Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall.

s / Placenta 36 (2015) A1eA14 A5 expression. These cells were cultured in the presence of human serum with or without complement inactivation and cell proliferations were evaluated by WST assay. Results: In immunohistochemistry, CD59 expression is higher on eEVT than on EVT. RT-PCR, immunocytochemistry, and flow cytometry showed that isolated EVT and Swan71 express CD59 intensely, whereas the expressions in BeWo, JAR and JEG are hardly detectable. In WST assay, there are no significant differences between two groups. JPA2015-15. SHORT TERM EXPOSURE OF BMP4 CREATED NOVEL TROPHOBLAST PROGENITOR CELLS FROM HUMAN PLURIPOTENT STEM CELLS Katsuyuki Adachi , Ying Yang , Toshihiko Ezashi , Andrei Alexenko , Megan Sheridan , D.J. Schust , L.C. Schulz , Mitsuyoshi Amita , R.M. Roberts , Kei Kawana , Yutaka Osuga , Tomoyuki Fujii . Department of OB/GYN School of Medicine University of Tokyo, Japan; Division of Animal Sciences, University of Missouri, Columbia, USA; Department of Obstetrics, Gynecology, and Women's Health, University of Missouri, Columbia, USA; Department of Biochemistry, University of Missouri, Columbia, USA Introduction: Preeclampsia is characterized by a lack of spiral artery remodeling in early placentation. Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited, there is a need for model systems for the study of trophoblast differentiation. Objective:To establish unique stem cells that allow efficient trophoblast differentiation from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) METHODS: hESC and iPSC were transiently exposed to BMP4 (10 ng/ml), A83-01 (1 mM) an activin A signaling inhibitor and PD173074 (0.1 mM) an FGF receptor inhibitor in culture medium lacking FGF2 for 24-36 h and then followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. RESULTS: Acquired self-renewing cell lines can be propagated clonally on gelatin and are morphologically distinct from human PSC progenitors but still meet standard in vitro criteria for pluripotency. The cells expressed weakly for CDX2 and strongly for NANOG and have a distinct transcriptome profile from the human PSCs fromwhich they were derived. The colonies spontaneously differentiated along multiple lineages, including trophoblast when cultured in nonconditioned medium lacking FGF2. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. They formwelldifferentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. CONCLUSION: The cell we established that BMP4 can prime ESC and iPSC to a self-renewing alternative state permissive for trophoblast development. JPA2015-16. INDUCED PLURIPOTENT STEM CELLS (IPS CELL) DIFFERENTIATES TO TROPHOBLAST Junya Kojima , Hidenori Akutsu , Hirotaka Nishi , Naoaki Kuji , Keiichi Isaka . Department of Obstetrics and Gynecology, Tokyo Medical University, Japan; National Center for Child Health and Development, Japan Objective: The induced pluripotent stem cells (iPS cell) was derived from somatic cells. The iPS cell had pluripotency which could differentiate to almost all cells, and had self-renewal. iPS cell was different from embryonic stem cells, because we could make from various patient’s somatic cells. There were many reports trying to differentiate this iPS cell into various cells of the body like osteocyte, nerve cells, and hepatocyte cells. In this study, we used iPS cell, and induced differentiation of trophoblast that formed placenta. Methods: We used iPS cells derived from human endometrial cells, and made embryoid body (EB) on the suspension culture plate. We cultured EB for one week and adhered it on the dish coated with an extracellular matrix ( ). Then we add bone morphogenetic protein 4 (BMP4) 10ng/ml, 100ng/ml and cultured for one week. After that we collected cells, extracted RNA and made cDNA. The expression of undifferentiation marker (Oct3/4), trophoblast marker (CDX2, GCM1, syncytinI, syncytinII, KLF6, and PPAR-g) were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Results: The expression of CDX2, GCM1, and syncytinI slightly increased in the BMP4 10ng/ml addition group compared with the control group. But there was no significant difference. In BMP4 100ng/ml addition group, all gene expression decreased compared with the control group. Conclusion: In this study, we used BMP4 and tried differentiation to trophoblast from iPS cells based on various reports, but could not differentiate effectively. Differentiation to trophoblast contributed to clinical elucidation and treatment of obstetrical disease, like preeclampsia, fetal growth restriction, and trophoblastic disease. We wanted to develop a more effective cultural method in future. JPA2015-17. PLACENTAMEGALY: FREQUENCY, CAUSES, AND COMPLICATIONS Arizawa Masayoshi. Tokyo Metropolitan Ohtsuka Hospital, Japan Introduction: The normal weight for a trimmed placenta is between 300 and 500 grams. If the weight is over 600 grams this is known as placentamegaly. This is usually associated with gestational diabetes (GDM), gigantism, virus infection, and placental hemangioma. However, we did not previously know the frequency of placentamegaly. This study looked at frequency, and complications with mother’s health and baby’s development. Method: I measured the weight of 1549 singleton placentas over a period of five years. I measured this by taking off the umbilical cord and membrane and measuring only the placental weight. Results: I found 63 placentamegaly cases out of 1549 high-risk cases sent to the department of pathology, which is 4.1% I studied the problems in three divisions; mothers, fetus and placenta A) Mothers’ problems From 63 cases there were 10 GDM, 4 unexamined pregnancies, and 1 mother’s anemia B) Babies’ problems From 63 cases we found 10 cases of 21 trisomy, and 4 cases of gigantism over 4000g C) Placenta problem From 63 cases we found 10 cases of chorangiosis, 9 cases of CAM, 7 cases of meconium stain, 2 cases of hemangioma, placental abnormal shape, and one case of abruptio placentae. Conclusion: Frommy study of 63 cases of placentamegaly (over 600 gram placental weight), I found that we should not only consider gigantism or GDM but also unexamined pregnancy, chromosomal abnormality, placental tumor, virus and bacterial infection, foetus hypoxia, and placental abnormal shapes. JPA2015-18. PREDICTION OF ADHERENT PLACENTA IN PATIENTS WITH PLACENTA PREVIA USING ULTRASONOGRAPHY AND MAGNETIC RESONANCE IMAGING Kenji Tanimura , Yui Yamasaki , Yoshiko Ueno , Tetsuo Maeda , Yasuhiko Ebina , Masashi Deguchi , Mayumi Morizane , Hideto Yamada . Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Japan; Department of Radiology, Kobe University Graduate School of Medicine, Japan Objective: Adherent placenta is a life-threatening condition in pregnancy, and is often complicated by placenta previa. The aim of this

