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Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase‐3 (z‐DEVD‐fmk) could improve post‐thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled‐rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1–3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 μM) and z‐DEVD‐fmk (0, 50, 100 or 200 μM), respectively. Post‐thaw survival was assessed at 24, 48 and 72 h. For experiments 4–5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post‐thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p <.05) re‐expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post‐thaw cryosurvival was not affected by the addition of either DTT or z‐DEVD‐fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p <.001) levels of intracellular ROS and fewer (p <.001) cells with DNA fragmentation. In conclusion, post‐thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Heat stress (HS) causes hyperthermia, and at its most severe form, can lead to death. More commonly, HS reduces feed intake, milk yield, growth rate and reproductive function in many mammals and birds, including the important cattle breeds in Japan. Rectal temperatures greater than 39.0°C and respiration rates greater than 60/min indicate cows are undergoing HS sufficient to affect milk yield and fertility. HS compromises oocyte quality and embryonic development, reduces expression of estrus and changes secretion of several reproductive hormones. One of the most effective ways to reduce the magnitude of HS is embryo transfer, which bypasses the inhibitory effects of HS on the oocyte and early embryo. It may also be possible to select for genetic resistance to HS. Cooling can also improve reproductive performance in cows and heifers, and probably, the most effective cooling systems currently in use are those that couple evaporative cooling with tunnel ventilation or cross ventilation. Its effect on improving reproductive performance in Japan remains to be evaluated. [ABSTRACT FROM AUTHOR]

Loureiro B, Oliveira LJ, Favoreto MG, Hansen PJ. Colony-stimulating factor 2 inhibits induction of apoptosis in the bovine preimplantation embryo. Am J Reprod Immunol 2011; 65: 578-588 Addition of colony-stimulating factor 2 (CSF2) to culture medium increases post-transfer survival of bovine embryos. Here we provide evidence that one mechanism by which CSF2 affects the embryo is through inhibition of apoptosis. In the first experiment, genes and pathways whose expression were regulated by CSF2 were identified by microarray analysis. Embryos were treated with 10 ng/ml CSF2 or vehicle at Day 5 after insemination; morulae were selected for microarray analysis at Day 6. In a second experiment, antiapoptotic effects of CSF2 were determined. Embryos were treated with CSF2 or vehicle at Day 5. On Day 6 (24 h after treatment), morulae were cultured for 15 h at either 42°C (a temperature that induces apoptosis) or 38.5°C (cow body temperature). In the first experiment, a total of 214 genes were differentially regulated and 160 of these could be annotated (67 upregulated genes and 93 downregulated genes). Differentially expressed genes could be placed in 13 biological process ontologies in four functional groups (development and differentiation process, cell communication, apoptosis and cell adhesion). Antiapoptotic effects of CSF2 were confirmed in the second experiment because the magnitude of the increase in TUNEL positive cells caused by heat shock was reduced by CSF2. CSF2 blocks apoptosis in bovine embryos through actions associated with regulation of genes controlling apoptosis. [ABSTRACT FROM AUTHOR]

Citation Padua MB, Hansen PJ. Evolution and function of the uterine serpins (SERPINA14). Am J Reprod Immunol 2010 Uterine serpins (recently designated as SERPINA14) are hormonally induced proteins secreted in large quantities by the endometrial epithelium during pregnancy. The SERPINA14 proteins belong to the ine roteinase hibitor (serpin) superfamily, but their apparent lack of inhibitory activity toward serine proteinases suggests that these proteins evolved a different function from the anti-proteinase activity typically found in most members of the serpin superfamily. The gene is present in a limited group of mammals in the Laurasiatheria superorder (ruminants, horses, pigs, dolphins and some carnivores) while being absent in primates, rodents, lagomorphs and marsupials. Thus, the gene is likely to have evolved by gene duplication after divergence of Laurasiatheria and to play an important role in pregnancy. That role may vary between species. In sheep, SERPINA14 probably serves an immunoregulatory role to prevent rejection of the fetal allograft. It is inhibitory to lymphocyte proliferation and natural killer cell function. In the pig, SERPINA14 is involved in iron transport to the fetus by binding to and stabilizing the iron-binding protein uteroferrin. It is possible that SERPINA14 has undergone divergence in function since the original emergence of the gene in a common ancestor of species possessing SERPINA14. [ABSTRACT FROM AUTHOR]

