Steroidal but not embryonic regulation of mucin 1 expression in bovine endometrium.

In cow herd management, inadequate embryo implantation leads to pregnancy loss and causes severe economic losses. Thus, it is crucial to understand the molecular mechanisms underlying endometrial receptivity and subsequent embryo implantation. Transmembrane glycocalyx mucin 1 (MUC1) has a large and highly glycosylated extracellular domain known to inhibit embryo implantation via steric hindrance. The role of MUC1 in the bovine endometrium remains to be explored. Herein, we used simple but reliable in vivo and in vitro experiments to investigate the expression and regulation of MUC1 in the bovine endometrium. MUC1 gene expression was analyzed in endometrial epithelial cells collected by the cytobrush technique using reverse transcription-quantitative polymerase chain reaction. MUC1 protein expression was evaluated by immunohistochemical analysis of endometrial samples collected from slaughtered cows. We used an in vitro cell culture model to study the regulation of MUC1 expression by treating cells with sex steroidal hormones or co-culturing cells with a blastocyst. The results revealed that MUC1 was expressed and localized to the apical surface of luminal epithelial cells in the bovine endometrium. MUC1 expression disappeared during the luteal phase of the estrous cycle and during pregnancy. 17ß-estradiol induced MUC1 expression, whereas progesterone inhibited its increase and co-culturing with blastocysts did not affect the expression. A long postpartum interval is a known risk factor for reduced fertility, and MUC1 expression was higher in this compromised condition. Our results demonstrated the MUC1 regulation by steroid hormones in bovine endometrium for embryo implantation, and we observed a negative correlation between MUC1 expression and fertility.

Pregnancy-associated changes of peroxisome proliferator-activated receptor delta (PPARD) and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) expression in the bovine corpus luteum.

We investigated gene expression profiles of the corpus luteum (CL) at the time of maternal recognition to evaluate the functional changes of the CL during early pregnancy in cows and help improve reproductive efficiency and avoid defective fetuses. Microarray analyses using a 15 K bovine oligo DNA microarray detected 30 differentially expressed genes and 266 differentially expressed genes (e.g., PPARD and CYP21A2) in the CL on pregnancy days 15 (P15) and 18 (P18), respectively, compared with the CL on day 15 (NP15) of non-pregnancy (n = 4 for each group). PPARD expression was the highest while the CYP21A2 expression was the lowest in P15 and P18 compared with that of NP15. These microarray results were validated by quantitative real-time PCR analysis. The addition of interferon-τ and supernatants derived from homogenized fetal trophoblast increased ISG15 and MX1 expressions in the cultured luteal tissue (P < 0.01), but did not affect PPARD and CYP21A2 expressions. PPARD expression in the luteal tissue was stimulated (P < 0.05) by GW0742, known as a selective PPARD agonist, and PPARD ligands (i.e., arachidonic, linoleic and linolenic acids). In contrast, CYP21A2 mRNA expression was not affected by both agonist and ligands. The concentration of prostaglandin (PG) E2 and PGF2α decreased after GW0742 stimulation and increased after arachidonic acid stimulation (P < 0.05). The addition of GW0742 and arachidonic acid increased progesterone (P4) concentration. Collectively, these findings suggest that high expression levels of PPARD and low expression levels of CYP21A2 in the CL during early pregnancy may support P4 production by bovine luteal cells.

Characterisation of bovine embryos following prolonged culture in embryonic stem cell medium containing leukaemia inhibitory factor.

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos invitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in invivo embryos, hypoblast cells formed just inside the trophoblast cells of the invitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in invitro-cultured embryos, levels of both genes were significantly lower in the invitro than invivo embryos. Similar to invivo embryos, binucleate cell clusters seen in Day 23invitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells invivo.

Differential gene expression profiling of endometrium during the mid-luteal phase of the estrous cycle between a repeat breeder (RB) and non-RB cows.

