Bovine Uterine Epithelial Cells Research Articles

Progesterone increases metabolism via the pentose phosphate pathway in bovine uterine epithelial cells.

During early pregnancy, glucose is essential for the uterine epithelium and the developing embryo. In cows, progesterone increases the secretion of glucose into the uterine lumen. The uterine epithelium can convert glucose to fructose, but other fates of glucose in the uterine epithelium have been sparsely investigated. Therefore, our objective was to investigate how progesterone influences glucose metabolism in immortalized bovine uterine epithelial (BUTE) cells. BUTE cells were grown to 80% confluence and treated with vehicle (DMSO) or 10 µM progesterone for 24h. Cells were collected and analyzed. Immunohistochemistry was performed on endometrial samples collected from the bovine endometrium on days 1 and 11 of the reproductive cycle. Progesterone treatment increased glucose consumption of BUTE cells. RNAseq identified 3,072 genes regulated by progesterone. KEGG analysis indicated that progesterone altered genes associated with metabolic pathways and glutathione metabolism. Manually examining genes unique to specific glucose metabolic pathways identified an increase in the rate-limiting enzyme in the pentose phosphate pathway-glucose-6-phosphate dehydrogenase. Functionally, a major product of the pentose phosphate pathway is NADPH, and progesterone treatment increased NADPH levels in BUTE cells. In cows, immunohistochemistry confirmed that glucose-6-phosphate dehydrogenase levels were higher in the uterine epithelium in the luteal phase when progesterone concentrations are high. Progesterone increased glucose-6-phosphate dehydrogenase expression and metabolism via the pentose phosphate pathway in the bovine uterine epithelium. This metabolism could provide substrates for cell proliferation, molecules to be secreted into the uterine lumen, or maintain reduction/oxidation balance in the uterine epithelium.

Membrane progesterone receptors mediate progesterone-stimulated glycogenolysis in the bovine uterine epithelium.

In livestock, the amount of glucose needed by the endometrium and embryo increases during early pregnancy. Yet, how glucose concentrations in the endometrium are regulated remains unclear. The bovine uterine epithelium can store glucose as glycogen, and glycogen content decreases in the luteal phase. Our objective was to elucidate the role of progesterone in glycogen breakdown in immortalized bovine uterine epithelial (BUTE) cells. After 48 h of treatment, progesterone decreased glycogen abundance in BUTE cells (P < 0.001) but did not alter glycogen phosphorylase levels. RU486, a nuclear progesterone receptor (nPR; part of the PAQR family) antagonist, did not block progesterone's effect, suggesting that progesterone acted through membrane progesterone receptors (mPRs). RT-PCR confirmed that BUTE cells express all five mPRs, and immunohistochemistry showed that the bovine uterine epithelium expresses mPRs in vivo. An mPRα agonist (Org OD 02-0) reduced glycogen abundance in BUTE cells (P < 0.001). Progesterone nor Org OD 02-0 affected cAMP concentrations. Progesterone increased phosphorylated AMP-activated protein kinase (pAMPK) levels (P < 0.001), indicating that progesterone increases intracellular AMP concentrations. However, AMPK did not mediate the effect of progesterone. AMP allosterically activates glycogen phosphorylase, and D942 (which increases intracellular AMP concentrations) decreased glycogen abundance in BUTE cells. A glycogen phosphorylase inhibitor partially blocked the effect of progesterone (P < 0.05). Progesterone and Org OD 02-0 had similar effects in Ishikawa cells (P < 0.01), a human cell line that lacks nPRs. In conclusion, progesterone stimulates glycogen breakdown in the uterine epithelium via mPR/AMP signaling. Glucose released from glycogen could support embryonic development or be metabolized by the uterine epithelium.

In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Effect of estradiol and IGF1 on glycogen synthesis in bovine uterine epithelial cells.

Glucose is an important nutrient for the endometrium and embryo during pregnancy. This study shows that estradiol (E2)/IGF1 signaling stimulates glycogen synthesis in the uterine epithelium of cows, which could provide glucose when needed. Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of E2 and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1-10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/mL) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than on day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased the levels of phospho-AKT, phospho-GSKβ, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

12-Oxoeicosatetraenoic acid, a candidate signal for placenta separation, activates matrix metalloproteinase and induces apoptosis in bovine trophoblast cells.