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

Background Signs of severe oxidative stress are evident in term placentae of infants born to mothers with preeclampsia (PE), but it is unclear whether this is a cause or consequence of the disease. Here fibroblast lines were established from umbilical cords (UC) delivered by mothers who had experienced early onset PE and from controls with the goal of converting these primary cells to induced pluripotent stem cells and ultimately trophoblast. Contrary to expectations, the oxidative stress responses of these non-placental cells from PE infants were more severe than those from controls. Methods and Findings Three features suggested that UC-derived fibroblasts from PE infants responded less well to oxidative stressors than controls: 1) While all UC provided outgrowths in 4% O2, success was significantly lower for PE cords in 20% O2; 2) PE lines established in 4% O2 proliferated more slowly than controls when switched to 20% O2; 3) PE lines were more susceptible to the pro-oxidants diethylmaleate and tert-butylhydroquinone than control lines, but, unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few genes differentially regulated between PE lines and controls in 4% O2 conditions. However, a more severely stressed phenotype than controls, particularly in the unfolded protein response, was evident when PE lines were switched suddenly to 20% O2, thus confirming the greater sensitivity of the PE fibroblasts to acute changes in oxidative stress. Conclusions UC fibroblasts derived from PE infants are intrinsically less able to respond to acute oxidative stress than controls, and this phenotype is retained over many cell doublings. Whether the basis of this vulnerability is genetic or epigenetic and how it pertains to trophoblast development remains unclear, but this finding may provide a clue to the basis of the early onset, usually severe, form of PE.

Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7(+) within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7(+) and in which the majority of cells are transiently CDX2(+). Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts.

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent “primed” induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after ≈22 days, providing an overall reprogramming efficiency of ≈0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of ≈17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.

