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Results have been inconsistent as to whether addition of colony stimulating factor 2 (CSF2) to culture medium improves embryo competence for establishment of pregnancy in cattle and humans. The purpose of the current study was to use all available experiments in cattle concerning effects of CSF2 on pregnancy success after transfer into recipient cattle. The approach was to perform a meta-analysis of all published data sets as well as data from an unpublished experiment described for the first time here. Meta-analysis failed to support the hypothesis that addition of CSF2 to embryo culture medium improves competence of bovine blastocysts to increase pregnancy or calving rates after transfer into recipient females. Thus, its general use as a culture medium additive to increase pregnancy success after embryo transfer is not recommended., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Procedures for in vitro embryo production in cattle have not been optimized. In the current experiment, we utilized a 3 × 3 factorial design to test whether the proportion of embryos becoming blastocysts in culture and the pregnancy rate after embryo transfer are affected by type of serum in the medium [no serum; 3% (v/v) KnockOut Serum Replacement (SR); 3% (v/v) fetal bovine serum (FBS)] and addition of specific embryokines [vehicle; 10 ng/mL colony stimulating factor 2 (CSF2); 100 ng/mL dickkopf related protein 1 (DKK1)] at day 5 of culture. Embryos were produced using abattoir-derived ovaries and Y-sorted semen from two Angus sires. The percent of putative zygotes and cleaved embryos becoming blastocysts was improved by SR and FBS. Pregnancy rate at day 30 was determined for 1426 Nelore recipients and calving rate for 266 recipients. In the absence of CSF2 or DKK1, pregnancy rates were lower for embryos cultured with SR or FBS. CSF2 and DKK1 reduced pregnancy rate for embryos cultured without serum but had no detrimental effect in the SR or FBS groups. Indeed, CSF2 blocked the negative effect of FBS on pregnancy rate. Data on birth weights were available for 67 bull calves. There were no effects of treatment. The sire used to produce embryos had significant and large effects on development to the blastocyst stage, pregnancy rate at day 30, calving rate and pregnancy loss between day 30 and calving. Results indicate that (1) SR and FBS can improve embryonic development in vitro while also compromising competence of embryos to survive after transfer, (2) actions of CSF2 and DKK1 depend upon other characteristics of the embryo production system, and (3) sire can have a large effect on embryonic development before and after transfer., (© 2022. The Author(s).)