BACKGROUND: Repeat breeding directly affects reproductive efficiency in cattle due to an increase in services per conception and calving interval. This study aimed to investigate whether changes in endometrial gene expression profile are involved in repeat breeding in cows. Differential gene expression profiles of the endometrium were investigated during the mid-luteal phase of the estrous cycle between repeat breeder (RB) and non-RB cows using microarray analysis. METHODS: The caruncular (CAR) and intercaruncular (ICAR) endometrium of both ipsilateral and contralateral uterine horns to the corpus luteum were collected from RB (inseminated at least three times but not pregnant) and non-RB cows on Day 15 of the estrous cycle (4 cows/group). Global gene expression profiles of these endometrial samples were analyzed with a 15 K custom-made oligo-microarray for cattle. Immunohistochemistry was performed to investigate the cellular localization of proteins of three identified transcripts in the endometrium. RESULTS: Microarray analysis revealed that 405 and 397 genes were differentially expressed in the CAR and ICAR of the ipsilateral uterine horn of RB, respectively when compared with non-RB cows. In the contralateral uterine horn, 443 and 257 differentially expressed genes were identified in the CAR and ICAR of RB, respectively when compared with non-RB cows. Gene ontology analysis revealed that genes involved in development and morphogenesis were mainly up-regulated in the CAR of RB cows. In the ICAR of both the ipsilateral and contralateral uterine horns, genes related to the metabolic process were predominantly enriched in the RB cows when compared with non-RB cows. In the analysis of the whole uterus (combining the data above four endometrial compartments), RB cows showed up-regulation of 37 genes including PRSS2, GSTA3 and PIPOX and down-regulation of 39 genes including CHGA, KRT35 and THBS4 when compared with non-RB cows. Immunohistochemistry revealed that CHGA, GSTA3 and PRSS2 proteins were localized in luminal and glandular epithelial cells and stroma of the endometrium. CONCLUSION: The present study showed that endometrial gene expression profiles are different between RB and non-RB cows. The identified candidate endometrial genes and functions in each endometrial compartment may contribute to bovine reproductive performance.

Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy.

The aim of the present study was to determine the possible roles of chemokines in regulating bovine endometrial function during early pregnancy. The expression of six chemokines, including CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10, was higher in the endometrium at 15 and 18 days of pregnancy than at the same days in non-pregnant animals. Immunohistochemical staining showed that chemokine receptors (CCR1, CCR2, CCR3, and CXCR3) were expressed in the epithelial cells and glandular epithelial cells of the bovine endometrium as well as in the fetal trophoblast obtained from a cow on day 18 of pregnancy. The addition of interferon-τ (IFNT) to an endometrial tissue culture system increased CCL8 and CXCL10 expression in the tissues, but did not affect CCL2, CCL11, and CCL16 expression. CCL14 expression by these tissues was inhibited by IFNT. CCL16, but not other chemokines, clearly stimulated interferon-stimulated gene 15 (ISG15) and myxovirus-resistance gene 1 (MX1) expression in these tissues. Cyclooxygenase 2 (COX2) expression decreased after stimulation with CCL8 and CCL14, and oxytocin receptor (OTR) expression was decreased by CCL2, CCL8, CCL14, and CXCL10. Collectively, the expression of chemokine genes is increased in the endometrium during early pregnancy. These genes may contribute to the regulation of endometrial function by inhibiting COX2 and OTR expression, subsequently decreasing prostaglandin production and preventing luteolysis in cows.

Chick embryos can form teratomas from microinjected mouse embryonic stem cells.

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra-embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon-like or dark-red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad-range potential as an experimental host model.

Gene expression profiles in the bovine corpus luteum (CL) during the estrous cycle and pregnancy: possible roles of chemokines in regulating CL function during pregnancy.

To determine functional differences between the corpus luteum (CL) of the estrous cycle and pregnancy in cows, gene expression profiles were compared using a 15 K bovine oligo DNA microarray. In the pregnant CL at days 20-25, 40-45 and 150-160, the expressions of 138, 265 and 455 genes differed by a factor of > 2-fold (P < 0.05) from their expressions in the cyclic CL (days 10-12 of the estrous cycle). Messenger RNA expressions of chemokines (eotaxin, lymphotactin and ENA-78) and their receptors (CCR3, XCR1 and CXCR2) were validated by quantitative real-time PCR. Transcripts of eotaxin were more abundant in the CL at days 40-45 and 150-160 of pregnancy than in the cyclic CL (P < 0.01). In contrast, the mRNA expressions of lymphotactin, ENA-78 and XCR1 were lower in the CL of pregnancy (P < 0.05). Messenger RNAs of CCR3 and CXCR2 were similarly detected both in the cyclic and pregnant CL. Tissue protein levels of eotaxin were significantly higher in the CL at days 150-160 of pregnancy than in the CL at other stages, whereas the lymphotactin protein levels in the CL at days 20-25 of pregnancy were lower (P < 0.05). Immunohistochemical staining showed that CCR3 was expressed in the luteal cells and that XCR1 was expressed in both the luteal cells and endothelial cells. Collectively, the different gene expression profiles may contribute to functional differences between the cyclic and pregnant CL, and chemokines including eotaxin and lymphotactin may regulate CL function during pregnancy in cows.

Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta.

BACKGROUND: Adrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome. METHODS: For real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation. RESULTS: AM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium. CONCLUSIONS: Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.

Differential neutrophil gene expression in early bovine pregnancy.

