Bovine scavenger receptor class A (SR-A) exhibit specific patterns of regulation in the endometrium during the oestrous cycle and early pregnancy.

In mammals, tight regulation of maternal endometrial function is critical for pregnancy success. In bovine species, endometrial expression of members of the scavenger receptor class A (SR-A) has been listed in high-throughput analyses, but very little is known about the involvement of these immune factors during implantation in mammals. To provide first insights into the contribution of SR-A to endometrial physiology, we analysed the expression and regulation of all members of SR-A (SR-A1, SR-A3-SR-A6) during the oestrous cycle and early pregnancy in cattle. Levels of SR-A1 were increased on Day 20 of pregnancy, whereas SR-A3 levels were increased on Day 13 of the oestrous cycle and of the pregnancy. Although SR-A4 levels were reduced on Day 20 of the oestrous cycle, they remained high in pregnant animals. SR-A5 levels increased by Day 13 of the oestrous cycle and decreased on Day 20, but remained high in pregnant animals. Interferon-τ does not affect SR-A gene expression, whereas progesterone regulates the expression of the SR-A3 and SR-A5 transcripts. Endometrial SR-A3 appeared significantly higher in cows carrying invitro-produced embryos than in AI cows. Our data suggest that members of the SR-A family are involved in endometrial remodelling and regulation of endometrial gland physiology, both processes being critical for implantation in mammals.

The effect of exogenous glucose infusion on early embryonic development in lactating dairy cows.

The objective of this study was to examine the effect of intravenous infusion of glucose on early embryonic development in lactating dairy cows. Nonpregnant, lactating dairy cows (n = 12) were enrolled in the study (276 ± 17 d in milk). On d 7 after a synchronized estrus, cows were randomly assigned to receive an intravenous infusion of either 750 g/d of exogenous glucose (GLUC; 78 mL/h of 40% glucose wt/vol) or saline (CTRL; 78 mL/h of 0.9% saline solution). The infusion period lasted 7 d and cows were confined to metabolism stalls for the duration of the study. Coincident with the commencement of the infusion on d 7 after estrus, 15 in vitro-produced grade 1 blastocysts were transferred into the uterine horn ipsilateral to the corpus luteum. All animals were slaughtered on d 14 to recover conceptuses, uterine fluid, and endometrial tissue. Glucose infusion increased circulating glucose concentrations (4.70 ± 0.12 vs. 4.15 ± 0.12 mmol/L) but did not affect milk production or dry matter intake. Circulating ß-hydroxybutyrate concentrations were decreased (0.51 ± 0.01 vs. 0.70 ± 0.01 mmol/L for GLUC vs. CTRL, respectively) but plasma fatty acids, progesterone, and insulin concentrations were unaffected by treatment. Treatment did not affect either uterine lumen fluid glucose concentration or the mRNA abundance of specific glucose transporters in the endometrium. Mean conceptus length, width, and area on d 14 were reduced in the GLUC treatment compared with the CTRL treatment. A greater proportion of embryos in the CTRL group had elongated to all length cut-off measurements between 11 and 20 mm (measured in 1-mm increments) compared with the GLUC treatment. In conclusion, infusion of glucose into lactating dairy cows from d 7 to d 14 post-estrus during the critical period of conceptus elongation had an adverse impact on early embryonic development.

Interferon-tau and fertility in ruminants.

Establishment of pregnancy in domestic ruminants includes pregnancy recognition signalling by the conceptus, implantation and placentation. Despite the high fertilisation success rate in ruminants, a significant amount of embryo loss occurs, primarily during early gestation. Interferon-tau (IFNT), a type I interferon that is exclusively secreted by the cells of the trophectoderm of the ruminant conceptus, has been recognised as the primary agent for maternal recognition of pregnancy in ruminants. It produces its antiluteolytic effect on the corpus luteum by inhibiting the expression of oxytocin receptors in the uterine epithelial cells, which prevents pulsatile, luteolytic secretion of prostaglandin F2α by the uterine endometrium. While the importance of IFNT in maternal recognition of pregnancy and prevention of luteolysis in ruminants is unequivocal, important questions, for example, relating to the threshold level of IFNT required for pregnancy maintenance, remain unanswered. This paper reviews data linking IFNT with measures of fertility in ruminants.