The preimplantation period of embryonic development can be a key window for programming of postnatal development because extensive epigenetic remodeling occurs during this time. It was hypothesized that modification of one-carbon metabolism of the bovine embryo by addition of the methyl-donor choline to culture medium would change postnatal phenotype through epigenetic modification. Embryos produced in vitro were cultured with 1.8 mM choline chloride or control medium. Blastocysts were transferred into females and pregnancy outcomes and postnatal phenotype of the resultant calves determined. Exposure of embryos to choline increased gestation length and calf birth weight. Calves derived from choline-treated embryos were also heavier at weaning and had increased ratio of body weight to hip height than control calves. Choline altered muscle DNA methylation of calves 4 months after birth. A total of 670 of the 8149 CpG examined were differentially methylated, with the predominant effect of choline being hypomethylation. Among the genes associated with differentially methylated CpG were ribosomal RNAs and genes in AMPK, mTOR, integrin, and BEX2 canonical pathways and cellular functions involved in growth and proliferation. Results demonstrate that provision of the methyl-donor choline to the preimplantation embryo can alter its developmental program to increase gestation length, birth weight, and weaning weight and cause postnatal changes in muscle DNA methylation including those associated with genes related to anabolic processes and cellular growth. The importance of the nutritional status of the embryo with respect to one-carbon metabolism for ensuring health and well-being after birth is emphasized by these observations., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Alterations in the environment of the preimplantation embryo can affect competence to establish pregnancy and phenotype of resultant calves. In this study, the bovine embryo produced in vitro was used to evaluate postnatal programming actions of the embryokine colony-stimulating factor 2 (CSF2) and serum, which is a common additive of culture media. Oocytes were collected by ovum pick up from Brahman donors and fertilized with semen from Brahman bulls. Embryos were randomly assigned to one of the three treatments: vehicle, CSF2 10 ng/mL, or 1% (v/v) serum. Treatments were added to the culture medium from day 5 to 7 after fertilization. Blastocysts were harvested on day 7 and transferred into crossbred recipients. Postnatal body growth and Longissimus dorsi muscle characteristics of the resultant calves were measured. The percent of cleaved embryos becoming blastocysts was increased by serum and, to a lesser extent, CSF2. Treatment did not affect survival after embryo transfer but gestation length was shortest for pregnancies established with serum-treated embryos. Treatment did not significantly affect postnatal body weight or growth. At 3 mo of age, CSF2 calves had lower fat content in the Longissimus dorsi muscle and less subcutaneous fat over the muscle than vehicle calves. There was a tendency for cross-sectional area of the muscle to be smaller for serum calves than vehicle calves. Results confirm the importance of the preimplantation period as a window to modulate postnatal phenotype of resultant calves. In particular, CSF2 exerted actions during the preimplantation period to program characteristics of accumulation of intramuscular and subcutaneous fat of resultant calves. The use of a low serum concentration in culture medium from day 5 to 7 of development can increase the yield of transferrable embryos without causing serious negative consequences for the offspring., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Development of assisted reproductive technologies has been driven by the goals of reducing the incidence of infertility, increasing the number of offspring from genetically elite animals, facilitating genetic manipulation, aiding preservation and long-distance movement of germplasm, and generating research material. Superovulation is associated with reduced fertilization rate and alterations in endometrial function. In vitro production of embryos can have a variety of consequences. Most embryos produced in vitro are capable of establishing pregnancy and developing into healthy neonatal animals. However, in vitro production is associated with reduced ability to develop to the blastocyst stage, increased incidence of failure to establish pregnancy, placental dysfunction, and altered fetal development. Changes in the developmental program mean that some consequences of being produced in vitro can extend into adult life. Reduced competence of the embryo produced in vitro to develop to the blastocyst stage is caused largely by disruption of events during oocyte maturation and fertilization. Conditions during embryo culture can affect embryo freezability and competence to establish pregnancy after transfer. Culture conditions, including actions of embryokines, can also affect the postnatal phenotype of the resultant progeny.

The preimplantation embryo has a remarkable ability to execute its developmental program using regulatory information inherent within itself. Nonetheless, the uterine environment is rich in cell signaling molecules termed embryokines that act on the embryo during the morula-to-blastocyst transition, promoting blastocyst formation and programming the embryo for subsequent developmental events. Programming can not only affect developmental processes important for continuance of development in utero but also affect characteristics of the offspring during postnatal life. Given the importance of embryokines for regulation of embryonic development, it is likely that some causes of infertility involve aberrant secretion of embryokines by the uterus. Embryokines found to regulate development of the bovine embryo include insulin-like growth factor 1, colony stimulating factor 2 (CSF2), and dickkopf WNT signaling pathway inhibitor 1. Embryo responses to CSF2 exhibit sexual dimorphism, suggesting that sex-specific programming of postnatal function is caused by maternal signals acting on the embryo during the preimplantation period that regulate male embryos differently than female embryos., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