12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-KETE), a metabolite of arachidonic acid, is a strong candidate signal for placenta separation following calf discharge at delivery. In the present study, the effects of 12-KETE on bovine trophoblast cells were investigated to determine its function in the placentome at delivery. Bovine trophoblast cells derived from blastocysts were used. They were cocultured with or without fibroblasts derived from bovine placentome and/or bovine uterine epithelial cells. 12-KETE was added to the culture medium. Bovine trophoblast cells contained binucleate cells and strongly expressed caudal type homeobox 2 (CDX-2) genes. Addition of 12-KETE to the trophoblast cell colony without feeder cells or that on a fibroblast monolayer induced rapid exfoliation of the colony. After 12-KETE addition, trophoblast cells emitted strong fluorescence caused by the degradation of dye-quenched collagen, indicating that 12-KETE activated matrix metalloproteinase of the trophoblast cells. Exfoliated cell colonies were stained with YOPRO-1, but not propidium iodide (PI). Moreover, DNA fragmentation and Bcl-2 associated X protein (Bax) gene (apoptosis stimulator) upregulation were observed in exfoliated cells, indicating that 12-KETE induced trophoblast cell apoptosis. These results were consistent with previous in vivo observations; however, even a lower concentration of 12-KETE activated trophoblast protease. Meanwhile, fibroblasts derived from the bovine placentome converted arachidonic acid to 12-KETE. These observations indicate that 12-KETE may serve as a signal for placenta separation at delivery.

Anti-Müllerian hormone stimulates expression of the collagen-specific chaperone 47-kDa heat shock protein in bovine uterine epithelial cells.

Uterine collagen is the most abundant component of the uterine extracellular matrix and plays a critical role in pregnancy. The 47-kDa heat shock protein (HSP47) is the sole collagen-specific molecular chaperone. We investigated the mechanisms regulating the expression of HSP47 in the uterus by assessing the effect of anti-Müllerian hormone (AMH) stimulation on HSP47 expression in cultured bovine uterine epithelial cells. AMH receptor type 2 (AMHR2), AMH, and HSP47 expression was assessed by fluorescence immunocytochemistry in uterine epithelial layers of the uteri of Japanese Black cows. The effect of AMH on HSP47 expression was assessed in cultured epithelial cells. The effect of MEK/ERK inhibitor on AMH-induced HSP47 expression was also assessed. We confirmed the expression of AMHR2, AMH, and HSP47 in the uterine epithelial layers. We confirmed the expression of AMHR2, AMH, HSP47, and type IV collagen in cultured uterine epithelial cells. AMH treatment at 10 or 100 ng/ml promoted significant HSP47 expression (p < 0.05). MEK/ERK inhibitor U0126 pretreatment suppressed such AMH stimulation on HSP47. These findings indicate that AMH induced HSP47 protein expression through the ERK pathway in bovine uterine epithelial cells.

Sperm enter glands of preovulatory bovine endometrial explants and initiate inflammation.

We previously reported that sperm binding to cultured monolayers of bovine uterine epithelial cells induces an acute inflammatory response involving the Toll-like receptor (TLR2) signaling pathway. This response serves to clear the uterus of sperm and thereby prepares the endometrium for implantation. The endometrium is lined by surface epithelial cells; however, epithelial cells also line uterine glands. To investigate the source of the immune response, we used an explant model. Explants of bovine endometrium were incubated with bull sperm illuminated by JC1 fluorescent labeling in their mitochondria. The sperm glided over the surface epithelium until they encountered and entered uterine glands, where they remained. Scanning electron microscopy of explants revealed polymorphonuclear neutrophils (PMNs) in uterine glands along with sperm. In the absence of sperm, PMNs were not seen in glands. The incubation of sperm with explants resulted in an acute inflammatory response, seen as the upregulation of mRNA expression of IL8, TNFA, IL1B, PGES and TLR2 in whole explants, as well as increased TNFA protein expression in uterine glands. TLR1/2 antagonist reduced sperm numbers in the glands and inhibited the increase of TNFA. Our observations suggest that uterine glands serve as a site where sperm interact with the uterine epithelium to trigger the innate immune response to clear excess sperm from the uterus.

Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo.

A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro.

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (104 , 105 , and 106 sperm/ml) for 3 hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 106 /ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE2 release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 106 /ml washed sperm with BUECs for 0, 1, 3, and 6 hr. The number of detached sperm increased gradually toward 6 hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3 hr, while C3 continued to increase toward 6 hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE2 up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs.

Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator.

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus.

Regulation of Interferon-stimulated Gene (ISG)12, ISG15, and MX1 and MX2 by Conceptus Interferons (IFNTs) in Bovine Uterine Epithelial Cells.

