancy? SUMMARY ANSWER: PD-1 and Tim-3 promote Type 2 helper T cell (Th2) bias and pregnancy maintenance by regulating CD4+ T cell function at the maternal-fetal interface. WHAT IS KNOWN ALREADY: The maternal CD4+ T cell response to fetal antigens is thought to be an important component of maternal-fetal tolerance during pregnancy. PD-1 and Tim-3 are important for limiting immunopathology. The co-expression of PD-1 and Tim-3 on T cells identifies a T cell subset with impaired proliferation and cytokine production. Combined blockade of Tim-3 and PD-1 could restore T cell function to the greatest degree. STUDY DESIGN, SIZE, DURATION: The expression of PD-1 and Tim-3 on CD4+ T cells was analyzed by flow cytometry, and in vitro and in vivo analyses were used to investigate the role of PD-1/Tim-3 signal in the regulation of CD4+ T cells function and pregnancy outcome. PARTICIPANTS/ MATERIALS, SETTING, METHODS: A total of 88 normal pregnant women, 37 women with recurrent spontaneous abortion, 36 normal pregnant mice and 45 abortion-prone mice were included. We measure the expression of PD-1 and Tim-3 on CD4+ T cells and their relationship to the function of CD4+ T cells and pregnancy outcome, as well as the effects of blocking PD-1 and Tim-3 pathways on decidual CD4+ T (dCD4+ T) cells during early pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: PD-1 and Tim-3, by virtue of their up-regulation on dCD4+ T cells during pregnancy, define a specific effector/memory subset of CD4+ T cells and promote Th2 bias at the maternal-fetal interface. Using in vitro and in vivo experiments, we also found that combined targeting of PD-1 and Tim-3 pathways results in decreased production of Th2-type cytokines by dCD4+ T cells and increased fetal resorption of normal pregnant murine models. Moreover, decreased PD-1 and Tim-3 on dCD4+ T cells may be associated with miscarriage. LIMITATIONS AND LIMITS OF CAUTION: Further study is required to examine the mechanism of PD-1 and Tim-3 effects on Th2 cytokine production by CD4+ T cells during pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: These results have important implications for understanding the physiological mechanisms that promote maternal-fetal tolerance. Our study also indicates that targeting Tim-3 and PD-1 pathways may represent novel therapeutic strategies to prevent pregnancy loss. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Basic Research Program of China (2015CB943300); National Nature Science Foundation of China (81490744, 91542116, 31570920, 81070537, 31171437, 81370770, 31270969, 31570920, 91542116); the Key Project of Shanghai Municipal Education Commission (14ZZ013) and the Key Project of Shanghai Basic Research from Shanghai Municipal Science and Technology Commission (12JC1401600). None of the authors have any conflict of interest to declare.

Pregnancy losses in cattle: potential for improvement.

For heifers, beef and moderate-yielding dairy cows, it appears that the fertilisation rate generally lies between 90% and 100%. For high-producing dairy cows, there is a less substantive body of literature, but it would appear that the fertilisation rate is somewhat lower and possibly more variable. In cattle, the major component of embryo loss occurs in the first 16 days following breeding (Day 0), with emerging evidence of greater losses before Day 8 in high-producing dairy cows. In cattle, late embryo mortality causes serious economic losses because it is often recognised too late to rebreed females. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival, with evidence of either excessive or insufficient concentrations being negatively associated with survival rate. The application of direct progesterone supplementation or treatments to increase endogenous output of progesterone to increase embryo survival cannot be recommended at this time. Energy balance and dry matter intake during the first 4 weeks after calving are critically important in determining pregnancies per AI when cows are inseminated at 70-100 days after calving. Level of concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates, although sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy must be developed. There is genetic variability within the Holstein breed for fertility traits, which can be exploited. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but also, in the future, could identify genes responsible for improved embryo survival. Such information could be incorporated into breeding objectives in order to increase the rate of genetic progress for embryo survival. In addition, there is a range of easily adoptable management factors, under producer control, that can either directly increase embryo survival or ameliorate the consequences of low embryo survival rates. The correction of minor deficits in several areas can have a substantial cumulative positive effect on herd reproductive performance.

Maternal N-Carbamylglutamate Supplementation during Early Pregnancy Enhances Embryonic Survival and Development through Modulation of the Endometrial Proteome in Gilts.

