Bovine Oviduct Epithelium Research Articles

An approach to study the local embryo effect on gene expression in the bovine oviduct epithelium in vivo.

This study aimed to examine the local embryo effect on the transcriptomic response of the epithelial cells of the oviduct in vivo. Fifteen heifers were synchronized and artificially inseminated to a standing heat. All heifers were slaughtered on Day 2.5 after oestrus. The oviducts from 13 animals were isolated, trimmed free of tissue and divided between ampulla/isthmus. The ipsilateral isthmus was divided into smaller sections (2cm). Each section was sequentially flushed until the embryo was located (4/13) and then opened and scraped longitudinally to obtain the epithelial cells. Cells were snap-frozen in LN2 for gene expression analysis. All recovered embryos were found at the beginning of the isthmus. The 2cm sections selected for the transcriptomic analysis were as follows: embryo section (in which the embryo was found); proximal section (through which the embryo had passed); distal section (on the uterine side of the embryo); and contralateral section (section from the contralateral isthmus). The expression pattern of eight genes (STK32A, KERA, QRFPR, MCTP1, PRELP, VAT1L, SOCS3 and CCL20) differentially expressed between the isthmus of pregnant (multiple embryo model) and cyclic heifers were assessed by RT-qPCR. One-way ANOVA and t test was used for statistical analysis. Comparisons between ipsilateral and contralateral oviduct or along the ipsilateral oviduct resulted in no differences for all genes. Despite the failure to detect a site-specific response of a single embryo on the abundance of distinct transcripts in the bovine oviduct in vivo on Day 2.5, the current methodology with proposed modifications would be useful for future studies to examine the local embryo effect.

Relative effects of location relative to the corpus luteum and lactation on the transcriptome of the bovine oviduct epithelium

BackgroundLactation and associated metabolic stresses during the post-partum period have been shown to impair fertility in dairy cows. The oviduct plays key roles in embryo development and the establishment of pregnancy in cattle. The aim of this study was to investigate the effects of lactation and location relative to the corpus luteum (CL) on the transcriptome of the bovine oviduct epithelium.ResultsAn original animal model was used. At 60 days post-partum, Holstein lactating (n = 4) and non-lactating (i.e. never milked after calving; n = 5) cows, as well as control nulliparous heifers (n = 5), were slaughtered on Day 3 following induced estrus, and epithelial samples from the oviductal ampulla and isthmus ipsilateral and contralateral to the corpus luteum (CL) were recovered for RNA sequencing. In the oviduct ipsilateral to the CL, differentially expressed genes (DEGs) were identified between heifers compared with both postpartum cow groups. However, only 15 DEGs were identified between post-partum lactating and non-lactating cows in the ipsilateral isthmus and none were identified in the ipsilateral ampulla. In contrast, 192 and 2583 DEGs were identified between ipsilateral and contralateral ampulla and isthmus, respectively. In both regions, more DEGs were identified between ipsilateral and contralateral oviducts in non-lactating cows and heifers than in lactating cows. Functional annotation of the DEGs associated with comparisons between metabolic groups highlighted a number of over-represented biological functions and cell pathways including immune response and cholesterol/steroid biosynthesis.ConclusionsGene expression in the oviduct epithelium, particularly in the isthmus, was more affected by the location relative to the CL than by lactation at Day 3 post-estrus. Furthermore, the effect of the proximity to the CL was modulated by the metabolic status of the cow.

Understanding the hidden relations between pro- and anti-inflammatory cytokine genes in bovine oviduct epithelium using a multilayer response surface method