Various endometrial genes in ruminant ungulates are regulated by conceptus interferon tau (IFNT). However, the effect of each IFNT isoform has not been carefully evaluated. In this study, the effects of 2 IFNT isoforms, paralogs found in utero, and interferon alpha (IFNA) on uterine epithelial and Mardin-Darby bovine kidney (MDBK) cells were evaluated. Expression vectors of the bovine interferon (bIFNT) genes bIFNT1, bIFNTc1, and bIFNA were constructed, and recombinant bIFNs (rbIFNs) were produced by 293 cells. Bovine uterine epithelial or MDBK cells were cultured in the presence or absence of increasing concentrations of each rbIFN for 24, 48, or 72 h. Transcript levels of the IFN-stimulated genes (ISGs) ISG12, ISG15, MX1, and MX2 were analyzed using quantitative reverse transcription-polymerase chain reaction. These messenger RNAs were up-regulated by rbIFN in a time- and concentration-dependent manner. In the epithelial cells, the ISG12 transcript level increased at 48 h after rbIFN treatment but slightly decreased at 72 h, whereas the transcript level of ISG15 increased at 24 h and was maintained through 72 h. Expressions of MX1 and MX2 increased at 72 h after rbIFN treatment. MX1 expression increased in all treatment groups, but MX2 increased only by bIFNTc1. In MDBK cells, the expression of ISG12 was increased by bIFNT1 and bIFNTc1 after 24 and 72 h; however, it was unchanged by rbIFNA. ISG15 increased following the same pattern as that seen in uterine epithelial cells, and MX1 showed a similar expression pattern. MX2 expression was increased by bIFNTc1 treatment in uterine epithelial cells, and its expression was increased by both bIFNT1 and bIFNTc1 in MDBK cells. These results show that epithelial and MDBK cell responses to IFNs differ, suggesting that IFNs possess common functions, but may have acquired different functions following gene duplication.

Down‐regulation of interferon tau gene transcription with a transcription factor, EOMES

Interferon tau (IFNT), produced for a short interval during early pregnancy by the ruminant embryonic trophectoderm, is essential for the maintenance of early pregnancy, but the mechanism by which it is transcriptionally regulated has not been fully determined. To identify a transcription factor(s) involved in the down-regulation of IFNT genes, mRNAs for various known transcription factors were investigated by reverse-transcriptase and real-time PCR in conceptus tissues collected on Days 15, 17, and 21, or Days 17, 20, and 22 of ovine or bovine pregnancy, respectively. In particular, the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in Day 17 or 22 ovine or bovine conceptuses. Interaction between EOMES and the identified transcription factors was studied using transient transfection, revealing that ovine/bovine IFNT-reporter transactivation was down-regulated by EOMES. Transcription factor interactions with EOMES were further studied through immunoprecipitation, demonstrating an association between EOMES and cAMP-response element binding protein (CREB)-binding protein (CREBBP). Uterine flushing media collected from cyclic or early pregnancy animals were added to bovine trophoblast CT-1 cells cultured on type-I collagen (monoculture) or bovine uterine epithelial cells (coculture) in an attempt to regulate EOMES expression. In the coculture, but not the monoculture, addition of uterine flushing from Day 17 pregnant animals resulted in increased EOMES expression in CT-1 cells. These results suggest that as conceptuses attach to the uterine epithelium, IFNT gene transcription is down-regulated by an increase in EOMES expression and EOMES-CREBBP binding in the attached trophoblast cells.

Modulatory Effects of Bovine Seminal Plasma on Uterine Inflammatory Processes

In this study, a simple model to simulate a uterine environment affected by subclinical endometritis was established by culturing isolated primary bovine uterine epithelial cells (pbUEC). Co-incubation of pbUEC and polymorphonuclear (PMN) granulocytes derived from peripheral bovine blood samples, was performed before testing the cell culture supernatant for production of interleukin-8 (IL-8) via ELISA. Cytokine secretion was only detectable after co-incubation of pbUEC with PMN, whereas neither pbUEC nor PMN alone generated IL-8 in relevant chemo attractive doses. Another objective was to examine the influence of bovine seminal plasma (SP) and vesicular gland fluid (VGF) on various functional parameters of PMN including cell viability, production of reactive oxygen species and chemotaxis. Analysis of these effects was conducted by flow cytometry. Viability of PMN was determined by staining the cells with propidium iodide. Seminal plasma was added to suspensions of PMN in increasing increments and resulted in a significant increase of cell membrane damaged PMN when using SP concentrations above 0.2%. The reactive oxygen species production of PMN suspensions, stimulated with phorbol-12-myristate-13-acetate, was significantly decreased by 30% up to 90% when adding 0.06-30‰ of either SP or VGF. The PMN transmigration induced by IL-8 was diminished by 50% when 0.4‰ of either SP or VGF were added. The results of this study indicate a potential regulatory impact of SP and VGF on inflammatory processes in the bovine uterus.