BACKGROUND: Early pregnancy loss is a major concern in humans and animals. N-carbamylglutamate (NCG) has been found to enhance embryonic survival during early pregnancy in rats. However, little is known about the key factors in the endometrium involved in the improvement of embryonic implantation and development induced by maternal NCG supplementation. OBJECTIVES: Our objectives were to investigate whether NCG supplementation during early gestation enhanced embryonic survival and development in gilts and to uncover the related factors using the approach of endometrium proteome analysis with isobaric tags for relative and absolute quantification (iTRAQ). METHODS: Uteruses and embryos/fetuses were obtained on days 14 and 28 of gestation from gilts fed a basal diet that was or was not supplemented with 0.05% NCG. The iTRAQ-based quantitative proteomics approach was performed to explore the endometrium proteome altered by NCG supplementation. RESULTS: Maternal NCG supplementation significantly increased the number of total fetuses and live fetuses on day 28 of gestation by 1.32 and 1.29, respectively (P < 0.05), with a significant decrease in embryonic mortality (P < 0.05). iTRAQ results indicated that a total of 59 proteins showed at least 2-fold differences (P < 0.05), including 52 proteins that were present at higher abundance and 7 proteins present at lower abundance in NCG-supplemented gilts. The differentially expressed proteins primarily are involved in cell adhesion, energy metabolism, lipid metabolism, protein metabolism, antioxidative stress, and immune response. On day 14 of gestation, several proteins closely related to embryonic implantation and development, such as integrin-αv, integrin-ß3, talin, and endothelial nitric oxide synthase, were upregulated (3.7-, 4.1-, 2.4-, and 5.4-fold increases, respectively) by NCG supplementation. CONCLUSION: To our knowledge, our results provide the first evidence that altered abundance of the endometrial proteome induced by NCG supplementation is highly associated with the improvement of embryonic survival and development in gilts.

Oocyte maturation and embryo survival in nulliparous female pigs (gilts) is improved by feeding a lupin-based high-fibre diet.

Inclusion of high levels of the high-fibre ingredient sugar-beet pulp in pre-mating diets has been shown to increase gonadotrophin concentrations and improve oocyte quality in nulliparous pigs (gilts). This study evaluated the effects of two alternative fibre sources on reproductive performance in gilts. Gilts received one of three diets from 3 weeks before puberty stimulation until Day 19 of the first oestrous cycle: control (39 g kg⁻¹ fibre), bran (500 g kg⁻¹ wheat bran, 65 g kg⁻¹ fibre) or lupin (350 g kg⁻¹ lupin, 118 g kg⁻¹ crude fibre). Diet did not affect circulating LH concentrations or ovarian follicle size. However, a higher percentage of oocytes collected from lupin-supplemented gilts reached metaphase II in vitro compared with those collected from bran-fed or control gilts (89±5% versus 72±5% and 66±5%, respectively; P<0.05). Furthermore, in a second experiment, gilts fed the same lupin-based diet before mating had improved embryo survival (92±5%) on Day 28 after mating compared with control gilts (76±4%; P<0.05). Therefore, feeding a high-fibre diet before mating can improve oocyte quality in gilts without changes in circulating LH, but this effect is dependent on the fibre source.

Folic acid and safflower oil supplementation interacts and protects embryos from maternal diabetes-induced damage.

Maternal diabetes increases the risk of embryo malformations. Folic acid and safflower oil supplementations have been shown to reduce embryo malformations in experimental models of diabetes. In this study we here tested whether folic acid and safflower oil supplementations interact to prevent embryo malformations in diabetic rats, and analyzed whether they act through the regulation of matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and nitric oxide (NO) and reactive oxygen species production. Diabetes was induced by streptozotocin administration prior to mating. From Day 0.5 of pregnancy, rats did or did not receive folic acid (15 mg/kg) and/or a 6% safflower oil-supplemented diet. Embryos and decidua were explanted on Day 10.5 of gestation for further analysis of embryo resorptions and malformations, MMP-2 and MMP-9 activities, TIMP-1 and TIMP-2 levels, NO production and lipid peroxidation. Maternal diabetes induced resorptions and malformations that were prevented by folic acid and safflower oil supplementation. MMP-2 and MMP-9 activities were increased in embryos and decidua from diabetic rats and decreased with safflower oil and folic acid supplementations. In diabetic animals, the embryonic and decidual TIMPs were increased mainly with safflower oil supplementation in decidua and with folic acid in embryos. NO overproduction was decreased in decidua from diabetic rats treated with folic acid alone and in combination with safflower oil. These treatments also prevented increases in embryonic and decidual lipid peroxidation. In conclusion, folic acid and safflower oil supplementations interact and protect the embryos from diabetes-induced damage through several pathways related to a decrease in pro-inflammatory mediators.