Citation Hansen PJ. Medawar redux – an overview on the use of farm animal models to elucidate principles of reproductive immunology. Am J Reprod Immunol 2010 Farm animals have been important models for the development of reproductive immunology. Two of the major concepts underpinning reproductive immunology, the idea of the fetal allograft and progesterone’s role in regulation of uterine immunity, were developed using the bovine as a model. This volume of the American Journal of Reproductive Immunology is composed of review articles that highlight the continued relevance of farm animals as models for research in mammalian biology. It is important that a diverse array of genotypes are used to elucidate biological principles relevant to mammalian biology and human health because the nature of mammalian evolution has resulted in a situation where the genome of the most commonly used animal model, the laboratory mouse, is less similar to the human than other species like the cow. Moreover, the evolution of placental function has been accompanied by formation of new genes during recent evolution so that orthologs do not exist in any but closely related species. Given the infrastructure needs to study farm animal species, optimal utilization of these animals as models for biomedical research will require significant increases in funding to reverse a historical erosion of resources devoted to animal agricultural research. [ABSTRACT FROM AUTHOR]

Uterine serpins (USs), designated as SERPINA14, are expressed in the endometrium in response to progesterone. All species identified as having USs exhibit epitheliochorial placentation and are in the Ruminantia and Suidae orders of the Laurasiatheria superorder. The objective was to identify US genes in species within and outside Laurasiatheria and evaluate whether evolution of the US gene was associated with development of the epitheliochorial placenta. Through queries of nucleotide and genomic databases, known US genes were identified (caprine, bovine, porcine, water buffalo), and new US coding sequences were found in dolphins, horses, dogs, and cats. The cat sequence contained several stop codons. No sequence was found in completed genomic sequences for primates, rodents, rabbits, opossums, or duck-billed platypuses. Reverse transcription-polymerase chain reaction confirmed expression of the US gene in the uterus of pregnant horses and dogs. The ratio of nonsynonymous/synonymous substitutions suggests that the US gene evolved under positive selection. In conclusion, the US gene evolved within the Laurasiatheria super-order to play a role in pregnancy for species with epitheliochorial placentation and some but not all Laurasiatheria species that have a different form of placentation. The positive selection taking place in die gene suggests development of species-specific functions. [ABSTRACT FROM AUTHOR]

Problem Macrophages are recruited in large number to the interplacentomal endometrium of the cow during pregnancy. We evaluated whether endometrial macrophages also accumulate in placentomal regions of endometrium during pregnancy and whether endometrial macrophages are regionally differentiated. Method of study Interplacentomal endometrium and placentomes were subjected to dual-color immunofluorescence using CD68 as a pan-macrophage marker. Results CD68+ cells were abundant in stroma of the interplacentomal endometrium and caruncular septa of the placentomes. CD68+ cells were not present in fetal villi of the placentomes or in the interplacentomal chorion. Regardless of location, the majority of CD68+ cells also expressed CD14. In interplacentomal endometrium, CD68+CD11b+ cells were present in deeper areas of the stroma but not in shallow endometrial stroma. In caruncular septa of the placentome, CD68+ cells were negative for CD11b. CD68+ cells in the interplacentomal endometrium were negative for MHC class II while most CD68+ cells in caruncular septa were positive for MHC class II. Conclusion CD68+CD14+ macrophages present in the stroma of the interplacentomal endometrium and caruncular septa of the placentome are regionally differentiated with regard to expression of CD11b and MHC class II. [ABSTRACT FROM AUTHOR]