In vitro cytopathic effects of a cysteine protease of Tritrichomonas foetus on cultured bovine uterine epithelial cells

To evaluate the cytopathic effects of Tritrichomonas foetus and a purified cysteine protease (ie, CP30) of T foetus on cultured bovine uterine epithelial cells (BUECs) in vitro. 10 reproductive tracts were obtained from late-term bovine fetuses at a commercial abattoir. An in vitro culture system of BUECs was developed to study the cytopathic effects of T foetus and purified CP30 of T foetus on host cells. Cytotoxicity of T foetus or CP30 on exposed BUECs was determined. Fluorescence microscopy and flow cytometry analyses were used to detect apoptosis. A fluorometric assay was used to detect BUEC caspase 3 activation. The CP inhibitor E-64 and a caspase inhibitor were used to inhibit apoptosis. Cytopathic effects were observed in BUECs treated with parasites or CP30 and were concentration and time dependent. The BUECs underwent apoptosis in the presence of parasites or CP30. The specific CP inhibitor E-64 abolished the induction of apoptosis in BUECs by CP30. The caspase inhibitor reduced the amount of apoptosis in BUECs. T foetus and its CP30 induce apoptosis in cultured BUECs in vitro. Induction of apoptosis by CP30 is correlated with protease activity. Endometrial cell death as a result of a T foetus infection is likely to be more important in mediating infertility than a direct effect on the conceptus. Provoking an apoptotic reaction in the host may mitigate an inflammatory reaction or immune response and therefore favor survival of the parasite in a chronic infection.

Differential effects of interferon-tau on the prostaglandin synthetic pathway in bovine endometrial cells treated with phorbol ester.

Rationale for these experiments was to evaluate the dose effects of bovine interferon-tau (IFN-tau) on the prostaglandin secretory pathway of immortalized bovine endometrial (BEND) cells in response to phorbol 12, 13-dibutyrate (PdBu) and to characterize similar responses in primary bovine uterine epithelial cells as a biomonitor of embryo-induced antiluteolytic effects on the endometrium. The BEND cells were treated with PdBu (0 or 100 ng/mL) and IFN-tau (0 or 50 ng/mL) for 6 h. The PdBu stimulated secretions of PGF2alpha and prostaglandin E2 (PGE2). Co-treatment of cells with IFN-tau blocked PdBu-induced secretion of both PGF2alpha and PGE2. Treatment with PdBu for 6 h induced expression of prostaglandin H synthase-2 mRNA, prostaglandin H synthase-2 protein, and prostaglandin E synthase mRNA, which were blocked with concurrent addition of IFN-tau. Doses of IFN-tau (0.05, 0.5, 1, 5, 10, and 20 microg/mL) were used with PdBu (0 and 100 ng/mL). The IFN-tau alone failed to stimulate secretion of PGF2alpha and PGE2, whereas IFN-tau doses <5 microg/mL suppressed PdBu-stimulated secretions of PGF2alpha and PGE2. Uterine epithelial cells were isolated from cows at d 17 after estrus and were cultured to confluence in serum-free medium. Cells were treated with IFN-tau (0, 50, or 500 ng/mL) and PdBu (0 or 100 ng/mL) before media were collected after 24 h for PGF2alpha and PGE2 analyses. Treatment of primary uterine epithelial cells with PdBu induced PGF2alpha secretion, and IFN-tau (50 and 500 ng/mL) caused a reduction in PGF2alpha secretion induced by PdBu. In the absence of PdBu, IFN-tau increased basal secretion of PGF2alpha. Concentrations of PGE2 increased in response to PdBu, and the 50-ng/mL dose of IFN-tau had a stimulatory effect on PGE2 concentrations compared with the 500-ng/mL dose in the absence of PdBu. Phorbol ester-induced gene transcription as related to prostaglandin synthesis is regulated by IFN-tau in vitro.

Progesterone-modulated induction of apoptosis by interferon-tau in cultured epithelial cells of bovine endometrium.