Anatomical integration and rich-club connectivity in euthymic bipolar disorder.

BACKGROUND: Although repeatedly associated with white matter microstructural alterations, bipolar disorder (BD) has been relatively unexplored using complex network analysis. This method combines structural and diffusion magnetic resonance imaging (MRI) to model the brain as a network and evaluate its topological properties. A group of highly interconnected high-density structures, termed the 'rich-club', represents an important network for integration of brain functioning. This study aimed to assess structural and rich-club connectivity properties in BD through graph theory analyses. METHOD: We obtained structural and diffusion MRI scans from 42 euthymic patients with BD type I and 43 age- and gender-matched healthy volunteers. Weighted fractional anisotropy connections mapped between cortical and subcortical structures defined the neuroanatomical networks. Next, we examined between-group differences in features of graph properties and sub-networks. RESULTS: Patients exhibited significantly reduced clustering coefficient and global efficiency, compared with controls globally and regionally in frontal and occipital regions. Additionally, patients displayed weaker sub-network connectivity in distributed regions. Rich-club analysis revealed subtly reduced density in patients, which did not withstand multiple comparison correction. However, hub identification in most participants indicated differentially affected rich-club membership in the BD group, with two hubs absent when compared with controls, namely the superior frontal gyrus and thalamus. CONCLUSIONS: This graph theory analysis presents a thorough investigation of topological features of connectivity in euthymic BD. Abnormalities of global and local measures and network components provide further neuroanatomically specific evidence for distributed dysconnectivity as a trait feature of BD.

Imprinted and DNA methyltransferase gene expression in the endometrium during the pre- and peri-implantation period in cattle.

The endometrium plays a key role in providing an optimal environment for attachment of the preimplantation embryo during the early stages of pregnancy. Investigations over the past 2 decades have demonstrated that vital epigenetic processes occur in the embryo during the preimplantation stages of development. However, few studies have investigated the potential role of imprinted genes and their associated modulators, the DNA methyltransferases (DNMTs), in the bovine endometrium during the pre- and peri-implantation period. Therefore, in the present study we examined the expression profiles of the DNMT genes (3A, 3A2 and 3B) and a panel of the most comprehensively studied imprinted genes in the endometrium of cyclic and pregnant animals. Intercaruncular (Days 5, 7, 13, 16 and 20) and caruncular (Days 16 and 20) regions were analysed for gene expression changes, with protein analysis also performed for DNMT3A, DNMT3A2 and DNMT3B on Days 16 and 20. An overall effect of day was observed for expression of several of the imprinted genes. Tissue-dependent gene expression was detected for all genes at Day 20. Differences in DNMT protein abundance were mostly observed in the intercaruncular regions of pregnant heifers at Day 16 when DNMT3A, DNMT3A2 and DNMT3B were all lower when compared with cyclic controls. At Day 20, DNMT3A2 expression was lower in the pregnant caruncular samples compared with cyclic animals. This study provides evidence that epigenetic mechanisms in the endometrium may be involved with implantation of the embryo during the early stages of pregnancy in cattle.

Insights into conceptus elongation and establishment of pregnancy in ruminants.

