SOCS genes expression during physiological and perturbed implantation in bovine endometrium.

In mammals, suppressor of cytokine signalling (CISH, SOCS1 to SOCS7) factors control signalling pathways involved in the regulation of numerous physiological processes including pregnancy. In order to gain new insights into the biological functions of SOCS in the endometrium, a comprehensive analysis of SOCS gene expression was carried out in bovine caruncular (CAR) and intercaruncular (ICAR) tissues collected i) during the oestrous cycle, ii) at the time of maternal recognition of pregnancy and at implantation in inseminated females, iii) following uterine interferon-tau (IFNT) infusion at day 14 post-oestrus, iv) following a period of controlled intravaginal progesterone release and v) following transfer of embryos by somatic-cell nuclear transfer (SCNT). The regulatory effects of IFNT on in vitro cultured epithelial and stromal cells were also examined. Altogether, our data showed that CISH, SOCS4, SOCS5 and SOCS7 mRNA levels were poorly affected during luteolysis and pregnancy. In contrast, SOCS1, SOCS2, SOCS3 and SOCS6 mRNA levels were strongly up-regulated at implantation (day 20 of pregnancy). Experimental in vitro and in vivo models demonstrated that only CISH, SOCS1, SOCS2 and SOCS3 were IFNT-induced genes. Immunohistochemistry showed an intense SOCS3 and SOCS6 staining in the nucleus of luminal and glandular epithelium and of stromal cells of pregnant endometrium. Finally, SOCS3 expression was significantly increased in SCNT pregnancies in keeping with the altered immune function previously reported in this model of compromised implantation. Collectively, our data suggest that spatio-temporal changes in endometrial SOCS gene expression reflect the acquisition of receptivity, maternal recognition of pregnancy and implantation.

Effects of exposure to environmental chemicals during pregnancy on the development of the male and female reproductive axes.

There is a large body of literature describing effects of environmental chemicals (ECs), many of them anthropogenic with endocrine-disrupting properties, on development in rodent laboratory species, some of which lead to impaired reproduction and adverse health. This literature joins extensive human epidemiological data and opportunistic wildlife findings on health effects of ECs. In contrast, the effect of endocrine disruption on foetal development and reproductive performance in domestic species is less extensively documented. This applies both to domestic farm and to companion species even though the former is critical to food production and the latter share our homes and many aspects of the modern developed human lifestyle. In domestic species, the nature of chemicals exposure in utero and their consequences for animal health and production are poorly understood. A complication in our understanding is that the pace of development, ontogeny and efficiency of foetal and maternal hepatic and placental activity differs between domestic species. In many ways, this reflects the difficulties in understanding human exposure and consequences of that exposure for the foetus and subsequent adult from epidemiological and largely rodent-based data. It is important that domestic species are included in research into endocrine disruption because of their (i) wide variety of exposure to such chemicals, (ii) greater similarity of many developmental processes to the human, (iii) economic importance and (iv) close similarities to developed world human lifestyle in companion species.

Dietary omega-3 and -6 polyunsaturated fatty acids affect the composition and development of sheep granulosa cells, oocytes and embryos.

The evidence that omega-3 (n-3) and -6 (n-6) polyunsaturated fatty acids (PUFAs) have differential effects on ovarian function, oocytes and embryo quality is inconsistent. We report on the effects of n-3 versus n-6 PUFA-enriched diets fed to 36 ewes over a 6-week period, prior to ovarian stimulation and follicular aspiration, on ovarian steroidogenic parameters and embryo quality. Follicle number and size were unaltered by diet, but follicular-fluid progesterone concentrations were greater in n-3 PUFA-fed ewes than in n-6 PUFA-fed ewes. The percentage of saturated FAs (mostly stearic acid) was greater in oocytes than in either granulosa cells or plasma, indicating selective uptake and/or de novo synthesis of saturated FAs at the expense of PUFAs by oocytes. High-density lipoproteins (HDLs) fractionated from sera of these ewes increased granulosa cell proliferation and steroidogenesis relative to the FA-free BSA control during culture, but there was no differential effect of n-3 and n-6 PUFAs on either oestradiol or progesterone production. HDL was ineffective in delivering FAs to embryos during culture, although n-6 PUFA HDL reduced embryo development. All blastocysts, irrespective of the treatment, contained high levels of unsaturated FAs, in particular linoleic acid. Transcripts for HDL and low-density lipoprotein (LDL) receptors (SCARB1 and LDLR) and stearoyl-CoA desaturase (SCD) are reported in sheep embryos. HDL reduced the expression of transcripts for LDLR and SCD relative to the BSA control. The data support a differential effect of n-3 and n-6 PUFAs on ovarian steroidogenesis and pre-implantation development, the latter in the absence of a net uptake of FAs.