Interferon-tau (IFN-tau) is produced by the trophoblast prior to implantation in ruminants. It is involved in maternal recognition of pregnancy, and is a pleiotropic molecule that can alter the synthesis of endometrial proteins and inhibit proliferation of some cells. We have observed that IFN-tau reduces the DNA content in cultures of bovine endometrial epithelial cells; therefore, the objective of this study was to determine whether IFN-tau would induce apoptosis in bovine endometrial cells. Epithelial cells were prepared, cultured to confluence, and then incubated for 24 or 48 h in the presence or absence of 10 ng/ml progesterone, 100 ng/ml IFN-tau, or 10 microg/ml cycloheximide (CHX; an apoptosis inducer used as a positive control). Cells undergoing apoptosis exhibit such characteristics as the appearance of apoptotic bodies and DNA fragmentation. The incidence of apoptosis was assessed by using TUNEL, DNA fragmentation analysis, and Western blot analysis of Bax-alpha protein expression. The results showed that IFN-tau and CHX significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment of the cells significantly inhibited the ability of IFN-tau to induce apoptosis (14.6%) compared with IFN-tau alone (33.6%; P < 0.05). DNA fragmentation analysis showed that INF-tau and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures. Western blot analysis showed that IFN-tau and CHX treatment resulted in a greater expression of the proapoptotic protein Bax-alpha compared with that in control cultures. These data demonstrate that IFN-tau can induce apoptosis in bovine uterine epithelial cells and that this effect is modulated by progesterone. We speculate that IFN-tau might play a critical role in the remodeling of the endometrium around the time of implantation.

Genetic and environmental determinants of interferon-τ secretion by in vivo- and in vitro-derived bovine blastocysts

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-τ secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo’s ability to develop to the blastocyst stage and of subsequent interferon-τ secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-τ. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-τ was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-τ by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.

Retention of functional characteristics by bovine oviduct and uterine epithelia in vitro.

The composition of fluids within the bovine oviduct and uterine lumen, important in fertilisation and early embryonic development, is ultimately determined by the transport properties of the epithelial cells which line the lumen. A preparation has therefore been devised to study the role of these cells in oviduct and uterine fluid formation. Pure preparations of epithelial cells, as judged immunocytochemically, were isolated by enzyme digestion, and grown on collagen filters in primary culture. The cells re-establish intercellular junctions to form a confluent epithelial layer. Serial samples from the apical and basal media were analysed for K+, Na+, Ca2+, glucose and lactate. Bovine oviduct epithelial cells maintained gradients of K+ and Ca2+ (apical > basal) for up to 14 days after confluence, while bovine uterine epithelial cells maintained apical > basal gradients of K+. Both types of epithelium exhibited a small transepithelial electrical potential difference and a higher uptake of glucose and production of lactate in the basal, as opposed to apical medium. There were no consistent differences in any of these parameters with the stage of the oestrous cycle at which the cells were removed. The data indicate that bovine oviduct and uterine epithelia may be isolated and grown as polarised layers in primary culture. The preparations will now enable the mechanisms underlying the secretion of ions and non-electrolytes to be determined.

Fertilization and early embryology: Effect of different co-culture systems in early human embryo development

The objective of this study was to examine the effects of different culture systems on the development of early human embryos in vitro. A total of 460 fertilized oocytes from 82 cycles of patients was transferred into one of four systems: (1) into droplets of Ham's F10 medium + 12% normal human serum (NHS); (2) co-cultured on a human granulosa monolayer; (3) co-cultured with bovine oviductal epithelial cells (BOEC); or (4) co-cultured with bovine uterine epithelial cells (BUEC). The percentage of cleavage and the morphological appearance of embryos were recorded daily for 72 h in each system using an inverted phase-contrast microscope. The results showed that the proportions of the fertilized oocytes which developed to the four-cell stage 48 h after retrieval were, by culture system: (1) 70% (84/120); (2) 74% (85/115); (3) 78% (91/117); and (4) 76% (82/108). At 72 h after retrieval, the proportions of the eight-cell stage were, by culture system: (1) 45% (38/84); (2) 62% (53/85); (3) 75% (68/91); and (4) 70% (57/82). We concluded that a higher proportion of fertilized oocytes developed to embryos at the eight-cell stage in systems 2, 3 and 4 than in system 1. This indicates the beneficial effect of co-culture of human embryos with granulosa cell, BOEC and BUEC monolayers, which may be due to various factors.

From embryo to calf after transfer of in vitro produced bovine embryos

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-τ secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo’s ability to develop to the blastocyst stage and of subsequent interferon-τ secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-τ. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-τ was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-τ by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.