Mesenchymal stem cells therapy protects fetuses from resorption and induces Th2 type cytokines profile in abortion prone mouse model.

The imbalance of Th1/Th2 cytokines is well known in recurrent spontaneous abortion (RSA) mouse model. Mesenchymal stem cells (MSCs) possess potent immunoregulatory properties that could modulate the Th1 cytokine responses in benefit of Th2 types. In this study, we aimed to analyze the local and systemic balance of Th1/Th2 cytokines following MSCs therapy. Syngeneic adipose derived MSCs were administered to abortion prone mice during the implantation window. The abortion rate was determined and IL-4, IL-6, IL-12, IL-2, IFN-γ and GM-CSF gene expression was evaluated by Real-Time-PCR in decidual and placental tissues of pregnant mice at day 13.5 of pregnancy. Splenocytes of pregnant mice were co-cultured with mitomycin C treated paternal splenocytes and IL-2, IL-4, IL-10 and IFN-γ cytokines were measured in co-cultures supernatants by ELISA method. Proliferation response of female splenocytes to paternal antigens was also evaluated using the CFSE method. Our results showed a significant reduction in abortion rate following MSCs administration in abortion prone mice. We also observed a significant down-regulation of IL-2 and IFN-γ as well as up-regulation of IL-4 and IL-10 production from pregnant mouse splenocytes following MSCs therapy along with a significant reduction of splenocytes proliferation against paternal antigens. Our findings revealed that MSCs therapy increased the IL-4, IL-6, IL-10 and GM-CSF and at the same time decreased the IL-12, IL-2 and IFN-γ gene expression at feto-maternal interface. Here, we showed that MSCs therapy could modulate the systemic as well as local Th1/Th2 cytokines production along with protection of fetus from resorption in abortion prone mice. The fine balance of Th1/Th2 cytokine response could be considered as one of the possible mechanisms for fetal protection following MSCs therapy.

Administration of high-dose intact immunoglobulin has an anti-resorption effect in a mouse model of reproductive failure.

Administration of high-dose intact human immunoglobulin (IH-Ig) has been applied to treat a variety of inflammatory and autoimmune diseases, and is expected to have beneficial effects on human fecundity. In the present study, we investigated whether Ig had anti-resorption effects using polyinosinic-polycytidylic acid sodium salt [poly (I:C)]-induced enhancement of fetal resorption in the mating of CBA/J x DBA/2J resorption-prone mouse model. Furthermore, we investigated the mechanism of the effect by examining the mRNA expression of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-10, IL-4 and TGF-beta(1) in spleens and placentas from the resorption-prone model treated with IH-Ig, by reverse transcription (RT)-polymerase chain reaction (PCR). Administration of high-dose IH-Ig significantly reduced the fetal resorption rate from 55% to 10%. This anti-resorption effect, however, was not detected in mice administered with Fab fragments of human Ig. We then performed adoptive transfer experiments to examine whether cellular components could transfer the effect. A remarkable anti-resorption effect was seen in poly (I:C)-injected pregnant recipients transferred with spleen cells from IH-Ig-treated donor mice. The RT-PCR study showed that IH-Ig reduced the expression of IFN-gamma and TNF-alpha mRNA in placentas of poly (I:C)-injected pregnant mice. The present findings demonstrate that intact Ig, particularly its Fc portion, possesses anti-resorption activity. The effect might be attributed to the suppressed production of pro-inflammatory cytokines at the maternofetal interface.

Beneficial effect of supplemental lipoic acid on diabetes-induced pregnancy loss in the mouse.