The objective of these experiments was to determine the effect of l-carnitine during oocyte maturation or embryo culture on embryo development and cryosurvival. For Experiments 1-3, embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). At d 7 after insemination, embryo development was assessed, and blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Post-thaw cryosurvival was determined by re-expansion and hatching rates at 24, 48 and 72 h post-thaw. In Experiment 1, COCs were matured with or without 3.03 mM l-carnitine. There was no effect of l-carnitine supplementation during maturation on embryo development or post-thaw cryosurvival. In experiment 2, presumptive zygotes were cultured in medium supplemented with or without 5% (v/v) fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5 and 3.03 mM. There was no effect of l-carnitine treatment on embryo development, but embryos treated with l-carnitine had increased (P ≤ 0.05) post-thaw re-expansion rates, irrespective of concentration. In experiment 3, presumptive zygotes were cultured with or without 0.75 mM l-carnitine from d 1 to d 4, from d 4 to d 7 or for the entire culture period. There was no effect of l-carnitine during culture on embryo development or post-thaw cryosurvival, regardless of the timing of addition. In Experiment 4, COCs were harvested by ovum pick-up from virgin dairy heifers (n = 24) and subjected to in-vitro embryo production with presumptive zygotes cultured with or without 0.75 mM l-carnitine. At d 7 after insemination, morula and blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Lactating Holstein cows (n = 102) were used as recipients and synchronized for timed embryo transfer. At d 7 after anticipated ovulation, a single embryo was thawed and transferred to the ipsilateral uterine horn of each recipient with a corpus luteum. Pregnancy was diagnosed at d 33, 44 and 72 of gestation. l-carnitine had no effect on the percentage of cows pregnant per embryo transfer (P/ET) after transfer of a frozen-thawed embryo. In conclusion, media supplementation with l-carnitine during in vitro embryo production can improve post-thaw cryotolerance as assessed in vitro but had no effect on P/ET after transfer of frozen-thawed embryos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Prostate androgen-regulated mucin-like protein 1 (PARM1) is a pro-proliferative and anti-apoptotic glycoprotein involved in the endoplasmic reticulum (ER) stress response. A single nucleotide polymorphism in the coding region of PARM1 has been associated with competence of bovine embryos to develop to the blastocyst stage. Here we tested the importance of PARM1 for development by evaluating consequences of reducing PARM1 mRNA abundance on embryonic development and differentiation, gene expression and resistance to ER stress., Results: Knockdown of PARM1 using an anti-PARM1 GapmeR did not affect competence of embryos to develop into blastocysts but decreased the number of trophectoderm (TE) cells in the blastocyst and tended to increase the number of cells in the blastocyst inner cell mass (ICM). Treatment of embryos with anti-PARM1 GapmeR affected expression of 4 and 3 of 90 genes evaluated at the compact-morula and blastocyst stage of development at days 5.5 and 7.5 after fertilization, respectively. In morulae, treatment increased expression of DAB2, INADL, and STAT3 and decreased expression of CCR2. At the blastocyst stage, knockdown of PARM1 increased expression of PECAM and TEAD4 and decreased expression of CCR7. The potential role of PARM1 in ER stress response was determined by evaluating effects of knockdown of PARM1 on development of embryos after exposure to heat shock or tunicamycin and on expression of ATF6, DDIT3 and EIF2AK3 at the compact morula and blastocyst stages. Both heat shock and tunicamycin reduced the percent of embryos becoming a blastocyst but response was unaffected by PARM1 knockdown. Similarly, there was no effect of knockdown on steady-state amounts of ATF6, DDIT3 or EIF2AK3., Conclusion: PARM1 participates in formation of TE and ICM cells in early embryonic development but there is no evidence for the role of PARM1 in the ER stress response.

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF, and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Analyses of biological pathways, key players, and ligand-receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus-endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development, and ripple effects of dysregulated conceptus-endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy., Competing Interests: Conflict of interest statement: Editor R.M.R. has not published any research papers with any of the authors within the last four years and does not have collaborative research grants with them.