Skewing of the sex ratio towards males occurs among pups born to mice fed a very high saturated fat (VHF) diet. in the present study, we tested whether the fat content of the VHF diet rather than the number of calories consumed is responsible for this effect. Eight-week-old NIH Swiss mice were placed,on the VHF diet either ad libitum (VHF) or in a restricted manner (VHF-R). The VHF-R mice gained weight at a similar rate to controls fed a standard chow diet. Mice were bred at 15 wk and subsequently at 26 wk and 35 wk of age. Overall, the VHF, VHF-R, and control groups delivered 244, 242, and 274 pups, respectively, with male proportions of 0.60, 0.43, and 0.48, respectively. The pup sex ratios of the VHF group (favoring males) and VHF-R group (favoring females) each differed from 0.5 (P < 0.01). The sex ratios also differed (P < 0.0001) between the VHF and control groups, and between the VHF and VHF-R groups. Within the diet groups, maternal body weight had no effect on sex ratio. Serum leptin concentrations among the dams were similar in the VHF and VHF-R groups but higher than in the control group, while the IGF1 and corticosterone levels were comparable in all three groups. Therefore, the atypical sex ratios of offspring born to dams on the VHF diet seem to be influenced by the amount of fat consumed. Since males fed the VHF diet had neither more Y-sperm nor sired more sons than daughters, the dietary effects are manifested exclusively through the female.

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17beta-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 +/- 3.0 pg/ml), compared with LF mice (5.2 +/- 2.3 pg/ml; P< or = 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 +/- 3.0 pg/ml) versus the LF group (32.7 +/- 8.4 pg/ml; P< or = 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 +/- 6.7 ng/ml) versus the 5015 group (60.1 +/- 4.9 ng/ml; P< or = 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 +/- 0.2 mmol/l) than LF and control mice (0.5 +/- 0.1 and 0.6 +/- 0.1 mmol/l respectively; P< or = 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 +/- 0.1 mmol/l) than LF and control females (0.4 +/- 0.1 and 0.6 +/- 0.1 mmol/l; P< or = 0.05). Mean vaginal pH of VHF females (6.41 +/- 0.09) was lower than 5015 females (6.76 +/- 0.10; P< or = 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

Changing environmental conditions govern adaptive skewing of offspring sex ratio in birds, insects, and in some mammalian species. However, the causes of sex ratio skewing and significance are not well understood. Female mice fed a very high fat (VHF) diet, for example, produce litters that are skewed towards males, while restricting access to this diet to 7 h/day (VHF-R) reduces the fraction of males. Conversely, mice fed a nutritionally balanced but low fat/high carbohydrate (LF) diet produce more daughters than sons. Sex ratio deviation from the expected 0.5 value was not related to the dam weight and no difference in litter size was observed among the dietary groups. Here, we tested whether these diets influence certain metabolic hormones (IGF1, corticosterone, and leptin), and glucose concentrations, thereby possibly altering conditions that might favor either the conception or development of one sex over the other. Experiment 1 used 5-week old mice that were placed permanently on one of three diets: VHF diet (caloric density 5.2 kcal/g; 54 % from lard) ad libitum (VHF, n=11); a restricted VHF diet (VHF-R, n=9) in which the mice had only 7 h access to food during their night; and Purina 5015 (caloric density 4.4 kcal/g; 26 % from fat) ad libitum (5015 Control, n=12), which produces approximately 0.5 offspring sex ratio. After three pregnancies (at 45–48 weeks of age), the mice were sacrificed and serum collected. Experiment 2 consisted of two groups of mice fed continuously from 5 to 17 weeks of age on either the VHF (n=13) or LF (caloric density 3.9 kcal/g; 10% from fat) (n=11) diets. These mice were then sacrificed and serum collected. In the third experiment, mice were fed ad libitum on the VHF (n=6), LF (n=6), or Control (n=4) diets for fifteen weeks beginning at 5 weeks of age. After 5 weeks on the experimental diets, blood glucose concentrations were determined at biweekly intervals over a period of 10 weeks. No differences were detected in serum IGF1 concentrations among the dietary groups in Experiment 1 (636.4 ± 44.3, 642.6 ± 49.0, and 615.6 ± 42.5 ng/ml for VHF, VHF-R, and 5015, respectively) and Experiment 2 (706.2 ± 25.6 and 659.8 ± 27.8 ng/ml for VHF and LF, respectively). Serum corticosterone concentrations were not different between the VHF and VHF-R groups (43.6 ± 6.6 vs 29.8 ± 7.7 ng/ml), but were higher than the controls (9.1 ± 7.7 ng/ml) in Experiment 1. No difference existed between the VHF and LF groups (36.3 ± 11.6 vs 61.6 ± 13.4 ng/ml) in Experiment 2. Leptin concentrations did not differ between the VHF and VHF-R groups (20.7 ± 3.8 vs 24.6 ± 4.9 ng/ml), but were higher than the 5015 and LF groups (9.8 ± 3.5 and 3.2 ± 3.2 ng/ml, respectively). VHF mothers show sex ratio skewing towards males. In contrast, the VHF-R females produce more daughters than sons. Thus, leptin seems unlikely to play a role in offspring sex selection. A significant interaction existed between time and diet (P<0.05) for both blood glucose concentrations and body weight. The overall glucose concentrations, however, did not differ among the three groups suggesting that maternal blood glucose does not influence pup sex ratio. While the blood chemistries displayed differences related to diet, these changes most likely do not provide an explanation for the skewing of offspring sex ratio. Supported by NIH Grant HD 044042. (poster)