An understanding gene-gene interaction helps users to design the next experiments efficiently and (if applicable) to make a better decision of drugs application based on the different biological conditions of the patients. This study aimed to identify changes in the hidden relationships between pro- and anti-inflammatory cytokine genes in the bovine oviduct epithelial cells (BOECs) under various experimental conditions using a multilayer response surface method. It was noted that under physiological conditions (BOECs with sperm or sex hormones, such as ovarian sex steroids and LH), the mRNA expressions of IL10, IL1B, TNFA, TLR4, and TNFA were associated with IL1B, TNFA, TLR4, IL4, and IL10, respectively. Under pathophysiological + physiological conditions (BOECs with lipopolysaccharide + hormones, alpha-1-acid glycoprotein + hormones, zearalenone + hormones, or urea + hormones), the relationship among genes was changed. For example, the expression of IL10 and TNFA was associated with (IL1B, TNFA, or IL4) and TLR4 expression, respectively. Furthermore, under physiological conditions, the co-expression of IL10 + TNFA, TLR4 + IL4, TNFA + IL4, TNFA + IL4, or IL10 + IL1B and under pathophysiological + physiological conditions, the co-expression of IL10 + IL4, IL4 + IL10, TNFA + IL10, TNFA + TLR4, or IL10 + IL1B were associated with IL1B, TNFA, TLR4, IL10, or IL4 expression, respectively. Collectively, the relationships between pro- and anti-inflammatory cytokine genes can be changed with respect to the presence/absence of toxins, sex hormones, sperm, and co-expression of other gene pairs in BOECs, suggesting that considerable cautions are needed in interpreting the results obtained from such narrowly focused in vitro studies.

The urokinase plasminogen activator system components are regulated by vascular endothelial growth factor D in bovine oviduct.

SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.

Modelling oviduct fluid formation in vitro.

Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage, and genome activation. However, the composition and regulation of this critical environment remains rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium, to investigate the formation and composition of in vitro derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes, and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility, and optimising embryo culture media.

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50% by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

77 OVIDUCT - EMBRYO INTERACTIONS: TWO-WAY TRAFFIC OR A ONE-WAY STREET? TRANSCRIPTOMIC RESPONSE OF THE BOVINE OVIDUCT TO THE PRESENCE OF AN EMBRYO

Despite clear evidence of a two-way interaction between the developing conceptus and the uterine endometrium in early pregnancy, the evidence for reciprocal cross-talk during the transit of the embryo through the oviduct is less clear. The aims were (1) to characterise the transcriptome of the bovine oviduct at the initiation of embryonic genome activation (EGA), (2) to examine the effect, if any, of the presence of an embryo on the oviduct transcriptome, and (3) to compare gene expression in the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum. The oestrous cycles of cross-bred beef heifers were synchronized and those recorded in standing oestrus were randomly allocated to control group, nonbred (n = 7), or AI group (n = 11). All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut in half (ampulla and isthmus). Each portion was flushed with 500 μL of PBS to confirm the presence of an oocyte/embryo and was then opened and scraped longitudinally to obtain epithelial cells. Cells were snap-frozen in liquid nitrogen for microarray analysis. All recovered oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. The recovery rate was 72.7% (8/11) and 83.3% (5/6) for pregnant and cyclic animals, respectively. The stage of the recovered embryos was as follows: 4-cell stage (n = 1), 8-cell stage (n = 5), and 8–16 cell stage (n = 2), whereas in the cyclic group all recovered structures were unfertilized oocytes. The cells of the isthmus from ipsilateral and contralateral oviducts from 5 cyclic and 5 pregnant animals (8-cell embryos) and the ipsilateral ampulla cells from the pregnant animals were used for microarray analysis (Affymetrix Bovine ST array, Affymetrix, Santa Clara, CA, USA). Array data were analysed using BioConductor packages in R and custom CDF files downloaded from MBNI. Preprocessing of raw data was performed with RMA, and differential expression was assessed by linear modelling implemented in the limma package. Genes displaying P < 0.05 after adjustment for multiple testing were considered differentially expressed. A total of 18 809 probe sets were assessed for differential expression. Comparison of pregnant and cyclic oviduct epithelium revealed no significantly altered genes. However, comparison of the isthmus and ampulla of the ipsilateral oviduct in pregnant animals revealed 4011 (P < 0.05) and 2327 (P < 0.01) differentially expressed genes. Some of the gene ontologies involved in biological processes included fatty acid metabolism, cell adhesion, cell morphogenesis, cellular developmental process, and reproduction. In conclusion, we have characterised the transcriptome of the bovine oviduct epithelium at the initiation of embryonic genome activation on Day 3 post-oestrus in pregnant and cyclic heifers. Although large differences in gene expression were observed between the isthmus and ampulla, data suggest that the presence of an 8-cell embryo had no effect on the transcriptome of the cells of the isthmus, although a local effect at the precise position of the embryo cannot be ruled out.