Eosinophils in chronically inflamed human upper airway tissues express transforming growth factor beta 1 gene (TGF beta 1).

Transforming growth factor beta (TGF beta) is a multifunctional protein which has been suggested to play a central role in the pathogenesis of chronic inflammation and fibrosis. Nasal polyposis is a condition affecting the upper airways characterized by the presence of chronic inflammation and varying degrees of fibrosis. To examine the potential role of TGF beta in the pathogenesis of this condition, we investigated gene expression and cytokine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGF beta 1 cDNA probe, we detected TGF beta 1-specific mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By the combination of tissue section staining with chromotrope 2R with in situ hybridization using the same TGF beta 1 probe, we found that approximately 50% of the eosinophils infiltrating the polyp tissue express the TGF beta 1 gene. In addition, immunohistochemical localization of TGF beta 1 was detected associated with extracellular matrix as well as in cells in the stroma. These results suggest that in nasal polyposis where eosinophils are the most prevalent inflammatory cell, TGF beta 1 synthesized by these cells may contribute to the structural abnormalities such as stromal fibrosis and basement membrane thickening which characterize this disease.

Disturbed development of the preimplantation embryo in the insulin-dependent diabetic BB/E rat.

Although improved regulation of maternal IDDM during pregnancy has resulted in a major fall in the stillbirth rate, the rates for other problems, such as spontaneous preterm labor, fetuses small for gestational age, congenital malformation, and the incidence of large placentas, remain raised. This has suggested the possibility that the damaging effect of conventionally treated but poorly regulated IDDM may operate primarily at the earliest stages of gestation, even before the diagnosis of pregnancy has been made. This study shows that spontaneous autoimmune IDDM in the Bio Breeding/Edinburgh (BB/E) rat is associated with severe disturbance in the development of the preimplantation embryo in a majority of pregnancies, as indicated by a fivefold increase in the incidence of degenerate fragmented embryos and a 33% reduction in the number of expanded blastocysts and in those blastocysts that reach the expanded stage a 20% cellular deficit in the inner-cell mass without any change in trophectoderm cell number. In addition, we find that blastocysts removed from diabetic rats and cultured in vitro for 24 h show no sign of "catch-up" growth of the inner-cell mass, although under these conditions, the trophectoderm exhibits a 25% cellular accretion. It is tempting to speculate that these phenomena are a presage of the characteristic combination of fetal growth retardation and large placentas, which are a feature of both BB/E rat and human IDDM pregnancy.

Human fetal testis: second trimester proliferative and steroidogenic capacities.

