The expression and potential function of bone morphogenetic proteins 2 and 4 in bovine trophectoderm

<p>Abstract</p> <p>Background</p> <p>Bone morphogenetic proteins (BMPs) were first described for their roles in bone formation, but they now also are known to possess additional activities, including those relating to embryogenesis. The objectives of this work were to 1) determine if peri-attachment bovine conceptuses and bovine trophoblast cells (CT1) contain transcripts for <it>BMP2 </it>and <it>4</it>, an innate inhibitor noggin (<it>NOG</it>), and BMP2/4 receptors (<it>BMPRII</it>, <it>ACVR1</it>, <it>BMPR1A</it>, <it>BMPR1B</it>), and 2) determine if BMP2 or 4 supplementation to CT1 cells affects cell proliferation, differentiation or trophoblast-specific gene expression.</p> <p>Methods</p> <p>RNA was isolated from day 17 bovine conceptuses and CT1 cells. After RT-PCR, amplified products were cloned and sequenced. In other studies CT1 cells were treated with BMP2 or 4 at various concentrations and effects on cell viability, cell differentiation and abundance of IFNT and CSH1 mRNA were evaluated.</p> <p>Results</p> <p>Transcripts for <it>BMP2 </it>and <it>4 </it>were detected in bovine conceptuses and CT1 cells. Also, transcripts for each BMP receptor were detected in conceptuses and CT1 cells. Transcripts for <it>NOG </it>were detected in conceptuses but not CT1 cells. Cell proliferation was reduced by BMP4 but not BMP2 supplementation. Both factors reduced <it>IFNT </it>mRNA abundance but had no effect on <it>CSH1 </it>mRNA abundance in CT1 cells.</p> <p>Conclusions</p> <p>The BMP2/4 ligand and receptor system presides within bovine trophectoderm prior to uterine attachment. BMP4 negatively impacts CT1 cell growth and both BMPs affect IFNT mRNA abundance.</p

Cytokines That Serve as Embryokines in Cattle.

The term embryokine has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos. Embryokines are being explored for their ability to overcome the poor development rates of IVP embryos and to limit post-transfer pregnancy retention efficiencies that exist in IVP embryos. This review will focus on describing two of the best-characterized cytokines, colony-stimulating factor 2 and interleukin 6, for their ability to modify bovine embryo quality and confirmation, promote normal fetal development, and generate healthy calves. Additional cytokines will also be discussed for their potential to serve as embryokines

Autoimmunization of Ewes Against Pregnancy-associated Glycoproteins Does Not Interfere with the Establishment and Maintenance of Pregnancy

doi:10.1017/S1751731109004145Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n522) or with KLH alone (n59), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.662.2 of pregnancy. Those ewes immunized against PAGs (n57), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.961.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n515), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1615.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.This work was supported by NIH Grant HD21896 and the Animal Reproductive Biology Group of the University of Missouri Food for the 21st Century Program

Sexually dimorphic gene expression in bovine conceptuses at the initiation of implantation

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X-chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium

Effect of dietary energy source on pregnancy rates and reproductive physiology of pastured beef heifers