Uncontrolled diabetes mellitus (DM) is an etiological factor for recurrent pregnancy loss, fetal growth disorders, and major congenital malformations in the offspring. Antioxidant therapy has been advocated to overcome the oxidant-antioxidant disequilibrium inherent in diabetes. The objective of this article was to evaluate the beneficial effects of alpha-lipoic acid (LA) on fetal outcome in a mouse model of streptozotocin (STZ)-induced DM. Timed pregnant mice were made diabetic by intraperitoneal (IP) injection of a single dose of STZ (200 mg/kg) on gestation day (GD) 2. Diabetic animals were supplemented daily with an IP injection of 15 mg/kg of LA starting on GD 4 and continued through GD 12. Fetuses were examined on GD 18 for malformations and growth restriction. Some diabetic mice injected with Evans blue were examined on GD 3.5 and GD 6.5 to evaluate frequency of implantations. STZ-treated mice had all cardinal signs of DM. LA treatment did not normalize blood glucose levels of DM mice. Rates of pregnancy in saline control, DM, and DM + LA groups were 90%, 28%, and 64%, respectively, indicating that LA promotes pregnancy in DM animals. However, postimplantation resorption showed a threefold increase in the DM + LA group. Rates of intrauterine growth restriction and major congenital malformations were also augmented thus indicating that the interaction between DM and LA has deleterious effects on postimplantation embryos.

Metformin prevents embryonic resorption induced by hyperandrogenisation with dehydroepiandrosterone in mice.

The present study examined the mechanism by which metformin prevents dehydroepiandrosterone (DHEA)-induced embryonic resorption in mice. Treatment with DHEA (6 mg/100 g bodyweight, 24 and 48 h post implantation) induced 88 +/- 1 % embryonic resorption and the diminution of both serum oestradiol (E) and progesterone (P) levels. However, when metformin (50 mg/kg bodyweight) was given together with DHEA, embryo resorption (43 +/- 3% v. 35 +/- 5% in controls) and both serum E and P levels were not significantly different from controls. Glucose and insulin levels were increased in the DHEA-treated mice but when metformin was administered together with DHEA these parameters were similar to control values. Treatment with DHEA increased ovarian oxidative stress and diminished uterine nitric oxide synthase (NOS) activity; however, when metformin was administered together with DHEA, both ovarian oxidative stress and uterine NOS activity were not different from controls. Metformin treatment did not modify the percentage of CD4(+) and CD8(+) T cells from both axillar and retroperitoneal lymph nodes but prevented the increase of serum tumour necrosis factor +/- produced in DHEA-treated mice. These results show that metformin acts in DHEA-induced embryonic resorption in mice by modulating endocrine parameters, ovarian oxidative stress and uterine NOS activity.

Role of Paternal Antigen-Specific Treg Cells in Successful Implantation.

Maternal lymphocytes recognize fetal antigens, so tolerance is necessary to prevent rejection. Seminal plasma is important for induction of paternal antigen-specific Treg cells in the uterine draining lymph nodes and the pregnant uterus. Elimination of Treg cells during implantation or early pregnancy induces implantation failure or fetal resorption in mice. Immunosuppressive therapy with an anti-TNF antibody or the immunosuppressive agent tacrolimus improves the pregnancy rate in women with repeated implantation failure and recurrent pregnancy loss of unknown etiology, suggesting that Treg cells play an essential role in successful implantation and pregnancy in humans.

Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134.

Based on previous in vitro studies that have illustrated prevention of ethanol-induced cell death by antioxidants, using an in vivo model, we have tested the anti-teratogenic potential of a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134. The developing limb of C57BL/6J mice, which is sensitive to ethanol-induced reduction defects, served as the model system. On their ninth day of pregnancy, C57BL/6J mice were administered ethanol (two intraperitoneal doses of 2.9 g/kg given 4 h apart) alone or in combination with EUK-134 (two doses of 10 mg/kg). Pregnant control mice were similarly treated with either vehicle or EUK-134, alone. Within 15 h of the initial ethanol exposure, excessive apoptotic cell death was observed in the apical ectodermal ridge (AER) of the newly forming forelimb buds. Forelimb defects, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies, occurred in 67.3% of the ethanol-exposed fetuses that were examined at 18 days of gestation. The right forelimbs were preferentially affected. No limb malformations were observed in control fetuses. Cell death in the AER of embryos concurrently exposed to ethanol and EUK-134 was notably reduced compared with that in embryos from ethanol-treated dams. Additionally, the antioxidant treatment reduced the incidence of forelimb malformations to 35.9%. This work illustrates that antioxidants can significantly improve the adverse developmental outcome that results from ethanol exposure in utero, diminishing the incidence and severity of major malformations that result from exposure to this important human teratogen.