The period of Leydig cell hyperplasia (14-18 weeks gestation) in human fetal testis is crucial for normal gonad development. We have studied the spatio-temporal distribution of key developmental and functional markers in human fetal testis between 13-19 weeks gestation. Proliferating cell nuclear antigen-positive cells were immunolocalized to both interstitium and tubules. Image analysis confirmed an increase in positive interstitial cells during Leydig cell hyperplasia (P: < 0.05). c-Myc was localized to the interstitium with no gestational changes. The steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase (protein) and cytochrome P450 17alpha-hydroxylase/C(17-20)-lyase (P450c17; messenger ribonucleic acid and protein) were confined to the Leydig cells. The number of immunopositive cells increased between 13 and 19 weeks (P: < 0.001). P450c17 mRNA (in situ hybridization) and protein were localized to the same population of interstitial Leydig cells. Androgen receptor and Bcl-2 protein (anti-apoptotic) were gradually restricted to the peritubular myoid cells as gestation progressed. Conversely, Bax protein (pro-apoptotic) was predominantly localized to the tubule Sertoli cells, whereas the germ cells were Bax immunonegative. In conclusion, human fetal Leydig cell hyperplasia is characterized by increasing numbers of proliferating cells and increased expression of steroidogenic enzymes. The Bcl-2-positive, Bax-negative status of the peritubular myoid cells may be a strategy for cell survival.

Role of inflammatory mediators in human endometrium during progesterone withdrawal and early pregnancy.

The role of progesterone (P4) in the regulation of inflammatory mediators interleukin-8 (IL-8), monocyte chemoattractant protein-1, and cyclooxygenase-2 (COX-2) and in the recruitment of leukocyte subpopulations in the endometrium has been examined, by employing a model of P4 withdrawal and maintenance in vivo. Messenger RNA and protein expression have been investigated in endometrial biopsies: 1) during the midsecretory phase (LH+8 to 10); during the maintained luteal phase (P4 administered vaginally for 4 days from LH+8) and biopsies collected 2) 24 h and 3) 48 h post withdrawal of P4; and 4) during pseudo pregnancy (lifespan of corpus luteum extended by 7 days with CG; (decidua collected from women with 5) an ectopic gestation and 6) from women undergoing first-trimester termination of pregnancy). CD56+ large granular lymphocytes remain the major leukocyte subtype, both 24 and 48 h after P4 withdrawal, and in decidua (CG supported or ectopic). Higher numbers (P < 0.05) of macrophages (CD68+) were present in endometrium 48 h post P4 withdrawal and in pseudo pregnant endometrium, compared with normal decidua. Significantly more macrophages (P < 0.01) were present in decidua from an ectopic pregnancy. A significant elevation of IL-8 (P < 0.01) and COX-2 (P < 0.05) messenger RNA was detected 48 h post P4 withdrawal. Evidence is provided for up-regulation of IL-8 and COX-2 in response to P4 withdrawal.

The effect of ceruloplasmin on iron release from placental (BeWo) cells; evidence for an endogenous Cu oxidase.

The mechanism of iron release from the placenta into the fetal circulation is not well understood. Ceruloplasmin, a plasma ferroxidase, has been implicated in iron efflux from a variety of cell types. The hypothesis is that circulating ceruloplasmin facilitates iron efflux by oxidizing the released Fe(II) to Fe(III) for incorporation into transferrin. We tested whether this mechanism mediates iron release from placental cells into the fetal circulation, using the BeWo cell line, a choriocarcinoma which can differentiate into a syncytium.(59)Fe release from undifferentiated or differentiated cells and from cells grown on porous filters was not stimulated by extracellular ceruloplasmin. Instead, we found that BeWo cells express an endogenous ferroxidase. The protein is membrane bound and cross-reacts with an anti-ceruloplasmin antibody, but has a different size; 100 and 140 kDa. Similar immunoreactivity was identified in first- and third-trimester human placentae. In BeWo cells, the protein has a perinuclear localization but does not entirely co-localize with markers for the endoplasmic reticulum or Golgi apparatus. We propose that this oxidase performs the same function as serum ceruloplasmin and is involved in iron release into the fetal circulation.

The effect of horse placental tissue extracts and equine chorionic gonadotrophin on the proliferation of horse lymphocytes stimulated in vitro.