The morula-stage embryo is transformed into a blastocyst composed of epiblast, hypoblast, and trophectoderm (TE) through mechanisms that, in the mouse, involve the Hippo signaling and mitogen-activated kinase (MAPK) pathways. Using the cow as an additional model, we tested the hypotheses that TE and hypoblast differentiation were regulated by the Hippo pathway regulators, yes-associated protein 1 (YAP1) and angiomotin (AMOT), and MAPK kinase 1/2 (MAPK1/2). The presence of YAP1 and CDX2 in the nucleus and cytoplasm of MII oocytes and embryos was evaluated by immunofluorescence labeling. For both molecules, localization changed from cytoplasmic to nuclear as development advanced. Inhibition of YAP1 activity, either by verteporfin or a YAP1 targeting GapmeR, reduced the percent of zygotes that became blastocysts, the proportion of blastocysts that hatched and numbers of CDX2+ cells in blastocysts. Moreover, the YAP1-targeting GapmeR altered expression of 15 of 91 genes examined in the day 7.5 blastocyst. Treatment of embryos with an AMOT targeting GapmeR did not affect blastocyst development or hatching but altered expression of 16 of 91 genes examined at day 7.5 and reduced the number of CDX2+ nuclei and YAP1+ nuclei in blastocysts at day 8.5 of development. Inhibition of MAPK1/2 with PD0325901 did not affect blastocyst development but increased the number of epiblast cells. Results indicate a role for YAP1 and AMOT in function of TE in the bovine blastocyst. YAP1 can also affect function of the epiblast and hypoblast, and MAPK signaling is important for inner cell mass differentiation by reducing epiblast numbers.

The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm:ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01nM and expression of S100A10 at 1.0nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

Purpose: Implantation of the mammalian embryo in the uterus is preceded by escape from the zona pellucida. In some species, hatching from the zona occurs preferentially from one or the other poles of the embryo. The situation for the bovine embryo, in which hatching precedes attachment to the uterus by more than a week, is unclear. The purpose was to describe whether hatching of the bovine embryo from the zona pellucida occurs preferentially from the embryonic or abembryonic pole., Methods: Bovine blastocysts undergoing hatching were examined by light microscopy (n = 84) and epifluorescence imaging using antibodies for markers of epiblast, hypoblast, and trophectoderm (TE) (n = 26). The location of hatching was classified as being at the embryonic pole, if hatching occurred ipsilateral to the inner cell mass (ICM), or abembryonic, if hatching occurred contralateral to the ICM., Results: A total of 55% of blastocysts exited the zona pellucida through an opening at the embryonic pole. In these cases, 68% of the cells emerging through the zona pellucida were derived from the ICM. The remainder of blastocysts hatched from an opening either contralateral or to the side of the ICM. In these cases, 87% of hatched cells were TE., Conclusion: For the bovine embryo, there is nearly equal probability of hatching from the embryonic or abembryonic poles. Given that the surface area of the zona pellucida in contact with the TE overlying the ICM is less than for the remainder of the blastocyst, there is some preference for hatching through the embryonic pole. Thus, the bovine embryo is distinct from the mouse and human, where hatching occurs preferentially at the abembryonic pole.

The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of β-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1 Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1., (© 2017 Society for Reproduction and Fertility.)

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine., (© 2016 Society for Reproduction and Fertility.)

The microenvironment of a preimplantation embryo can influence changes in development that affect postnatal phenotypes. One of the potential mediators of this effect in many species is colony-stimulating factor (CSF2), which can increase an embryo's ability to establish pregnancy after its transfer into recipients. Exposure of embryos to CSF2 during early development can also affect the pattern of development later in pregnancy in a sex-dependent manner. We therefore hypothesized that treatment of in vitro-produced embryos with CSF2 in culture would alter birth weight and postnatal growth of the resultant calf. Body weight and withers height were measured for Holstein heifer calves produced in vitro with or without 10 ng/ml CSF2 and for calves produced by artificial insemination. There were no differences in birth weight between groups; thereafter, however, calves from the CSF2-treated group experienced greater increases in body weight through 13 months of age, with only small differences in withers height. These results support the model that an embryo's postnatal characteristics can be programmed during the preimplantation period, and that CSF2 is one of the embryokines through which programming is directed. Mol. Reprod. Dev. 82: 892-897, 2015. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos., (© 2014 by the Society for the Study of Reproduction, Inc.)