123 GENISTEIN TRANSPORT ACROSS THE BOVINE OVIDUCT EPITHELIUM

The oviduct plays a vital role in regulating the environment surrounding the gametes and early mammalian embryo. However, the permeability of the oviduct to circulating dietary-derived compounds remains relatively unknown. The present study has investigated the barrier properties of the oviduct epithelium in vitro to the movement of genistein, a soya isoflavone and analogue of 17β-estradiol that naturally occurs in the diet and has been reported to exert teratogenic effects. Bovine oviduct epithelial cells (BOECs) were isolated from abattoir-derived reproductive tracts at the mid-luetal phase. The purity of the cell isolate was confirmed using flow cytometry to determine the proportion of cells that expressed CK18 (epithelia), and Vimentin (fibroblasts). Cells were seeded at a density of 1 × 106 mL–1 on polyethylene terephthalate transwell porous supports (Corning) and maintained between two media-filled chambers for 10 to 12 days, until they formed a polarised confluent monolayer, as confirmed by transepithelial resistance (TEER) greater than 700 Ωcm2. To assess the rate of transport, genistein was added to the basal compartment at physiologically relevant levels (100 µM) and the apical compartment was sampled at regular time points for 120 mins. The concentration of genistein in the apical and basal media was measured by HPLC. Furthermore, we compared the rate of genistein transport at physiological (39°C) and room temperature to indicate whether transport was temperature dependent. Rates of transport are expressed as mean apparent permeability coefficient and were compared between groups by a Student’s t-test. Genistein crossed the bovine oviduct epithelium at a linear rate that was higher than spontaneous diffusion across a blank membrane support (12.7 × 10–3 cm–2 µM–1 v. 7.32 × 10–3 cm–2 µM–1, n = 4; P = 0.0075). The rate of genistein transport by epithelial cells was unchanged when cells were assayed at room temperature (12.7 × 10–3 cm–2 µM–1 v. 13.11 × 10–3 cm–2 µM–1, n = 3; P = 0.76), respectively. No significant difference in the directionality of transport was found. Furthermore, TEER was maintained at approximately 700 Ωcm2, indicating that the cells remained confluent for the duration of the experiment. These data suggest that the bovine oviduct epithelial cell monolayer facilitates genistein movement from the basal to the apical compartment in vitro. Furthermore, the observation that the rate of transport is unchanged by temperature suggests a passive, trans-cellular, physicochemical mechanism, rather than an active biological process. Regardless of the mechanism, the oviduct epithelium is permeable to genistein, and may even facilitate its transport into the lumen, suggesting that gametes and early embryos could be exposed to this compound in vivo. This is relevant given the previously reported finding indicating that this naturally occurring dietary isoflavone has detrimental effects on early development (Newbold et al. 2001 Cancer Res. 61, 4325–4328). Furthermore, the results demonstrate the potential use of this epithelial model in characterising the transport or barrier properties of the oviduct epithelium towards a range of circulating xenobiotics.

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.

Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines.

Cellular Expression and Localization of Estrogen Receptor α and Progesterone Receptor mRNA in the Bovine Oviduct Combining Laser-Assisted Microdissection, Quantitative PCR, and In Situ Hybridization

The importance of using techniques that allow the study of pure populations of cells has been increasingly recognized. The authors used laser-assisted microdissection (LAM) in combination with quantitative real-time PCR (qPCR) to assess the relative expression of mRNAs encoding estrogen receptor α (ERα) and progesterone receptor (PR) in the different compartments of the bovine oviduct (epithelium, stroma, smooth muscle coat) during the follicular and mid-luteal phases of the estrus cycle. The localization of receptor mRNA was further studied using non-radioactive in situ hybridization (NISH). A special focus was on whether formalin fixation and paraffin embedding influence the quality and quantity of mRNA obtained from microdissected material. Distinct cyclic changes of the mRNA in the bovine oviduct were observed with elevated levels of PR mRNA transcripts in the epithelium and smooth muscle coat during the follicular phase. The expression of PR mRNA did not vary significantly in the stroma of the bovine oviduct during follicular and mid-luteal phases. In conclusion, the authors found that LAM with qPCR can precisely locate and accurately quantify mRNA expression in specific cell populations from formalin-fixed and paraffin-embedded oviductal tissue.