BACKGROUND: In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT)-stimulated gene expression in peripheral blood leukocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. METHODS: PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q) PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (MX) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analyzed in each fraction through day 28 of gestation using qPCR. RESULTS: Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and with density gradient separation. CONCLUSIONS: The expression profiles of ISG15, MX1, MX2, and OAS1 could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of these genes in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination.

Dynamics of CD3⁺ T-cell distribution throughout the estrous cycle and gestation in the bovine endometrium.

T cells are the dominant lymphocytes in the endometrium and are considered to play a crucial role in implantation and in the maintenance of gestation through cytokine production and immune regulation. The mechanisms underlying immunoregulation at the feto-maternal interface are still obscure for this complex system. Understanding the role of T cells is a key factor in understanding the endometrial immune system. In this study, the distribution of endometrial CD3⁺ T cells in bovines was examined by immunohistochemical analysis. The estrous cycle and gestation was divided into 4 stages, and the number of CD3⁺-positive T cells was counted in each stage. CD3⁺ cells were found in the endometrium in significant numbers throughout the estrous cycle and were mostly located in the subepithelial area. The number of CD3⁺ cells significantly increased in the early and mid-luteal phases but decreased after implantation with the progression of gestation. No T cells were found in the placentome or specifically in the tissues near the fetus, including the trophoblastic area. In addition, very few T cells were found in stromal regions close to the myometrium of the endometrium. These findings suggest that downregulation of bovine endometrial CD3⁺ T-cell functions is closely related to the successful maintenance of gestation in a spatiotemporal manner.

Bovine trophoblastic cell differentiation and binucleation involves enhanced endogenous retrovirus element expression.

BACKGROUND: Endogenous retrovirus (ERV) envelope (env) genes are involved in the differentiation of trophoblastic cells in humans and mice. However, there is limited information about their roles in ruminant trophoblastic cells. Thus, we attempted to explore the possible roles of ERV elements in the binucleation of bovine trophoblastic cells using in vitro bovine trophoblastic (BT) cell lines. METHODS: In this study, blastocysts and elongated embryos were obtained from Japanese Black cows, and endometrial and fetal membrane tissues were collected from day 17 to 37 of gestation. The gene expression levels of four ERV elements, bERVE (bovine endogenous retrovirus envelope element-like transcript) -A, bERVE-B, BERV (bovine endogenous retrovirus) -K1 env, and BERV-K2 env, were analyzed in the fetal and endometrial tissue and cultured BT cell lines using quantitative RT-PCR. On-Matrigel gel and on-collagen gel culturing were used to induce binucleate cell (BNC) formation in the BT cell lines. How the culture conditions affected the expression of BNC-specific genes and ERV elements was examined by quantitative RT-PCR and immunocytochemistry. RESULTS: bERVE-A, bERVE-B, BERV-K1 env, and BERV-K2 env were expressed in almost all BT cell lines; however, only bERVE-A and BERV-K1 env were detected in trophoblastic tissues during the peri-implantation period. In the on-Matrigel cultures, the expression levels of BNC-specific genes and molecules were enhanced in the BT cells. The expression levels of bERVE-A and BERV-K1 env were also increased in the BT cells during on-Matrigel culturing. The BT cell expression levels of these ERV elements were consistent with those of BNC-specific genes during on-Matrigel culturing (P < 0.01). CONCLUSIONS: These results suggest that bERVE-A and BERV-K1 env are involved in the expression of BNC-specific genes and the progression of bovine trophoblastic cell binucleation, as their expression levels increased during periods of increased BNC-specific molecule expression, which is strongly suggestive of the development of BNC from mononucleate trophoblastic cells. The on-Matrigel culture system is a convenient in vitro tool for studying bovine trophoblastic cell lineages.

Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia.