Commercial preparations of equine chorionic gonadotrophin (eCG) and extracts of horse placenta taken at 80 days gestation were incorporated into mixed lymphocyte culture and mitogen stimulation assays of horse peripheral blood mononuclear cells. A dose-related inhibition of lymphocyte proliferation, indicative of immunosuppressive activity, was observed in both systems, both with commercial eCG preparations and tissue extracts. Negligible inhibitory activity was observed with an extract of term placenta. The inhibitory activity of the placental samples was not related to their eCG content as measured by immunoassay. The fact that one pregnant horn allantochorion extract containing 50 IU/ml of eCG showed an identical dose-dependent lymphoproliferation-inhibitory activity to an extract of a second such allantochorion containing only 1.7 IU/ml of eCG suggests that this hormone does not inhibit lymphocyte proliferation and that commercially available eCG contains inhibitory contaminants.

Identification of low molecular weight immunosuppressor molecules in human in vitro fertilization supernatants predictive of implantation as a polyamine--possibly spermine.

Suppressor activity in in vitro fertilization (IVF) culture medium correlates with successful implantation. High performance liquid chromatography fractionation revealed peak(s) of inhibitory activity in the 1,000- to 5,000-Da molecular weight range. Inhibitory activity was dependent on the presence of fetal bovine serum (FBS) and was abrogated both by heat treatment of the serum and by pretreatment with monoamine oxidase inhibitors. Spermine becomes toxic when oxidized by monoamine oxidase to spermine dialdehyde or acrolein. Spermine also shows suppressive activity in the same molecular weight range as IVF supernatants. These data suggest IVF-associated inhibitory activity may be attributable to oxidation of a polyamine, possibly spermine, by monoamine oxidase in FBS. The biological significance is discussed.

The detection of spermine and spermidine in human in vitro fertilization supernatants and their relation to early embryo-associated suppressor activity.

Suppressor activity in human IVF supernatants is dependent on the activity of monoamine oxidase present in nonheat-treated FBS and is therefore likely a polyamine. Pooled suppressive growth phase IVF supernatants were concentrated and subjected to HPLC fractionation. The inhibitory peak in the 1,000 to 2,000 d range was neutralized by antibody reactive with spermine and spermidine, and the presence of both polyamines was confirmed by TLC and by ELISA. These data suggest that IVF supernatant polyamines (spermine and spermidine) are responsible for the early embryo-associated suppressor activity correlating with pregnancy success.

Novel gonadal characteristics in an aged bovine freemartin.

The gonads from a five-year-old freemartin Holstein animal were subjected to morphological analysis and to immunohistochemistry using antibodies against developmental and functional markers. We demonstrate, for the first time, the retention of anti-mullerian hormone (AMH) producing intratubular cells (Sertoli cells) in the context of abundant steroidogenic interstitial cells, and structures consistent with clusters of luteal cells. This novel report describes the clinical, gross and histological findings accompanying this newly described gonadal immunophenotype, and its implication in the understanding of freemartin development.

Effects of environmental pollutants on the reproduction and welfare of ruminants.

Anthropogenic pollutants comprise a wide range of synthetic organic compounds and heavy metals, which are dispersed throughout the environment, usually at low concentrations. Exposure of ruminants, as for all other animals, is unavoidable and while the levels of exposure to most chemicals are usually too low to induce any physiological effects, combinations of pollutants can act additively or synergistically to perturb multiple physiological systems at all ages but particularly in the developing foetus. In sheep, organs affected by pollutant exposure include the ovary, testis, hypothalamus and pituitary gland and bone. Reported effects of exposure include changes in organ weight and gross structure, histology and gene and protein expression but these changes are not reflected in changes in reproductive performance under the conditions tested. These results illustrate the complexity of the effects of endocrine disrupting compounds on the reproductive axis, which make it difficult to extrapolate between, or even within, species. Effects of pollutant exposure on the thyroid gland, immune, cardiovascular and obesogenic systems have not been shown explicitly, in ruminants, but work on other species suggests that these systems can also be perturbed. It is concluded that exposure to a mixture of anthropogenic pollutants has significant effects on a wide variety of physiological systems, including the reproductive system. Although this physiological insult has not yet been shown to lead to a reduction in ruminant gross performance, there are already reports indicating that anthropogenic pollutant exposure can compromise several physiological systems and may pose a significant threat to both reproductive performance and welfare in the longer term. At present, many potential mechanisms of action for individual chemicals have been identified but knowledge of factors affecting the rate of tissue exposure and of the effects of combinations of chemicals on physiological systems is poor. Nevertheless, both are vital for the identification of risks to animal productivity and welfare.