Galectin 15 (LGALS15) is expressed specifically by the endometrial luminal epithelium (LE) of the ovine uterus in concert with blastocyst growth, elongation, and implantation. LGALS15 contains a predicted carbohydrate recognition domain (CRD) as well as LDV and RGD recognition sequences for integrin binding. Studies tested the hypothesis that LGALS15 is a secreted regulator of blastocyst development, as well as growth, migration, adhesion, and apoptosis of trophoblast. Bovine embryos were produced in vitro by standard conditions, and putative zygotes were cultured in the presence of recombinant ovine LGALS15. Rates of embryo cleavage and blastocyst formation were not affected by LGALS15. LGALSI5 moderately increased proliferation of ovine trophectoderm (oTr) cells. Staurosporine elicited apoptosis of oTr cells, which could be partially inhibited by LGALS15. Migration of oTr cells was stimulated by LGALS15 that was dependent on Jun N-terminal kinase (JNK). A dose-dependent increase in oTr cell attachment to LGALS15 was found that could be inhibited by cyclic GRGDS, but not GRADS, peptides. Mutation of the LDVRGD integrin binding sequence of LGALS15 to LADRAD decreased its ability to promote oTr cell attachment, whereas mutation of the CRD had tittle effect. LGALS15 induced formation of robust focal adhesions in oTr cells that was abolished by mutation of the LDVRGD sequence. Collectively, these results support the hypothesis that LGALS15 stimulates trophectoderm cell migration and attachment via integrin binding and activation which are critical to blastocyst elongation and implantation. [ABSTRACT FROM AUTHOR]

Progesterone is critical for the establishment and the maintenance of pregnancy, both by its endocrine and immunological effects. The genomic actions of progesterone are mediated by the intracellular progesterone receptors; A and B. A protein called P-induced blocking factor (PIBF), by inducing a TH2 dominant cytokine production, mediates the immunological effects of progesterone. Progesterone plays a role in uterine homing of NK cells and up-regulates HLA-G gene expression, the ligand for various NK inhibitory receptors. At high concentrations progesterone is a potent inducer of Th2-type cytokines as well as of LIF and M-CSF production by T cells. Though a key role for progesterone in creating local immunosuppression has been conserved during the evolution of an epitheliochorial placenta, there has been some divergence in the pattern of endocrine-immunological cross talk in Bovidae. In sheep, uterine serpin, a progesterone-induced endometrial protein, mediates the immunosuppressive effects of progesterone. Epidemiological studies suggest the role of stress in premature pregnancy termination and exposure to stress induces abortion in mice via a significant reduction in progesterone levels, accompanied by reduced serum levels of PIBF. These effects are corrected by progesterone supplementation. These findings indicate the significance of a progesterone-dependent immuno-modulation in maternal tolerance of the fetus, which is discussed in this review. [ABSTRACT FROM AUTHOR]

Problem Semen is a rich source of transforming growth factor- β (TGF- β) and it has been proposed that this molecule promotes embryonic survival by modifying immune responses to promote tolerance toward paternal antigens and by inducing release of cytokines that promote embryonic development. The role of TGF- β was tested using pigs by evaluating whether its addition to washed sperm improves conceptus survival and fetal growth. Methods of study At estrus, gilts were artificially inseminated twice at 12-hr intervals with 100 mL of either washed semen resuspended in a commercial semen extender supplemented with 2 mg/mL of gelatin or washed semen in the same extender containing 65 ng/mL of TGF- β1. Three boars were used as semen donors. At day 80 (±4 days) of gestation, gilts were sacrificed and reproductive tracts harvested. Results Treatment had no effect ( P > 0.10) on total or live fetuses per litter, implantation rate, fetal survival or percentage of corpora lutea resulting in live fetuses at day 80. Insemination with TGF- β1 also did not affect total or average fetal weight or total placental weight. There was a tendency ( P = 0.09) for average placental weight of live fetuses to be lower for pregnancies established in gilts treated with TGF- β1. Also, placental efficiency (mass of fetus/mass of placenta) was greater ( P < 0.05) for pregnancies established in gilts treated with TGF- β1. The high fertility in control gilts (80% implantation rate and 11.5 live fetuses per litter) is indicative that soluble seminal factors are not necessary for the establishment of pregnancy. Conclusions Within the ranges tested, concentration of TGF- β in the fluid phase of the inseminate is not an important determinant of conceptus survival or fetal and placental growth to day 80 of gestation in the pig. [ABSTRACT FROM AUTHOR]