This review integrates established and new information on the factors and pathways regulating conceptus-endometrial interactions, conceptus elongation and establishment of pregnancy in sheep and cattle. Establishment of pregnancy in domestic ruminants begins at the conceptus stage (embryo or fetus and associated extra-embryonic membranes) and includes pregnancy recognition signalling, implantation and the onset of placentation. Survival and growth of the preimplantation blastocyst and elongating conceptus require embryotrophic factors (amino acids, carbohydrates, proteins, lipids and other substances) provided by the uterus. The coordinated and interactive actions of ovarian progesterone and conceptus-derived factors (interferon-τ and prostaglandins) regulate expression of elongation- and implantation-related genes in the endometrial epithelia that alter the uterine luminal milieu and affect trophectoderm proliferation, migration, attachment, differentiation and function. A comparison of sheep and cattle finds both conserved and non-conserved embryotrophic factors in the uterus; however, the overall biological pathways governing conceptus elongation and establishment of pregnancy are likely conserved. Given that most pregnancy losses in ruminants occur during the first month of pregnancy, increased knowledge is necessary to understand why and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency.

Effect of combined exogenous progesterone with luteotrophic support via equine chorionic gonadotrophin (eCG) on corpus luteum development, circulating progesterone concentrations and embryo development in cattle.

The aim was to examine the effect of a single intramuscular (i.m.) injection of equine chorionic gonadotrophin (eCG) on Day 3 after oestrus on corpus luteum (CL) development, circulating progesterone and conceptus development in cross-bred beef heifers. In Experiment 1, heifers received: (1) saline, or a single i.m. injection of eCG on Day 3 at (2) 250IU (3) 500IU (4) 750IU or (5) 1000IU. Administration of eCG resulted in increased luteal tissue area and progesterone and oestradiol concentrations compared with controls. In Experiment 2, heifers received (1) a progesterone-releasing intravaginal device (PRID Delta) from Day 3 to 5 or (2) a PRID Delta from Day 3 to 5 plus a single injection of 750IU eCG on Day 3. In vitro-produced blastocysts (n=10 per recipient) were transferred on Day 7 and heifers were slaughtered on Day 14 to assess conceptus development. Administration of eCG reduced the number of short cycles (6.3% vs 31.3%) and increased mean luteal tissue weight (P=0.02). Insertion of a PRID Delta on Day 3 resulted in an elevation (P<0.05) in serum progesterone until removal on Day 5. Administration of eCG at the time of PRID Delta insertion resulted in higher progesterone levels (P<0.05) from Day 10 onwards. Conceptus dimensions were not affected. In conclusion, a single injection of eCG on Day 3 increased CL size and progesterone concentrations and, when given in conjunction with a progesterone-releasing device, appeared to reduce the number of short cycles, presumably due to its luteotrophic nature. The implications of the elevated oestradiol concentrations for embryo quality require further study.

Analysis of STAT1 expression and biological activity reveals interferon-tau-dependent STAT1-regulated SOCS genes in the bovine endometrium.

Signal transducer and activator of transcription (STAT) proteins are critical for the regulation of numerous biological processes. In cattle, microarray analyses identified STAT1 as a differentially expressed gene in the endometrium during the peri-implantation period. To gain new insights about STAT1 during the oestrous cycle and early pregnancy, we investigated STAT1 transcript and protein expression, as well as its biological activity in bovine tissue and cells of endometrial origin. Pregnancy increased STAT1 expression on Day 16, and protein and phosphorylation levels on Day 20. In cyclic and pregnant females, STAT1 was located in endometrial cells but not in the luminal epithelium at Day 20 of pregnancy. The expression of STAT1 during the oestrous cycle was not affected by progesterone supplementation. In vivo and in vitro, interferon-tau (IFNT) stimulated STAT1 mRNA expression, protein tyrosine phosphorylation and nuclear translocation. Using chromatin immunoprecipitation in IFNT-stimulated endometrial cells, we demonstrated an increase of STAT1 binding on interferon regulatory factor 1 (IRF1), cytokine-inducible SH2-containing protein (CISH), suppressor of cytokine signaling 1 and 3 (SOCS1, SOCS3) gene promoters consistent with the induction of their transcripts. Our data provide novel molecular insights into the biological functions of STAT1 in the various cells composing the endometrium during maternal pregnancy recognition and implantation.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

[New impulses for tic research through international collaboration]. / Internationale samenwerking geeft nieuwe impulsen aan onderzoek naar stoornis van Gilles de la Tourette.