CXCR3 blockade protects against Listeria monocytogenes infection-induced fetal wastage.

Mammalian pregnancy requires protection against immunological rejection of the developing fetus bearing discordant paternal antigens. Immune evasion in this developmental context entails silenced expression of chemoattractant proteins (chemokines), thereby preventing harmful immune cells from penetrating the maternal-fetal interface. Here, we demonstrate that fetal wastage triggered by prenatal Listeria monocytogenes infection is driven by placental recruitment of CXCL9-producing inflammatory neutrophils and macrophages that promote infiltration of fetal-specific T cells into the decidua. Maternal CD8+ T cells with fetal specificity upregulated expression of the chemokine receptor CXCR3 and, together with neutrophils and macrophages, were essential for L. monocytogenes-induced fetal resorption. Conversely, decidual accumulation of maternal T cells with fetal specificity and fetal wastage were extinguished by CXCR3 blockade or in CXCR3-deficient mice. Remarkably, protection against fetal wastage and in utero L. monocytogenes invasion was maintained even when CXCR3 neutralization was initiated after infection, and this protective effect extended to fetal resorption triggered by partial ablation of immune-suppressive maternal Tregs, which expand during pregnancy to sustain fetal tolerance. Together, our results indicate that functionally overriding chemokine silencing at the maternal-fetal interface promotes the pathogenesis of prenatal infection and suggest that therapeutically reinforcing this pathway represents a universal approach for mitigating immune-mediated pregnancy complications.

Vaccination against spontaneous abortion in mice.

We report here that the high rate of spontaneous resorption observed in CBA/J female mice mated with DBA/2 J males can be dramatically reduced by vaccination with Balb/c male spleen cells, but not by CBA/J or DBA/2 J male spleen cells. This effect correlates with the differential ability of Balb/c spleen cells to induce MLR suppressor activity in CBA/J female mice, and should lead to a better understanding of the immunology of the materno-fetal relationship.

Maternal immunopotentiation affects the teratogenic response to hyperthermia.

Immune responses occurring between the embryo and mother have been shown to influence the embryo's tolerance to teratogens, including chemical teratogens and diabetes-induced teratogenic insult. In this study, we tried to evaluate whether maternal immunostimulation alters the embryo's response to heat shock, one of few teratogens which directly affect the embryo. In order to induce structural anomalies, both intact ICR female mice and mice which had been immunostimulated with xenogeneic rat splenocytes before mating, were exposed to two consecutive exposures to heat (43.6 +/- 0.2 degrees C) for 10 min on day 9 of pregnancy. The number of malformed fetuses, resorptions, and fetal weight were assessed on day 19 of pregnancy. Heat shock-induced apoptosis, and the level of heat shock protein (HSP) 60 expression, were examined in embryonic cells at different time points within 24 h after heating. All these indices differed dramatically in immunized and non-immunized heat shocked females. Heat shocked non-immunized females demonstrated an increased level of resorptions (approximately, 21% versus 8.6% in controls) and the proportion of fetuses with such anomalies as encephalocele and open eyes reached 28% and 21%, respectively. Maternal immunostimulation was associated with a significant decrease in the proportion of fetuses with encephalocele (12.8%), open eyes (8.9%), and resorptions (8%). The maximum level of heat shock-induced apoptosis in cell populations from the embryos of non-immunized females, was approximately, 30% versus 7% in cells of embryos of immunized mice. Heat shock was also followed by a significant increase in HSP60 expression, but only in the cells of embryos of non-immunized females. Together, these findings suggest that the tolerance of mouse embryos to a heat shock-induced teratogenic insult may, to some extent, depend on the character of the maternal immune responses.

Serum hormone characterization and exogeneous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat.