PROBLEM: Ovine uterine serpin (OvUS) is a member of the serine proteinase inhibitor superfamily and is the major protein produced by luminal and glandular epithelium of the sheep endometrium during mid to late pregnancy. The protein does not have prototypical proteinase inhibitory activity but can inhibit a wide variety of lymphocyte functions such as mitogen-induced proliferation and natural killer cell cytotoxicity. METHOD OF STUDY: The antiproliferative actions of OvUS were studied. RESULTS: It was demonstrated that, in addition to inhibiting lymphocyte proliferation, OvUS inhibits growth of two tumor cell lines (D17 and PC-3). The protein also interrupts development of pre-implantation embryos. Inhibition of cell proliferation is not universal, however, as OvUS did not inhibit growth of two non-tumorigenic cell lines (MDBK and BEND). The mechanism of action of inhibitory effects of OvUS is not known although experiments with inhibitors of protein kinase A indicate that the protein does not inhibit lymphocyte proliferation through this pathway. Moreover, the protein does not induce apoptosis. CONCLUSIONS: The finding that OvUS has antiproliferative activity is demonstrative of the wide range of functions exerted by members of the serpin superfamily. The antiproliferative property of OvUS may reflect the role of the protein during pregnancy and may be exploitable for design of new antiproliferative drugs. [ABSTRACT FROM AUTHOR]

Hansen PJ, Soto P, Natzke RP. Mastitis and fertility in cattle – possible involvement of inflammation or immune activation in embryonic mortality. AJRI 2004; 51:294–301 © Blackwell Munksgaard, 2004 Causes for pre-implantation embryo loss, which can be as high as 50% or more of fertilized embryos, are multifactorial and largely undescribed. Studies in cattle using mastitis as a model indicate that one cause of early embryonic loss is infectious disease or activation of immune responses at sites outside the reproductive tract. Infection of the mammary gland in dairy cattle is associated with a reduction in pregnancy rate (proportion of inseminated cows that become pregnant) and an increase in the number of inseminations required to establish pregnancy. Also, intravenous challenge with bacterial peptidoglycan and polysaccharide at ∼days 3–5 after breeding reduced subsequent pregnancy rate in sheep that had been previously immunized against the same material. The mechanism by which extrauterine activation of immune and inflammatory responses leads to embryonic loss is not clear although cytokines probably play a crucial role. Effects could be exerted at the level of the hypothalamic–pituitary axis, ovary, reproductive tract or embryo. Interferon (IFN)- α, for example, which can reduce pregnancy rate in cattle when injected around 13–19 days after breeding, increases body temperature, inhibits secretion of luteinizing hormone, and reduces circulating concentrations of progesterone. Other cytokines or products of cytokine activation could cause embryonic loss by causing hyperthermia (as elevated temperature blocks oocyte function and embryonic development), exerting toxic effects on the corpus luteum [for example, IFN- γ, tumor necrosis factor- α (TNF- α) and prostaglandin F2 α], stimulating endometrial prostaglandin synthesis [TNF- α and interleukin(IL)-1 β], reducing endometrial cell proliferation (IL-1 β), and interfering with oocyte maturation and embryonic development (TNF- α, nitric oxide, and prostaglandin F2 α). Although largely neglected by reproductive immunologists, study of the involvement of the immune system in pre-implantation embryonic loss is likely to lead to new methods for enhancing fertility. [ABSTRACT FROM AUTHOR]