Developmental programming of reproduction and fertility: what is the evidence?

The concept of the foetal/developmental origins of adult disease has been around for ~20 years and from the original epidemiological studies in human populations much more evidence has accumulated from the many studies in animal models. The majority of these have focused upon the role of early dietary intake before conception, through gestation and/or lactation and subsequent interactions with the postnatal environment, e.g. dietary and physical activity exposures. Whilst a number of theoretical models have been proposed to place the experimental data into a biological context, the underlying phenomena remain the same; developmental deficits (of single (micro) nutrients) during critical or sensitive periods of tissue growth alter the developmental pathway to ultimately constrain later functional capacity when the individual is adult. Ageing, without exception, exacerbates any programmed sequelae. Thus, adult phenotypes that have been relatively easy to characterise (e.g. blood pressure, insulin sensitivity, body fat mass) have received most attention in the literature. To date, relatively few studies have considered the effect of differential early environmental exposures on reproductive function and fecundity in predominantly mono-ovular species such as the sheep, cow and human. The available evidence suggests that prenatal insults, undernutrition for example, have little effect on lifetime reproductive capacity despite subtle effects on the hypothalamic-pituitary-gonadal axis and gonadal progenitor cell complement. The postnatal environment is clearly important, however, since neonatal/adolescent growth acceleration (itself not independent from prenatal experience) has been shown to significantly influence fecundity in farm animals. The present paper will expand these interesting areas of investigation and review the available evidence regarding developmental programming of reproduction and fertility. However, it appears there is little strong evidence to indicate that offspring fertility and reproductive senescence in the human and in farm animal species are overtly affected by prenatal nutrient exposure. Nevertheless, it is clear that the developing gonad is sensitive to its immediate environment but more detailed investigation is required to specifically test the long-term consequences of nutritional perturbations during pregnancy on adult reproductive well-being.

The developmental origins of health and disease: current theories and epigenetic mechanisms.

The retrospective cohort studies of David Barker and colleagues during the late 1980s established the principle that the incidence of certain adult diseases such as stroke, type 2 diabetes and dyslipidaemia may be linked to in utero development. Later termed the "Developmental Origins of Health and Disease (DOHaD)" hypothesis, there have been several more recent attempts to explain this phenomenon. Although a general conceptual framework has been established to explain how mechanisms may have evolved to facilitate rapid adaptations to changing ecological conditions, it doesn't identify the actual mechanisms responsible for such effects. Extensive covalent modifications to DNA and related proteins occur from the earliest stages of mammalian development. These determine lineage-specific patterns of gene expression and so represent the most plausible mechanisms by which environmental factors can influence development during the life course. In providing a contemporary overview of chromatin modifications during early mammalian development, this review highlights both the complexity and our current lack of understanding of how epigenetic alterations may contribute to in utero programming. It concludes by providing some thoughts to future research endeavours where the emphasis should be on bettering our understanding of epigenesis and devising more thoughtful experimental approaches that focus on specific environmental factors in appropriate animal and cellular models.

Effects of maternal undernutrition during early pregnancy on apoptosis regulators in the ovine fetal ovary.