BACKGROUND: A section of the UMCG Child and Adolescent Psychiatry Department is currently focusing much of its research on tic disorders. AIM: To provide an overview of the research projects on tic disorders being currently undertaken at our center. METHOD: We discuss our research projects giving particular attention to factors that are restricting current research. RESULTS: The first project is TIC Genetics, a project involving international collaboration. The researchers are looking for rare genetic variants in several family members who have tics and for new gene mutations in children who have tics but no family history of tics. TIC Genetics also investigates the interactions between genes and the environment. A second large-scale longitudinal project, the European Multicentre Tics in Children Study (EMTICS), is focusing on the interplay between genetics, auto-immunity, and environmental factors as a possible cause for the onset and exacerbation of tics. Finally, TS-EUROTRAIN is a European collaboration that concentrates on genetics, neuro-imaging and animal models. CONCLUSION: International collaborations are essential if we are to acquire a deeper understanding of the etiology of tic disorders.

Temporal regulation of fibroblast growth factors and their receptors in the endometrium and conceptus during the pre-implantation period of pregnancy in cattle.

We hypothesised that the expression pattern of members of the fibroblast growth factor (FGF) family would be altered in the endometrium as the oestrous cycle/early pregnancy progressed associated with changes in the expression pattern of their receptors in the developing embryo/conceptus. Expression of FGF1 and FGF10 transcript variants 1 and 2 increased significantly as the oestrous cycle/early pregnancy progressed. Neither progesterone (P4) supplementation nor pregnancy status significantly affected the expression of any of the FGF ligands studied. However, there was a significant interaction between day, pregnancy and P4 status on FGF2 expression (P<0.05) and a significant interaction between P4 status and day on FGF10_tv2 expression. FGF10 protein was localised in the luminal and glandular epithelium as well as the stroma but was not detected in the myometrium. By RNA sequencing, the expression of FGF ligands in the developing embryo/conceptus was found to be minimal. The expression of FGF receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, FGFRL1 and FRS3 was significantly affected by the stage of conceptus development. Interestingly, the expression of FGFR1 and FGFR4 was higher during early embryo development (days 7-13, P<0.05) but decreased on day 16 (P<0.05) while FGFR2 (P<0.001) expression was similar from day 7 through to day 13, with a significant increase by day 16 (P<0.05) that was maintained until day 19 (P>0.05). In conclusion, these data demonstrate that FGF ligands are primarily expressed by the endometrium and their modulation throughout the luteal phase of the oestrous cycle/early pregnancy are associated with alterations in the expression of their receptors in the embryo/conceptus.

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.

White matter microstructural abnormalities in families multiply affected with bipolar I disorder: a diffusion tensor tractography study.

BACKGROUND: White matter (WM) abnormalities are proposed as potential endophenotypic markers of bipolar disorder (BD). In a diffusion tensor imaging (DTI) voxel-based analysis (VBA) study of families multiply affected with BD, we previously reported that widespread abnormalities of fractional anisotropy (FA) are associated with both BD and genetic liability for illness. In the present study, we further investigated the endophenotypic potential of WM abnormalities by applying DTI tractography to specifically investigate tracts implicated in the pathophysiology of BD. METHOD: Diffusion magnetic resonance imaging (MRI) data were acquired from 19 patients with BD type I from multiply affected families, 21 of their unaffected first-degree relatives and 18 healthy volunteers. DTI tractography was used to identify the cingulum, uncinate fasciculus (UF), arcuate portion of the superior longitudinal fasciculus (SLF), inferior longitudinal fasciculus (ILF), corpus callosum, and the anterior limb of the internal capsule (ALIC). Regression analyses were conducted to investigate the effect of participant group and genetic liability on FA and radial diffusivity (RD) in each tract. RESULTS: We detected a significant effect of group on both FA and RD in the cingulum, SLF, callosal splenium and ILF driven by reduced FA and increased RD in patients compared to controls and relatives. Increasing genetic liability was associated with decreased FA and increased RD in the UF, and decreased FA in the SLF, among patients. CONCLUSIONS: WM microstructural abnormalities in limbic, temporal and callosal pathways represent microstructural abnormalities associated with BD whereas alterations in the SLF and UF may represent potential markers of endophenotypic risk.