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf-1 (DKK1), an antagonist of the wingless-related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal-embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro-produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony-stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies., (© FASEB.)

Fertilization and development of the preimplantation embryo is under genetic control. The present goal was to test 434 single nucleotide polymorphisms (SNPs) for association with genetic variation in fertilization and early embryonic development. The approach was to produce embryos from 93 bulls using in vitro procedures (n = 3-6 replicates per bull) and relate cleavage rate (CR) and development of cleaved embryos to the blastocyst stage (BDRC) to the genotype for each SNP. Bulls were selected to have either high or low estimates for predicted transmitted ability for daughter pregnancy rate (DPR), an estimate of female fertility. The repeatability was 0.84 for CR and 0.55 for BDRC. Semen extender affected CR, with lower results for milk extender than yolk extender. There was no significant correlation between DPR and either CR or BDRC. A total of 100 SNPs had a minor allele frequency sufficiently high (>5%) to allow association analysis. There were nine genes with SNPs associated with CR (AVP, DEPP, EPAS1, HSD17B6, NT5E, SERPINE2, SLC18A2, TBC1D24, and a noncharacterized gene) and 12 genes with SNPs associated with BDRC (C1QB, FAM5C, HSPA1A, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3, and WBP1). Results demonstrate that in vitro fertilization and blastocyst development are under genetic control and point out the potential importance of some previously unknown genes in these processes. Selection of cattle based on the genotype at one or more of these 19 loci may prove useful in conjunction with other genetic markers for improving genetic ability for fertility.

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.

Background: While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3' tag digital gene expression (3'DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance., Results: Using 3'DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT., Conclusions: Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.

The objective of this study was to examine the effect of treating pig oocytes during in vitro maturation (IVM) with a proteasome inhibitor, MG132, on oocyte maturation and embryonic development. In one series of experiments, oocytes from medium-sized follicles (3-8 mm in diameter) were untreated (MCO) or treated with MG132 during 0-22 hr (M0-22) or 30-42 hr (M30-42) of IVM. There was no significant effect of MG132 on nuclear maturation or cytoplasmic maturation (as assessed by intracellular amounts of glutathione and p34cdc2 kinase activity). Blastocyst formation after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), however, was increased for M30-42 (65.2% and 27.7% for PA and SCNT, respectively) compared to MCO (42.6% and 13.6%, respectively) and M0-22 (45.3% and 19.5%, respectively; P<0.05). Expression of PCNA and ERK2 was increased in M30-42 for IVM oocytes while transcript abundance for POUF51, DNMT1, FGFR2, and PCNA was increased in M30-42 for 4-cell SCNT embryos. When oocytes derived from small follicles (<3 mm in diameter) were untreated (SCO) or treated with MG132 during 0-22 hr (S0-22), 30-42 hr (S30-42) of IVM, or 0-22 and 30-42 hr of IVM (S0-22/30-42), expression of POU5F1, DNMT1, FGFR2, and PCNA and blastocyst formation were increased for SCNT embryos derived from S30 to 42 (16.5%) and S0-22/30-42 oocytes (20.8%) as compared to embryos from SCO (8.7%) or S0-22 oocytes (8.8%; P<0.05). Results demonstrate that treatment of oocytes with MG132 during the later stage of IVM improves embryonic development and alters gene expression in pigs., (Copyright © 2011 Wiley Periodicals, Inc.)

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine factors. The aim of this work is to determine whether fibroblast growth factor 10 (FGF10) is an oocyte competent factor. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus-oocyte complexes to FGF10 during in vitro maturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8-16-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression of CTSB and SPRY2 in cumulus cells and BMP15 in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact on in vitro embryo development implicates it as a noteworthy oocyte competent factor.