Proper production of steroid hormones, such as progesterone (P4), is essential for maintenance of pregnancy. Provision of gluconeogenic substrates from the diet may alter postabsorptive energy supplies and associated hormone signaling in a manner supportive of elevating P4 concentrations. Crossbred Angus heifers (n=27) were balanced for pre-trial bodyweight (BW) and body condition score (BCS), then assigned to isoenergetic diets leveraging starch (ES) or fat (EF) as the primary source of energy (45.7% starch vs 11.5% fat). The heifers were placed on the assigned diets 8 days prior to the initiation of estrous synchronization and continued the diets through the second pregnancy diagnosis, 52 days after the initial insemination. On day 28 post-breeding, pregnancy status was diagnosed via ultrasonography. Measurements of follicle size and number were collected via rectal palpation and ultrasonography on days −10, −8, −4, and day 0 relative to breeding. Blood samples were collected on days −10, 1 through 7, 10, 14, 18, 21, 24, and 28 to analyze plasma for P4 concentrations and placental-associated glycoprotein (PAG) concentrations. Data were analyzed using a linear mixed effects model with a fixed effect for treatment and a random effect for pen. Observations collected over time were analyzed using a repeated measures approach with fixed effects for treatment, time, initial condition, and a random effect for pen. Interactions between treatment and pregnancy status were also tested for PAG and P4 concentrations. No treatment differences were observed in final BCS (P=0.12) and total dry matter intake (DMI; P=0.51), though heifers on the ES treatment tended to reach an increased BW (P=0.10). Although heifers fed ES diets had numerically higher conception rates compared to heifers fed EF diets (71.4% vs. 66.7%), there was no statistically significant difference. Furthermore, there were no significant differences observed among diets in terms of pregnancy outcomes, follicle size, or number. A treatment by pregnancy status interaction was detected for both PAG and P4 concentrations. These results suggest that short-term dietary starch supplementation provided around the time of breeding can result in shifts to reproductive hormone abundance that may be conducive for pregnancy establishment

Bovine Blastocyst-Derived Trophectoderm and Endoderm Cell Cultures: Interferon Tau and Transferrin Expression as Respective In Vitro Markers

Continuous cultures of bovine trophectoderm (CT-1 and CT- 5) and bovine endoderm (CE-1 and CE-2) were initiated and maintained on STO feeder cells. CT-1 and CT-5 were derived from the culture of intact, 10- to 11-day in vitro-produced blastocysts. CE-1 and CE-2 were derived from the culture of immunodissected inner cell masses of 7- to 8-day in vitro-produced blastocysts. The cultures were routinely passaged by physical dissociation. Although morphologically distinct, the trophectoderm and endoderm both grew as cell sheets of polarized epithelium (dome formations) composed of approximately cuboidal cells. Both cell types, particularly the endoderm, grew on top of the feeder cells for the most part. Trophectoderm cultures grew faster, relative to endoderm, in large, rapidly extending colonies of initially flat cells with little or no visible lipid. The endoderm, in contrast, grew more slowly as tightly knit colonies with numerous lipid vacuoles in the cells at the colony centers. Ultrastructure analysis revealed that both cell types were connected by desmosomes and tight junctional areas, although these were more extensive in the trophectoderm. Endoderm was particularly rich in rough endoplasmic reticulum and Golgi apparatus indicative of cells engaged in high protein production and secretion. Interferon tau expression was specific to trophectoderm cultures, as demonstrated by reverse transcription-polymerase chain reaction, Western blot, and antiviral activity; and this property may act as a marker for this cell type. Serum protein production specific to endoderm cultures was demonstrated by Western blot; this attribute may be a useful marker for this cell type. This simple coculture method for the in vitro propagation of bovine trophectoderm and endoderm provides a system for assessing their biology in vitro

ISOLATION AND CHARACTERIZATION OF A BOVINE VISCERAL ENDODERM CELL LINE DERIVED FROM A PARTHENOGENETIC BLASTOCYST

A cell line, BPE-1, was derived from a parthenogenetic 8-d in vitro-produced bovine blastocyst that produced a cell outgrowth on STO feeder cells. The BPE-1 cells resembled visceral endoderm previously cultured from blastocysts produced by in vitro fertilization (IVF). Analysis of the BPE-1 cells demonstrated that they produced serum proteins and were negative for interferon-tau production (a marker of trophectoderm). Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions in conjunction with desmosomes. Rough endoplasmic reticulum was prominent within the cells as were lipid vacuoles. Immunocytochemistry indicated the BPE-1 cells had robust microtubule networks. These cells have been grown for over 2 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The BPE-1 cell line presumably arose from embryonic cells that became diploid soon after parthenogenetic activation and development of the early embryo. However, metaphase spreads prepared at passage 41 indicated that the cell population had a hypodiploid (2n = 60) unimodal chromosome content with a mode of 53 and a median and mean of 52. The cell line will be of interest for functional comparisons with bovine endoderm cell lines derived from IVF and nuclear transfer embryos