The endocannabinoid system in bull sperm and bovine oviductal epithelium: role of anandamide in sperm–oviduct interaction

Anandamide binds to cannabinoid receptors and plays several central and peripheral functions. The aim of this work was to study the possible role for this endocannabinoid in controlling sperm-oviduct interaction in mammals. We observed that bull sperm and bovine oviductal epithelial cells express cannabinoid receptors, CB1 and CB2, and fatty acid amide hydrolase, the enzyme that controls intracellular anandamide levels. A quantitative assay to determine whether anandamide was involved in bovine sperm-oviduct interaction was developed. R(+)-methanandamide, a non-hydrolysable anandamide analog, inhibited sperm binding to and induced sperm release from oviductal epithelia. Selective CB1 antagonists (SR141716A or AM251) completely blocked R(+)-methanandamide effects. However, SR144528, a selective CB2 antagonist, did not exert any effect, indicating that only CB1 was involved in R(+)-methanandamide effect. This effect was not caused by inhibition of the sperm progressive motility or by induction of the acrosome reaction. Overall, our findings indicate for the first time that the endocannabinoid system is present in bovine sperm and oviductal epithelium and that anandamide modulates the sperm-oviduct interaction, by inhibition of sperm binding and induction of sperm release from oviductal epithelial cells, probably by activating CB1 receptors.

Prenatal Differentiation of Bovine Oviductal Epithelium: An Electron Microscopic Study

In this study, we investigated the ultrastructural changes during the prenatal differentiation of oviductal epithelium in 16 bovine embryos and fetuses from CRL of 18.0 cm to a CRL of 94.0 cm. Ciliated and secretory cells of bovine uterine tube, a derivative of the Müllerian duct, differentiate to distinct development stages in the prenatal period. The typical cellular pattern, which is generally characteristic for the adult bovine oviduct, is also obtained during fetal life. In the early stages (CRL 18.0/20.4 cm), the bovine oviductal epithelium appears mostly undifferentiated. The epithelial cells show only a few mitochondria, some cisternae of rough endoplasmic reticulum (rER) and a small Golgi-complex. Most of the cytoplasm is filled with a large amount of glycogen, which decreases during later development. Interspersed between the undifferentiated epithelial cells, a few cells undergoing ciliogenesis can be observed. Ciliogenesis increased significantly during the later prenatal developmental stages. At a CRL of 55.0 cm, ciliated cells appear fully differentiated with mature cells covering their luminal surface. Formation of cilia usually use the acentriolar pathway. Fibrous granules occurred initially in association with the Golgi-apparatus and r(ER) in the supranuclear cytoplasm. Fibrous granules later fuse with deuterosomes and give rise to procentrioles, which are translocated to the luminal plasma membrane. There they become arranged in a line just beneath the apical cell membrane and further differentiate to basal bodies from which the formation of cilia and striated rootlets take place. Clear signs of differentiation of secretory cells were first seen in our material in fetuses with a CRL of 51.0 cm and 64.0 cm. These cells contain a well developed rER and Golgi-apparatus with dilated cisterns. In the supranuclear cytoplasm, the number of secretory granules continuously increases during later development and the cells adapt to the morphology of mature secretory cells at the CRL 94.0 cm.