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30-35 of gestation. Moreover, treatment with CSF2 from either d 1-7 or 5-7 after insemination reduced pregnancy loss after d 30-35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

Ceramide is a second messenger induced by various cellular insults that plays a regulatory role in apoptosis. The objective of the present study was to determine whether ceramide signaling can occur in the preimplantation embryo by testing (1) effects of ceramide on development, cytokinesis, and apoptosis and (2) whether heat shock, which can induce apoptosis in embryos, causes activation of neutral or acidic sphingomyelinases responsible for generation of ceramide. Treatment of embryos > or =16 cells collected at Day 5 after insemination with 50 microM C(2)-ceramide increased caspase-9 activity and the proportion of blastomeres undergoing apoptosis but did not increase caspase-8 activity. Induction of apoptosis was more extensive when culture with ceramide was for 24 hr than for 9 hr. Ceramide also reduced the proportion of embryos that developed to the blastocyst stage when exposure was for 24 hr. At the two-cell stage, a period in development when apoptosis responses are blocked, culture of embryos with ceramide did not increase caspase-9 activity or the proportion of blastomeres that were apoptotic. However, culture with ceramide for 24 hr reduced cell proliferation and caused an increase in multinucleated cells because of inhibition of cytokinesis. Exposure of Day 5 embryos to a heat shock of 41 degrees C for 15 hr increased neutral sphingomyelinase activity but did not change acid sphingomyelinase activity. In conclusion, ceramide can regulate embryo development and apoptosis in a time and stage-of-development dependent manner and ceramide generation can be activated by cellular insult. Thus, the ceramide signaling pathway is present in the preimplantation embryo.

Addition of insulin-like growth factor-1 (IGF-1) to culture medium increases the proportion of bovine embryos that develop to the blastocyst stage and increases embryo survival following transfer to heat-stressed, lactating dairy cows. The objective of the present study was to determine molecular and cellular correlates of these actions of IGF-1. Embryos were produced in vitro and cultured for 7 days with or without 100 ng/ml IGF-1. On d 7 after insemination, grade 1 expanded blastocysts were harvested and used to determine total cell number, percent apoptosis, cell allocation to the inner cell mass and trophectoderm, and the relative abundance of several developmentally important gene transcripts. There was no significant effect of IGF-1 treatment on blastocyst cell number, the proportion of blastomeres that were apoptotic, or the number of cells in the inner cell mass and trophectoderm. However, differences in the relative abundance of several mRNA transcripts were observed between control and IGF-1 treated embryos. Addition of IGF-1 increased (P < 0.02) amounts of mRNA for IGF binding protein-3 and desmocollin II and tended (P < 0.08) to increase amounts of mRNA for Na/K ATPase and Bax. Moreover, IGF-1 treatment decreased (P < 0.05) steady-state amounts of transcripts for heat shock protein 70 and tended (P < 0.08) to reduce amounts of IGF-1 receptor mRNA. In conclusion, increased survival of embryos treated with IGF-1 does not appear due to effects on cell number, percent apoptosis, or cell allocation. Addition of IGF-1 to culture can, however, alter expression of several transcripts which may be important for embryo development and survival following transfer., ((c) 2007 Wiley-Liss, Inc.)

Insulin-like growth factor (IGF-I) has been implicated as a thermoprotective molecule for the preimplantation bovine embryo. Here, it was shown that effects of heat shock (41 degrees C for 15 hr) on induction of apoptosis and reduction in cell number in bovine embryos collected at Day 5 after fertilization were blocked by addition of 100 ng/ml IGF-I at the initiation of heat shock. This action of IGF-I to block heat shock-induced apoptosis was eliminated if embryos were cultured with either a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) or an Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-o-methyl-3-o-octadecylcarbonate). Immunofluorescence microscopy confirmed the expression of phosphorylated Akt for IGF-I and control embryos. Immunoblotting using an antibody to Akt (phospho S473) indicated increased phosphorylation of Akt in IGF-I-treated embryos. In conclusion, short-term treatment of embryos with IGF-I can block induction of apoptosis caused by heat shock through signaling events requiring PI3K and Akt., ((c) 2007 Wiley-Liss, Inc.)

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.

Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into postnatal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on the maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.