Tekin Ş, Hansen PJ. Regulation of numbers of macrophages in the endometrium of the sheep by systemic effects of pregnancy, local presence of the conceptus, and progesterone. AJRI 2004; 51:56–62 © Blackwell Munksgaard, 2004 Many species exhibiting hemochorial placentation experience an accumulation of macrophages in the endometrium during pregnancy. For the present study, it was tested whether macrophages also accumulate in the endometrium of the sheep, which is a species undergoing an epitheliochorial placentation. An additional objective was to determine whether regulation of endometrial macrophage number occurs via systemic or local signals and whether progesterone is one of these signals. The approach was to evaluate presence of macrophages immunohistochemically using antibodies to CD68 and CD14. Tissues examined were from cyclic ewes in the luteal phase of the estrous cycle, unilaterally-pregnant ewes at day 140 of pregnancy in which pregnancy was surgically confined to one uterine horn, ovariectomized ewes, and ovariectomized ewes treated with progesterone for 44 days. Macrophages were localized predominately to the stromal compartment of the stratum compactum region of the endometrium. In non-pregnant ewes, macrophages were not abundant regardless of physiological status. Increased numbers of endometrial macrophages were seen for both the pregnant and non-pregnant uterine horns of unilaterally pregnant ewes. Numbers of macrophages were higher in the endometrium from the pregnant uterine horn than from endometrium from the non-pregnant uterine horn. Results indicate that macrophages accumulate in the endometrium by day 140 of pregnancy in the sheep and that this induction is because of both systemic and local signals. Progesterone appears not to be an important regulator of numbers of endometrial macrophages. [ABSTRACT FROM AUTHOR]

Problem: Semen is a rich source of transforming growth factor-beta (TGF-beta) and it has been proposed that this molecule promotes embryonic survival by modifying immune responses to promote tolerance toward paternal antigens and by inducing release of cytokines that promote embryonic development. The role of TGF-beta was tested using pigs by evaluating whether its addition to washed sperm improves conceptus survival and fetal growth., Methods of Study: At estrus, gilts were artificially inseminated twice at 12-hr intervals with 100 mL of either washed semen resuspended in a commercial semen extender supplemented with 2 mg/mL of gelatin or washed semen in the same extender containing 65 ng/mL of TGF-beta1. Three boars were used as semen donors. At day 80 (+/-4 days) of gestation, gilts were sacrificed and reproductive tracts harvested., Results: Treatment had no effect (P > 0.10) on total or live fetuses per litter, implantation rate, fetal survival or percentage of corpora lutea resulting in live fetuses at day 80. Insemination with TGF-beta1 also did not affect total or average fetal weight or total placental weight. There was a tendency (P = 0.09) for average placental weight of live fetuses to be lower for pregnancies established in gilts treated with TGF-beta1. Also, placental efficiency (mass of fetus/mass of placenta) was greater (P < 0.05) for pregnancies established in gilts treated with TGF-beta1. The high fertility in control gilts (80% implantation rate and 11.5 live fetuses per litter) is indicative that soluble seminal factors are not necessary for the establishment of pregnancy., Conclusions: Within the ranges tested, concentration of TGF-beta in the fluid phase of the inseminate is not an important determinant of conceptus survival or fetal and placental growth to day 80 of gestation in the pig.

Many species exhibiting hemochorial placentation experience an accumulation of macrophages in the endometrium during pregnancy. For the present study, it was tested whether macrophages also accumulate in the endometrium of the sheep, which is a species undergoing an epitheliochorial placentation. An additional objective was to determine whether regulation of endometrial macrophage number occurs via systemic or local signals and whether progesterone is one of these signals. The approach was to evaluate presence of macrophages immunohistochemically using antibodies to CD68 and CD14. Tissues examined were from cyclic ewes in the luteal phase of the estrous cycle, unilaterally-pregnant ewes at day 140 of pregnancy in which pregnancy was surgically confined to one uterine horn, ovariectomized ewes, and ovariectomized ewes treated with progesterone for 44 days. Macrophages were localized predominately to the stromal compartment of the stratum compactum region of the endometrium. In non-pregnant ewes, macrophages were not abundant regardless of physiological status. Increased numbers of endometrial macrophages were seen for both the pregnant and non-pregnant uterine horns of unilaterally pregnant ewes. Numbers of macrophages were higher in the endometrium from the pregnant uterine horn than from endometrium from the non-pregnant uterine horn. Results indicate that macrophages accumulate in the endometrium by day 140 of pregnancy in the sheep and that this induction is because of both systemic and local signals. Progesterone appears not to be an important regulator of numbers of endometrial macrophages.