Effect of osteopontin (OPN) on in vitro embryo development in cattle

Fertility-related phosphoprotein osteopontin (OPN) is present in the bovine oviduct epithelium and fluid. The objectives were to determine the effects of OPN on percentages of cleavage and embryo development in vitro in cattle, and to assess the ability of OPN to induce in vitro capacitation of bovine sperm. In vitro-matured bovine oocytes were fertilized in the presence of 0, 10, 20, or 40 μg/mL OPN. There were greater percentages ( P < 0.01) of cleavage and compact morulae-blastocysts (79.7 and 43.3%, respectively) with 10 μg/mL OPN than in the control group (without OPN; 71.2 and 32.1%, respectively). Furthermore, percentages of advanced blastocysts were greater in the group receiving 40 μg/mL OPN versus control (56.4% vs. 42.0%, P < 0.05). Capacitation was assessed by the ability of sperm to undergo the acrosome reaction after incubation with lysophosphatidylcholine. Semen from three bulls was incubated for 2 h in either TALP medium alone (control) or with TALP medium containing 0.01 mM heparin, or with TALP medium containing 10 or 20 μg/mL OPN. Incubation with 10 and 20 μg/mL OPN produced more ( P < 0.01) capacitated sperm (14.4 and 13.6%, respectively) than the untreated control group (8.3%), but both untreated sperm and those treated with OPN had significantly fewer capacitated sperm than those treated with 0.01 mM of heparin (30.5%). In conclusion, OPN improved the efficiency of bovine in vitro embryo production and influenced sperm capacitation.

Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal Reservoir1

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.

THE OVIDUCTAL SPERM RESERVOIR: OVIDUCTAL ANNEXINS AS RECEPTORS FOR BULL SPERM BSP PROTEINS

Mammalian sperm are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. In the bovine, we determined that seminal vesicle secretory proteins in the BSP family, namely PDC-109 (BSP A1/A2), BSP-A3, and BSP-30-kDa, coat the sperm head and enable sperm to bind to the epithelium. Our next objective was to identify the receptors for sperm on the oviductal epithelium. In order to capture oviductal ligands, a co-immunoprecipitation system was constructed using purified BSPs immobilized with corresponding antibodies covalently bound to Protein A agarose beads. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were passed through the beads packed into columns. Unbound proteins were washed from the beads and then bound proteins were eluted using EGTA, because sperm binding to oviductal epithelium is calcium-dependent. Protein bands of 34 and 38 kDa were detected in silver-stained SDS PAGE of the eluates from each of the three columns. Protein constituents of the bands were identified by tandem mass spectrometry as annexins (ANXA) 1, 2, 4, and 5. Subsequently, commercial antibodies to each of the ANXAs were found to inhibit binding of washed ejaculated sperm to explants of oviductal epithelium. Antibodies to ANXA 1, 2, 4, and 5 reduced binding by 76, 67, 70, and 84 %, respectively, compared to nonspecific IgGs (P<0.05). Anti-annexin antibodies also labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of annexins in apical plasma membranes, and 2-D blots demonstrated multiple forms of the annexins, suggesting post-translational modifications. Because fucose had been determined to be a critical component of the oviductal receptor for sperm, the ANXAs were immunoprecipitated from solubilized apical membranes and probed with fucose lectins to verify the presence of fucose. ANXA 1, 2, 4, and 5 exhibited staining with Anguilla anguilla lectin that was blocked by fucose. Annexins comprise a large multigene family of calcium-regulated phospholipid- and membrane-binding proteins whose diverse functions include cell adhesion, ion channel modulation, signal transduction, and modulation of membrane architecture. Thus, annexins are strong candidates for the sperm receptors on bovine oviductal epithelium. NRICGP Grant no. 2004-35203-14952 from USDA CREES. (platform)

Dissection of embryo- and progesterone-induced transcriptome changes in bovine endometrium during the pre-attachment stage