Previously, we demonstrated that bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss in F344 rats when given on gestational days (GD) 6-10, encompassing the luteinizing hormone (LH)-dependent period of pregnancy (GD 7-10). Pregnancy loss, i.e., full-litter resorption, was associated with reduced serum progesterone levels; however, we were unable to identify an effect on serum LH. Here, we reevaluated serum LH levels using the more sensitive technique, DELFIA(R). We further sought to better define the temporal pattern of endocrine disruption caused by BDCM during pregnancy with more frequent sampling. Lastly, we attempted to prevent BDCM-induced pregnancy loss using exogenous progesterone or human chorionic gonadotropin (hCG), an LH-agonist. BDCM, in 10% Alkamuls(R), was dosed at 75 mg/kg/day by gavage to F344 rats on GD 6-10 (plug day = GD 0). BDCM-induced pregnancy loss was associated with marked reductions in serum progesterone and LH on GD 10. The decrease in serum LH consistently preceded the decrease in progesterone. In the hormone replacement studies, BDCM and progesterone were administered on GD 6-10, hCG on GD 8-10. BDCM was delivered at 100 mg/kg/day, progesterone at 10 mg/kg twice daily, and hCG at 0.5 IU/0.2 ml/rat. Both progesterone and hCG prevented BDCM-induced pregnancy loss. Thus, BDCM-induced pregnancy loss was associated with marked GD-10 reductions in serum LH and corresponding decreases in progesterone. Furthermore, coadministration of an LH agonist prevented pregnancy loss, supporting the hypothesis that BDCM-induced pregnancy loss in the rat occurs via an LH-mediated mode of action.

Effectiveness of sodium 4,5-dihydroxybenzene-1,3-disulfonate (Tiron) in protecting against uranium-induced developmental toxicity in mice.

The effect of Tiron (sodium 4,5-dihydroxybenzene-1,3-disulfonate), a chelating agent used in the treatment of experimental poisoning by a number of heavy metals, on uranium-induced developmental toxicity was evaluated in Swiss mice. A series of four Tiron injections was administered intraperitoneally to pregnant mice immediately after a single subcutaneous injection of 4 mg/kg of uranyl acetate dihydrate given on day 10 of gestation and at 24, 48, and 72 h thereafter. Controls received 0.9% saline with or without uranyl acetate. Tiron effectiveness was assessed at 500, 1000 and 1500 mg/kg per day. Amelioration by Tiron of uranium-induced embryolethality was not noted at the two lower doses. The percentage of dead and resorbed fetuses in the Tiron-treated groups was not statistically different from that in the positive control group. However, treatment at 1500 mg/kg per day showed isolated protective effects against uranium fetotoxicity, such as that evidenced by the lack of differences in fetal body weight between this group and the uranium-untreated group, as well as by a decrease in the number of skeletal defects. According to these results, the ability of Tiron to protect the developing mouse fetus against uranium-induced developmental toxicity offers only modest encouragement with regard to its possible therapeutic potential for pregnant women exposed to this metal.

Prevention by L-arginine and polyamines of delayed development and embryotoxicity caused by chemically-induced diabetes in rats.

Diabetes mellitus induction with alloxan at a dose of 110 mg/kg i.p. in rats on Day 4 of pregnancy causes delayed development and resorptions as signs of embryotoxicity. In the present study, the administration of human NPH insulin at doses of 1 to 5 U/d to rats or 1.0 mL of 10 mM L-arginine for 8 d, starting the day following diabetes induction, prevented embryotoxicity and delayed development. Similar results were obtained when the polyamines putrescine, spermidine, or spermine were administered at doses of 1.0 mL of a 10 microM solution to each rat daily. However, even though L-arginine and polyamines prevented adverse effects of severe diabetes on the conceptus, and caused normalization of glucose, beta-hydroxybutyrate levels remained elevated. These results support the hypothesis that the mechanisms of normal and altered development could be mediated by the action of polyamines.

Antagonism of cocaine-induced fetal anomalies by prazosin and diltiazem in mice.