Paradoxical effect of supplementary progesterone between Day 3 and Day 7 on corpus luteum function and conceptus development in cattle.

The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRID® Delta; 1.55 g P4) treatment with administration of a prostaglandin F(2α) analogue (Enzaprost; CEVA Sante Animale) the day before PRID® Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRID® Delta from Day 3 to Day 7; (4) insertion of a PRID® Delta from Day 3 to Day 5; or (5) insertion of a PRID® Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2-5 on Day 7 (n=10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-τ (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P<0.05) in serum P4 that declined following removal. In Experiment 1, P4 supplementation from Day 3 to Day 5 (17.0±1.4 mm) or Day 3 to Day 7 (11.3±2.3 mm) increased conceptus length compared with placebo (2.1±1.8 mm). Serum P4 was significantly lower from Day 9 to Day 14 (P<0.05) and the weight of the Day 14 corpus luteum (CL) was lower in the PRID Day 3-7 group than the placebo or control groups. In Experiment 2, supplementation from Day 3 to Day 5 (94.0±18.8 mm) or Day 3 to Day 7 (143.6±20.6 mm) increased conceptus length on Day 16 compared with placebo (50.3±17.4 mm). Serum P4 was significantly lower in the two supplemented groups following PRID removal compared with placebo (P<0.05) and was associated with a lower CL weight in the Day 3-7 group. Conceptus length was strongly correlated with the IFNT concentration in the uterine flush (r=0.58; P=0.011) and spent culture medium (r=0.68; P<0.002). The findings of the present study highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrous period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.

Altered endometrial immune gene expression in beef heifers with retarded embryos.

The aim of the present study was to compare endometrial gene expression profiles in a group of beef heifers yielding viable or retarded embryos on Day 7 after oestrus as a means of potentially explaining differences in embryo survival rates. Heifers were classified as either: (1) viable, when the embryo collected on Day 7 after oestrus was at the correct developmental stage (i.e. morula/early blastocyst); or (2) retarded, when the embryo was arrested at the 2-16-cell stage. The focus of the present study was on genes that were associated with either the pro- or anti-inflammatory immune response. Endometrial gene expression was determined using quantitative real-time polymerase chain reaction analysis. Expression of the ß-defensin (DEFB1), interferon (IFN)-α (IFNA), IFN-γ (IFNG), interleukin (IL)-6 (IL6), IL-10 (IL10), forkhead box P3 (FOXP3) and natural cytotoxicity triggering receptor 1 (NCR1) genes was lower in endometria from viable than retarded heifers. Expression of the nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (NKFB1), transforming growth factor (TGF)-ß (TGFB), IFN-γ-inducible protein 16 (IFI16) and IL-21 (IL21) genes was higher in viable than retarded heifers. We propose that small disturbances in the expression of immune genes in the endometrium on Day 7 after oestrus can have detrimental effects on embryo survival.

Review: Endometrial function in pregnancy establishment in cattle.

The endometrium is fundamentally required for successful pregnancy in ruminants and species where the posthatching conceptus undergoes a protracted elongation and peri-implantation phase of pregnancy. Moreover, there are substantial waves of pregnancy loss during this pre- and peri-implantation period of pregnancy the precise source of which has not been clearly defined i.e., the maternal uterine contribution to this loss. Understanding the molecular interactions required for successful pregnancy in cattle will allow us to intervene to support pregnancy success during this vulnerable window. The endometrium contributes to most key developmental milestones of pregnancy establishment, including (1) contributing to the regulation of the oestrus cycle, (2) nourishing the preimplantation conceptus, (3) responding to the conceptus to create a more receptive microenvironment, (4) providing essential biophysical support, and (5) signalling and producing factors which affect the mother systemically. This review will summarise what we currently know about conceptus-maternal interactions as well as identify the gaps in our knowledge that could be filled with newer in vitro model approaches. These include the use of microfluidics, organ-on-a-chip devices, and bioinformatic approaches. This will help maximise food production efficiency (both meat and dairy) and decrease the environmental burden, while enhancing our understanding of the fundamental processes required for successful implantation in cattle.

