Jai Prakash Mehta

Changes in the Endometrial Transcriptome During the Bovine Estrous Cycle: Effect of Low Circulating Progesterone and Consequences for Conceptus Elongation

In cattle, elevated concentrations of circulating progesterone (P4) in the immediate postconception period are associated with advanced conceptus development, while low P4 is implicated as a causative factor in low pregnancy rates observed in dairy cows. This study aimed to: 1) describe the transcriptional changes that occur in the bovine endometrium during the estrous cycle, 2) determine how elevated P4 affects these changes, 3) identify if low P4 alters the expression of these genes, and 4) assess the impact that low P4 has on conceptus development. Relatively few differences occurred in endometrial gene expression during the early luteal phase of the estrous cycle (Day 5 vs. 7), but comparison of endometria from more distant stages of the luteal phase (Day 7 vs. 13) revealed large transcriptional changes, which were significantly altered by exogenous supplementation of P4. Induction of low circulating P4 altered the normal temporal changes in gene expression, and these changes were coordinate with a delay in the down-regulation of the PGR from the LE and GE. Altered endometrial gene expression induced by low P4 was associated with a reduced capacity of the uterus to support conceptus development after embryo transfer on Day 7. In conclusion, the present study provides clear evidence that the temporal changes in the transcriptome of the endometrium of cyclic heifers are sensitive to circulating P4 concentrations in the first few days after estrus. Under low P4 conditions, a suboptimal uterine environment with reduced ability to support conceptus elongation is observed.

Conceptus-Induced Changes in the Endometrial Transcriptome: How Soon Does the Cow Know She Is Pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

Sequential Analysis of Global Gene Expression Profiles in Immature and In vitro Matured Bovine Oocytes: Potential Molecular Markers of Oocyte Maturation

BackgroundWithout intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis.Results8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation.ConclusionUsing sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

Investigation of the molecular profile of basal cell carcinoma using whole genome microarrays

BackgroundSkin cancer accounts for 1/3 of all newly diagnosed cancer. Although seldom fatal, basal cell carcinoma (BCC) is associated with severe disfigurement and morbidity. BCC has a unique interest for researchers, as although it is often locally invasive, it rarely metastasises. This paper, reporting the first whole genome expression microarray analysis of skin cancer, aimed to investigate the molecular profile of BCC in comparison to non-cancerous skin biopsies. RNA from BCC and normal skin specimens was analysed using Affymetrix whole genome microarrays. A Welch t-test was applied to data normalised using dCHIP to identify significant differentially-expressed genes between BCC and normal specimens. Principal component analysis and support vector machine analysis were performed on resulting genelists, Genmapp was used to identify pathways affected, and GOstat aided identification of areas of gene ontology more highly represented on these lists than would be expected by chance.ResultsFollowing normalisation, specimens clustered into groups of BCC specimens and of normal skin specimens. Of the 54,675 gene transcripts/variants analysed, 3,921 were differentially expressed between BCC and normal skin specimens. Of these, 2,108 were significantly up-regulated and 1,813 were statistically significantly down-regulated in BCCs.ConclusionFunctional gene sets differentially expressed include those involved in transcription, proliferation, cell motility, apoptosis and metabolism. As expected, members of the Wnt and hedgehog pathways were found to be significantly different between BCC and normal specimens, as were many previously undescribed changes in gene expression between normal and BCC specimens, including basonuclin2 and mrp9. Quantitative-PCR analysis confirmed our microarray results, identifying novel potential biomarkers for BCC.

RNA Sequencing Reveals Novel Gene Clusters in Bovine Conceptuses Associated with Maternal Recognition of Pregnancy and Implantation

ABSTRACT Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatching embryo and elongating conceptus. This period of development is arguably the most important, as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle. Here, we describe the global transcriptome profile of the bovine conceptus at five key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using state-of-the-art RNA sequencing techniques. More than 287 million reads were generated at the five stages, and more than 22 700 unique transcripts were detected. Analysis of variance followed by self-organizing maps identified differentially regulated (P < 0.05) genes organized in nine gene clusters forming a sequential transcript dynamics across these developmental stages. Of particular interest, genes in clusters 3 (n = 236) and 6 (n = 1409) were significantly up-regulated on Days 16 and 19, suggesting a role in maternal recognition and initiation of implantation. This transcriptome analysis of the bovine conceptus will provide a blueprint of the dynamic changes in gene expression occurring during maternal recognition and implantation and will complement existing knowledge of the temporal changes in the endometrial transcriptome, thus facilitating a better understanding of conceptus-maternal cross talk during the peri-implantation period of pregnancy.

Evidence for an early endometrial response to pregnancy in cattle: both dependent upon and independent of interferon tau

The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endometria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status.