This study aimed to determine whether reduced fetal ovary folliculogenesis in ewes undernourished during early/midpregnancy is associated with altered ovarian cell proliferation and/or the expression of apoptosis-regulating genes. Groups of ewes (n = 11-19) were fed either 100% (high; H) or 50% (low; L) of metabolisable energy requirements for live-weight maintenance during selected windows of gestation. All animals were killed at days 50, 65 or 110 of gestation. Between mating and slaughter, control animals were fed the H ration, while animals of other subgroups were fed the L ration from (a) mating to slaughter at 50, 65 or 110 days; (b) 0 to 30 days; (c) 31 to 50 or 65 days; or (d), in the day 110 slaughter group only, from 66 to 110 days. Bouin's-fixed fetal ovaries were examined for (a) Ki67 immunoexpression (proliferation) and (b) Bax and Mcl-1 (apoptosis-regulating genes) expression by in situ hybridisation (day 110) and immunohistochemistry (days 50, 65 and 110). At day 50, maternal nutrition had no effect on Ki67, predominant in germ cells, or Bax and Mcl-1, predominant in the oocytes. Restricted maternal food intake from 0 to 30 days significantly reduced staining for Ki67 in germ cells at day 65 (P < 0.05) but increased staining in granulosa cells at day 110 (P < 0.05). In animals fed the L ration for 110 days, primordial follicle Bax and Mcl-1 were significantly increased (Bax: P < 0.01; Mcl-1: P < 0.05). Granulosa cell Bax was also increased (P < 0.05). When the L ration was fed from 66 to 110 days, granulosa cell Bax (P < 0.05) and primordial follicle Mcl-1 (P < 0.01) were also significantly increased. In the fetal ovarian vasculature, animals underfed for 0-110 days had significantly elevated perivascular Mcl-1 (P < 0.001) and endothelial Bax expression (P < 0.05). Moreover, at day 110, endothelial Mcl-1 was increased by underfeeding from 0 to 30 days (P < 0.05). These data indicate that maternal undernutrition alters proliferation and the expression of apoptosis-regulating genes in the developing fetal ovary. The precise mechanism depends on the window of maternal food restriction.

Macrophages and migratory cells in endometrium relevant to implantation.

The implantation of an appropriately developed embryo into a suitably conditioned uterine lining depends on the synchronous maturation of the preimplantation embryo and uterine lining. The pre- and postimplantation embryo also requires protection from immunocompetent maternal immune effectors. Preimplantation embryo development is affected by genotype, intercellular communication and autocrine growth factors (polyamines, TGF-alpha, TGF-beta 1, PAF). Factors of maternal origin may also enhance embryo development (EGF, TGF-alpha, TGF-beta 1, IGF, polyamines). The preimplantation embryo signals its presence to the mother by release of factor(s) such as IFN-alpha-II and a PAF-like factor. PAF may induce EPF in the mother and enhances vascular permeability at the implantation site. Uterine or peritoneal leukocytosis may inhibit development via toxic effects of lymphokines/monokines (IL-2, IL-1?, IFN-gamma, TNF-alpha). Immunoprotection of the preimplantation embryo is conferred by embryo derived maternal factors (EPF, T-cell suppressor factors). The uterus is receptive during a limited period of time (implantation window) and the substrate adhesion molecules produced by uterine and embryonic trophectoderm cells are crucial for the initial stages of implantation. At implantation, trophoblast expression of MHC and non-MHC antigens is shut off and both immunocompetent maternal cells (macrophages, dendritic cells, granulocytes, IELs, immunocytes) and lymphatics become sparse at implantation sites. Peri-implantation cytokines of maternal origin, such as CSF-1, GM-CSF and IGF-1 binding protein, are probably important for trophoblast growth and development. Immuno-protection of the embryo at this stage may be mediated by embryo derived factors that inactivate macrophages and by a population of large, hormone dependent Lyt 2+ (CD8+) suppressor cells. It is possible that these CD8+ cells respond to progesterone and secrete molecules that inactivate natural effector (NK-type) cells against trophoblast. Prostaglandins (PGE2) may play a brief role in immunosuppression at the time of implantation but its role is probably more important with respect to the decidual response. Defects in the pre- and peri-implantation stages of pregnancy may lead to delayed failure in the form of clinical miscarriage.