Temporal changes in endometrial gene expression and protein localization of members of the IGF family in cattle: effects of progesterone and pregnancy.

In the study presented, the hypothesis that ligands of the insulin-like growth factor (IGF) family, as well as their binding proteins (BPs), are temporally regulated and are altered by elevated progesterone (P4) and/or pregnancy was tested. Heifers detected in standing estrus following synchronization (n = 210, day 0), were artificially inseminated (n = 140) or left as noninseminated cyclic controls (n = 70). On day 3, half of each group were randomly assigned to receive a P4-releasing intravaginal device resulting in four treatment groups, pregnant and cyclic heifers with high and normal P4 concentrations on either day 5, 7, 13, or 16 of the estrous cycle/early pregnancy. Quantitative real-time PCR and immunohistochemistry were performed on endometrial homogenate and uterine cross sections to measure transcript abundance and protein localization respectively (n = 5 per treatment per time point). No effect of day, treatment, or their interactions was observed for IGF2, IGFBP4, and -5 (P > 0.05). IGF1 and IGFBP6 expression decreased, while IGF1R and IGFBP2 expression increased (P < 0.05) as the days of the cycle or pregnancy progressed. The expression of IGFBP2 was increased by elevated P4; in addition, localization of both IGF2 and IGFBP2 protein in the luminal and superficial glandular epithelium displayed a P4 × day interaction. In conclusion it is proposed that decreased IGF1 and IGFBP6 expression with a coordinate increase in IGF1R and IGFBP2 as the estrous cycle/early pregnancy progresses, along with other factors, are required to establish a uterine environment that promotes the growth and development of the conceptus prior to implantation. In addition, the increased protein abundance of both IGF2 and IGFBP2 observed in heifers supplemented with P4 contributes to the enhanced conceptus elongation observed in this model.

Endometrial response of beef heifers on day 7 following insemination to supraphysiological concentrations of progesterone associated with superovulation.

Ovarian stimulation is a routine procedure in assisted reproduction to stimulate the growth of multiple follicles in naturally single-ovulating species including cattle and humans. The aim of this study was to analyze the changes induced in the endometrial transcriptome associated with superovulation in cattle and place these observations in the context of our previous data on changes in the endometrial transcriptome associated with elevated progesterone (P4) concentrations within the physiological range and those changes induced in the embryo due to superovulation. Mean serum P4 concentrations were significantly higher from day 4 to day 7 in superovulated compared with unstimulated control heifers (P < 0.05). Between-group analysis revealed a clear separation in the overall transcriptional profile of endometria from unstimulated control heifers (n = 5) compared with superovulated heifers (n = 5). This was reflected in the number of differentially expressed genes (DEGs) identified between the two groups with 795 up- and 440 downregulated in superovulated endometria. Ten times more genes were altered by superovulation (n = 1,234) compared with the number altered due to elevated P4 within physiological ranges by insertion of a P4-releasing intravaginal device (n = 124) with only 22 DEGs common to both models of P4 manipulation. Fewer genes were affected by superovulation in the embryo compared with the endometrium, (443 vs. 1,234 DEGs, respectively), and the manner in which genes were altered was different with 64.5% of genes up- and 35.5% of genes downregulated in the endometrium, compared with the 98.9% of DEGs upregulated in the embryo. In conclusion, superovulation induces significant changes in the transcriptome of the endometrium which are distinct from those in the embryo.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of ß-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.