Pronounced skewing of the sex ratio towards males occurs among pups born to mature NIH Swiss dams fed continuously on a diet very high in saturated fats (VHF diet). Conversely, mice fed a nutritionally complete but low fat/high carbohydrate diet produce significantly more daughters than sons. Sex ratio deviation from the expected 0.5 value relates to the diets fed rather than dam weight and is observed with no change in litter size. Here, we tested the hypothesis that the high lard content of the VHF diet rather than the calories consumed by the dams is responsible for the increase in sex ratio among the pups. To accomplish this, mice eight weeks of age were placed permanently on the VHF diet (lard supplying 54 % of the calories; caloric density 5.2 kcal/g) either ad libitum (VHF group) or in a restricted manner (∼7 h access to food during their night: VHF-R group). Under the latter conditions, the VHF-R mice gained weight at a similar rate to controls fed Purina 5015 (calories from fat 26 %; caloric density 4.4 kcal/g). Mice (n=12/group) were first bred at 15 weeks of age and subsequently at 25.5, and 35 weeks. Pup sex was assessed at day 2 and confirmed at weaning by two independent observers. The VHF, VHFR and 5015 groups produced 27, 30 and 33 litters, respectively and 244, 242, and 274 total pups, with sex ratios (fraction males) of 0.54, 0.59, 0.79 (VHF); 0.45, 0.48, 0.35 (VHF-R), and 0.5, 0.5, 0.44 (Control, 5015 diet) respectively. The sex ratios in the VHF and 5015 and VHF and VHF-R groups differed significantly (each p< 0.0001). There was also a tendency of sex ratio to increase with parity in the VHF group, as had been noted by Rosenfeld et al. (2003). No difference was detected in sex ratio between the VHF-R and 5015 groups, although pup mortality in the immediate postnatal period was significantly higher in the VHF and particularly the VHF-R group, where the mothers failed to gain body mass at a comparable rate to the ad libitum fed controls as they began to lactate. These experiments confirm that the high fat diet strongly biases pup sex ratio towards males and suggests that the skewing of the sex ratio in favor of sons in VHF dams is a consequence of calories consumed rather than the high saturated fat content of the diet. However, an alternative explanation for the low sex ratio of pups from the VHF-R group is that stress induced by the restricted access to food may have favored a higher fraction of female pups than when the VHF diet was fed ad libitum. Supported by NIH Grant HD 044042 (poster)

Interferon-tau (IFN-tau) is the antiluteolytic factor released by concepti of ruminant ungulate species prior to implantation. All type I interferons, including IFN-tau, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c, but the distribution of the two polypeptides in uterine endometrium has not been examined. In situ hybridization and immunohistochemistry on sections from pregnant and nonpregnant ovine uteri at Days 14 and 15 after estrus and mating showed that both IFNAR1 and IFNAR2 mRNA and protein were strongly expressed in endometrial luminal epithelium (LE), superficial glandular epithelium (GE), and stromal cells, within but not outside caruncles. Similar staining patterns were noted in pregnant and nonpregnant uteri for both subunits. Western blot analysis of membrane fractions from cell lines derived from endometrial LE, GE, and stromal cells, and affinity cross-linking experiments with radioactively labeled IFN-tau performed on crude endometrial membranes indicated the presence of both high ( approximately 110 kDa) and low (75-80 kDa) molecular mass forms of the two receptor subunits. To localize where IFN-tau binds when it is introduced into the uterine lumen, immunohistochemistry with an antiserum against IFN-tau was performed on sections of uteri from Day 14 nonpregnant ewes whose uteri had previously been infused with IFN-tau. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues. These studies demonstrate the heavy concentration of IFNAR1 and IFNAR2 in cells of the LE and superficial GE, which appear to be the main targets for IFN-tau.