Effect of Elevated Circulating Progesterone Concentration on Bovine Blastocyst Development and Global Transcriptome Following Endoscopic Transfer of In Vitro Produced Embryos to the Bovine Oviduct

Elevated concentrations of circulating progesterone in the immediate postconception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the uterine tissues. Using state-of-the-art endoscopic techniques, this study examined the effect of elevated progesterone on the development of in vitro produced bovine zygotes transferred to the oviducts of heifers with high or normal circulating progesterone concentrations and on the transcriptome of blastocysts developing under such conditions. Simmental heifers (n = 34) were synchronized using a controlled internal drug release (CIDR) device for 8 days, with a prostaglandin F2alpha analogue administered 3 days before removal of the CIDR device. Only animals exhibiting a clear standing estrus (Day 0) were used. To produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the estrous cycle; the PRID was left in place until embryo recovery. All animals were sampled for blood daily from Day 0 to Day 7. Cleaved embryos were transferred by endoscopy to the ipsilateral oviduct of each recipient on Day 2 and then recovered by nonsurgically flushing the oviduct and the uterus on Day 7. The number of embryos developing to the blastocyst stage was recorded at recovery and following overnight culture in vitro. Potential effects of elevated progesterone on transcript abundance were examined using the Affymetrix GeneChip Bovine Genome Array. Insertion of a PRID on Day 3 resulted in a significant elevation of progesterone concentration (P < 0.05) from Day 3.5 until Day 6. Elevated progesterone did not affect the proportion of embryos developing to the blastocyst stage. Genomewide gene expression analysis identified 194 differentially expressed genes between embryos collected from heifers with normal or elevated progesterone, and quantitative real-time PCR validation with a subset of selected genes and an independent sample confirmed the microarray results. Interaction network analysis indicated a significant interaction between progesterone-regulated genes in the blastocyst and in the maternal endometrium. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo but do result in subtle changes to the transcriptome of the embryo that may be associated with advanced elongation posthatching.

Effects of Low Progesterone on the Endometrial Transcriptome in Cattle

ABSTRACT The objective of the present study was to determine how low progesterone (P4) affects the endometrial transcriptome, with specific emphasis on those changes that may impact conceptus elongation. Following estrous synchronization and detection (estrus = Day 0, n = 40), heifers were randomly assigned to a control group (n = 12) or a low P4 group (n = 28). Heifers in the low P4 group had consistently lower P4 concentrations compared to controls (P < 0.05). Microarray analysis of endometrial gene expression revealed low P4 altered the expression of 498 differentially expressed genes (DEGs; 215 up- and 283 down-regulated) on Day 7 and 351 DEGs (272 up- and 79 down-regulated) on Day 13. A similar number of temporal changes occurred between Day 7 and Day 13 in both groups (2212 in heifers with normal P4 compared with 2247 in heifers with low P4); of these DEGs, 1278 were common to both groups. Little overlap in the number of DEGs affected by high or low P4 was observed across days. Comparison of the temporal changes that occur during normal estrous cycle progression (i.e., from Day 7 to Day 13) to those affected by altered P4 found significant numbers of genes were modulated by elevated (4157) and decreased (809) P4 alone. Analysis of selected genes by quantitative real-time PCR and in situ hybridization revealed that expression of MEP1B, NID2, and PRSS23 increased on Day 13 compared to Day 7 (P < 0.05) and that the magnitude of increase was significantly diminished in heifers with low P4 compared to controls. MEP1B predominantly localized to the both the superficial and deep glandular epithelium (GE), NID2 localized to the deep GE, whereas PRSS23 localized only to the luminal epithelium. In conclusion, we have determined the global changes in the endometrial transcriptome induced by decreasing the output of P4 from the corpus luteum in vivo using a unique animal model. Placing these data into context with previous data in which P4 was supplemented or elevated after ovulation, we have identified a panel of genes that are truly regulated in the endometrium by circulating concentrations of P4 in vivo and that likely impact conceptus elongation.

Transcriptome Changes at the Initiation of Elongation in the Bovine Conceptus

The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of this study was to examine the temporal changes in transcriptional profile as the embryo develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo- and in vitro-derived blastocysts that may be associated with embryonic survival/mortality using the bovine Affymetrix microarray. All embryos were produced either in vitro by in vitro fertilization or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen, and the remainder were transferred (n = 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n = 25 per pool) and Day 13 conceptuses (n = 5 per pool) were used for microarray analysis. In Day 7 blastocysts, 50 genes were found to be differentially expressed (P < 0.05), of which 19 were up-regulated and 31 down-regulated in the in vivo compared to in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (P < 0.05), of which 133 were up-regulated and 155 down-regulated in the in vivo compared to in vitro embryos. The comparison between Day 7 and Day 13 embryos revealed significant temporal changes in transcript profile with 1806 and 909 transcripts differentially expressed in the in vitro- and in vivo-derived embryos, respectively. Across the three array comparisons between Day 7 and Day 13 embryos, 444 genes were consistently exclusively present in the in vivo embryos, whereas 1341 were exclusively present in the in vitro embryos. Regardless of the origin of the embryo, 465 differentially expressed genes between Day 7 and 13 were common to both in vivo- and in vitro-derived embryos; these genes are likely critical for the transition between the blastocyst (Day 7) and ovoid conceptus (Day 13) stages of embryo development. In order to validate the microarray findings, differences in the expression of six genes (CYP51A1, FADS1, TDGF1, HABP2, APOA2, and SLC12A2) were confirmed by quantitative real-time PCR on in vivo- and in vitro-derived embryos on Day 7 and Day 13 using independent samples from those used for the microarray. Subsequent mapping of these differentially expressed genes into relevant functional groups and pathways identified important pathways involved in conceptus elongation in cattle. In conclusion, this analysis has identified genes and pathways crucial for the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).

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 β-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.