Cocaine-induced uteroplacental and fetal vasoconstriction have been observed to cause fetal hypoxemia. The ability of cocaine to elevate norepinephrine (NE) levels has been proposed as one mechanism to explain the effect of cocaine on fetal development. Prazosin, a selective antagonist of alpha-1 adrenergic receptors, and diltiazem, a calcium channel blocker, were used to determine if antagonism of NE-induced vasoconstriction would reduce the effects of chronic cocaine administration on fetal development. The dose-response relationship of cocaine with fetal development was established in CF-1 mice by administering cocaine sc on days 5 to 18 of gestation followed by teratologic evaluation. Cocaine 2 mg/kg/day produced a significant incidence of fetal anomalies without significantly affecting food consumption or maternal and fetal weight gain. In subsequent experiments, prazosin (0.03, 0.3 mg/kg) or diltiazem (1.7, 5.1 mg/kg) were administered po 2 h prior to 2 mg/kg cocaine sc (gestation days 5 to 18) followed by teratologic evaluation. Diltiazem (5.1 mg/kg) produced a significant increase, whereas prazosin (0.3 mg/kg) produced a significant reduction, in the incidence of fetal anomalies compared with saline controls. While data from the pretreatment studies were inconclusive, comparisons between cocaine alone and the cocaine groups pretreated with the high doses of either prazosin or diltiazem seem worthy of further study with larger sample sizes.

Ethanol effects on postimplantation rat embryos: influence of zinc and methionine.

Organic zinc salts and thiols, administered simultaneously, protect mice synergistically against ethanol toxicity. Moreover, chronic ethanol consumption could affect the bioavailability of zinc and amino acids such as methionine. This could result in impaired embryonic growth and development. The influence of zinc and methionine on ethanol-induced embryopathy was investigated by simultaneous administration of ethanol, zinc and methionine to pregnant rats from gestational day 6 through 12. Ethanol was given in the form of a liquid diet; zinc administered i.p., and methionine was given by gavage. The ethanol group received the liquid ethanol diet; the ethanol + zinc and methionine group received the ethanol diet, zinc and methionine; and the pair-fed control group was given an isocaloric control diet. On day 12 of gestation, embryos of ethanol alone treated rats revealed a significantly reduced embryonic protein content, number of somites, crown-rump length, and lower morphological score (embryological differentiation) compared to the pair-fed control embryos. However, embryonic growth and developmental parameters in the ethanol, zinc and methionine treated group were not significantly different from those exposed to ethanol alone.

Stereoisomers of alpha-tocopheryl acetate--characterization of the samples by physico-chemical methods and determination of biological activities in the rat resorption-gestation test.

Stereoisomers of alpha-tocopheryl acetate (alpha-TA) have been identified and characterized by means of a recently developed gas-liquid chromatographic technique prior to the determination of their biopotency. It has been shown that totally synthetic all-rac-alpha-TA (dl-alpha-TA) is composed of four pairs of diastereomers (RRS-SSR, RRR-SSS, RSR-SRS, RSS-SRR) in virtually equal proportions. In addition, it has been verified that 2-ambo-alpha-TA (partially synthetic, ex phytol) is an equimolar mixture of RRR-alpha-TA and 2 epi-alpha-TA. Finally, it has been demonstrated that the RRR-alpha-TA (d-alpha-TA) tested was diastereomerically pure. The biopotencies of these alpha-tocopheryl acetate stereoisomers have been evaluated by standardized rat resorption-gestation test. Our first study concerned the comparison of all-rac-alpha-TA with RRR-alpha-TA and resulted in a potency ratio of 1:1.50 or 1:1.48. The first value was derived from the amounts weighed out while the second value was based on the analytically determined alpha-TA content of the stock solutions. Similarly, the comparison of 2-ambo-alpha-TA with RRR-alpha-TA yielded potency ratios of 1:1.32 (amount weighed out) and 1:11.36 (alpha-TA analysed). The influence of the configuration of the side-chain on biopotency has been analysed by a second series of investigations. Comparison of all-rac-alpha-TA with 2-ambo-alpha-TA resulted in a biopotency ratio of 1:1.09 (amount weight out) and 1:1.10 (alpha-TA analysed). However, these slightly higher values for 2-ambo-alpha-TA did not reach statistical significance. These studies demonstrate the validity of the established potency ratio of 1:1.36 for 2-ambo-alpha-TA to RRR-alpha-TA by direct comparison of these compounds. In addition, the experimental ratios for all-rac-alpha-TA to 2-ambo-alpha-TA and for all-rac-alpha-TA to RRR-alph-TA can be grouped into the expression (1:1.10)/(1:1.48) = 1.35. This indirect comparison further supports the accepted value of 1.36 USP Units for 1 mg d-alpha-tocopheryl acetate.