BACKGROUND: SERPINs (serine protease inhibitors) regulate proteases involving fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation and apoptosis. This study aimed to investigate differentially expressed genes of members of the SERPIN superfamily between healthy and atretic follicles using a combination of microarray and quantitative real-time PCR (QPCR) analysis. In addition, we further determined mRNA and protein localization of identified SERPINs in estradiol (E2)-active and E2-inactive follicles by in situ hybridization and immunohistochemistry. METHODS: We performed microarray analysis of healthy (10.7 +/- 0.7 mm) and atretic (7.8 +/- 0.2 mm) follicles using a custom-made bovine oligonucleotide microarray to screen differentially expressed genes encoding SERPIN superfamily members between groups. The expression profiles of six identified SERPIN genes were further confirmed by QPCR analysis. In addition, mRNA and protein localization of four SERPINs was investigated in E2-active and E2-inactive follicles using in situ hybridization and immunohistochemistry. RESULTS: We have identified 11 SERPIN genes expressed in healthy and atretic follicles by microarray analysis. QPCR analysis confirmed that mRNA expression of four SERPINs (SERPINA5, SERPINB6, SERPINE2 and SERPINF2) was greater in healthy than in atretic follicles, while two SERPINs (SERPINE1 and SERPING1) had greater expression in atretic than in healthy follicles. In situ hybridization showed that SERPINA5, SERPINB6 and SERPINF2 mRNA were localized in GCs of E2-active follicles and weakly expressed in GCs of E2-inactive follicles. SERPING1 mRNA was localized in both GCs and the theca layer (TL) of E2-inactive follicles and a weak hybridization signal was also detected in both GCs and TL of E2-active follicles. Immunohistochemistry showed that SERPINA5, SERPINB6 and SERPINF2 were detected in GCs of E2-active and E2-inactive follicles. SERPING1 protein was localized in both GCs and the TL of E2-active and E2-inactive follicles. CONCLUSIONS: Our results demonstrate a characteristic expression of SERPIN superfamily member genes in bovine healthy and atretic follicles. The cell-type-and stage-specific expression of SERPINs may be associated with bovine follicular growth and atresia.

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries.

BACKGROUND: It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. METHODS: Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and in situ hybridization. Immunohistochemical analysis was also performed. RESULTS: The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. CONCLUSIONS: Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle.

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.

Cloning and expression of SOLD1 in ovine and caprine placenta, and their expected roles during the development of placentomes.

BACKGROUND: The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions. RESULTS: Ovine and caprine SOLD1 mRNAs have 303 bp open reading frames and encode for deduced SOLD1 proteins with 100 amino acids, including a 22-aa-long signal peptide at the N-terminal. Both of the SOLD1 amino acid sequences have high similarities with the bovine sequence. Both SOLD1 mRNAs were also expressed in TMCs of cotyledons and intercotyledonary membranes. The mature SOLD1 proteins were localized in the mesenchymal villi of cotyledons after secretion. Bovine, ovine and caprine SOLD1 affected gene expression in mesenchymal fibroblasts in vitro; nucleoredoxin expression was upregulated and BCL2-like 13 was downregulated. Thus, we suggest that SOLD1 acts as a modulator of cell proliferation and apoptosis. CONCLUSION: Expressing cells and protein localization of SOLD1 coincided among the three ruminants. SOLD1 participated in regulating nucleoredoxin and BCL2-like 13 expression in chorionic fibroblasts. SOLD1 is produced specifically in the cotyledons and intercotyledonary membranes in ruminants and appears to be involved in the construction of the ruminant placenta.

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles.

BACKGROUND: Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes. METHODS: Global gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid. RESULTS: Hierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC. CONCLUSION: We demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle.

BACKGROUND: Extracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow. METHODS: In this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis. RESULTS: EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium. CONCLUSION: EMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions.

Temporal expression and localization of vascular endothelial growth factor family members in the bovine uterus during peri-implantation period.

The aim of this study was to determine endometrial mRNA expression patterns and uterine protein localizations of vascular endothelial growth factor (VEGF) ligands (VEGFA, VEGFB, VEGFC, and VEGFD) and their receptors (VEGFR1, soluble VEGFR1 (sVEGFR1), VEGFR2, and VEGFR3) during the peri-implantation period in cows. The number of blood and lymphatic vessels in the bovine uterus was also investigated. Bovine uterine tissues were collected from pregnant animals on days 15, 18, and 27 after artificial insemination and from non-pregnant animals on days 15 and 18 of the estrous cycle (day 0 = day of estrus). The mRNA expression level of VEGFA, VEGFR1, sVEGFR1, and VEGFR3 were higher on day 18 than on day 15 in the non-pregnant group. On day 18, the levels of mRNA expression of these genes were higher in the non-pregnant group than in the pregnant group. VEGFB mRNA expression levels was higher on day 15 than on days 18 and 27 of gestation and was higher in the pregnant group than in the non-pregnant group on day 15. Using immunohistochemistry, VEGF ligands and their receptors were found in luminal epithelium, glandular epithelium, stroma, and blood vessels of the endometrium. In addition, VEGFA, VEGFD, and VEGFR3 were also detected in the uterine myometrium. In the pregnant group, the number of blood vessels in the endometrium increased from day 15 to 18 and was greater than that of the non-pregnant group on day 18. Our results demonstrate that the VEGF family is expressed and regulated in the bovine uterus during the peri-implantation period, which may be associated with uterine functions, including vascular remodeling in maternal recognition of pregnancy and implantation.