The preparation of the endometrium for the process of embryo implantation is critical for the establishment of a pregnancy. There are two major effects on the bovine endometrium during the pre-implantation period important for successful implantation – embryo-derived signals (e.g. interferon tau) and ovarian progesterone. To distinguish between these two effects a biological model was analyzed comprised of three different groups: day 18 pregnant animals after artificial insemination (AI), day 18 controls with high serum progesterone levels (CPH) and day 18 controls with low serum progesterone levels (CPL). RNA was isolated from endometrial samples of the ipsilateral caudal uterine horns and analyzed with the Affymetrix Bovine Genome Array and a custom-made Bovine oviduct epithelium and endometrium (BOE) cDNA array. The BOE array consists of approximately 1000 mRNAs, which were identified as regulated in bovine endometrium or oviduct epithelium during the estrous cycle or during early pregnancy in previous studies. The normalized data (BioConductor packages plier for Affymetrix and vsn for BOE array) were analyzed with SAM (Significance Analysis of Microarrays) to detect significant changes of mRNA levels between groups (false discovery rate 1%, fold change at least twofold). The annotation of the significantly changing transcripts derived from the Bovine Genome Array was supplemented based on the current UniGene build #79, Ensembl gene IDs, and BLAST analyses. In comparison of the groups AI vs. CPH 460 mRNAs, AI vs. CPL 1340 mRNAs, and CPH vs. CPL 1370 mRNAs were obtained that met the significance threshold. The identified genes were assigned to Gene Ontology categories and to molecular pathways for each comparison. Most specific categories and pathways were investigated in more detail (e.g. using literature-based networks) to find molecular functions that are specifically regulated by progesterone or by embryonic signaling.

Bovine Seminal Plasma Proteins PDC-109, BSP-A3, and BSP-30-kDa Share Functional Roles in Storing Sperm in the Oviduct1

On ejaculation, sperm become coated with proteins secreted by the male accessory sex glands. In the bull, these proteins consist predominantly of the bovine seminal plasma family of proteins (BSPs): PDC-109 (BSP-A1/-A2), BSP-A3, and BSP-30-kDa. PDC-109 plays a role in forming an oviductal sperm reservoir by enabling sperm to bind to oviductal epithelium. Because PDC-109 has high sequence identity with the other BSPs, we tested BSP-A3 and BSP-30-kDa for the capacity to bind sperm to oviductal epithelium. BSP-A3 and BSP-30-kDa each increased binding of epididymal sperm to epithelium and were as effective as PDC-109 in competitively inhibiting binding of ejaculated sperm. Because binding extends the motile life of sperm, BSPs were tested for the ability to maintain sperm motility. BSP-treated epididymal sperm incubated with plasma membrane vesicles from bovine oviductal epithelium maintained progressive motility longer than untreated sperm. To our knowledge, this is the first report of this protective effect of BSPs. Similarities in function among the BSPs were reflected in their three-dimensional structure, whereas surface maps of electrostatic potential indicated differences in binding affinities and kinetics. Such differences may provide sperm with greater adaptability to variations among females. Altogether, these results indicate that BSPs play a crucial role in fertilization by maintaining sperm motility during storage.

Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct

In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher ( P = 0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5 ± 10.49 mg/L in ampullar ODF, compared to 43.2 ± 10.74 mg/L in isthmic ODF); least square mean (L.S.M.) ± standard error of the mean (S.E.M.). There was also a significantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5 ± 10.54 mg/L on the ovulation side, compared to 43.1 ± 10.71 mg/L in the oviduct on the contralateral side (L.S.M. ± S.E.M., P = 0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes, while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo.

Detection of the Hyaluronan Receptor CD44 in the Bovine Oviductal Epithelium

Hyaluronan is involved in fundamental reproductive events such as sperm storage in the female reproductive tract, fertilization, and early embryo development, these functions are presumably mediated by its major cell surface receptor, CD44. The present study was conducted to investigate the presence and localization of CD44 in the bovine oviductal epithelium, using immunohistochemical and Western blot methods on tissue sections and epithelial cell extracts collected from the uterotubal junction (UTJ), isthmus, and ampulla of animals in the oestrus or luteal phase of the oestrous cycle. While positive immunolabelling for CD44 was found on the ad-luminal surface and supra-nuclear region of epithelial cells in all tubal segments investigated, in the UTJ, there were epithelial cells in which the entire cytoplasm positively stained. We found no differences in terms of CD44-positive staining between the different stages of the oestrous cycle. Presence of CD44 was detected by Western blotting in the tubal epithelium as a single band at 200 kDa. Although it appeared in all tubal segments, the expression of CD44 protein was more accentuated in the sperm reservoir (UTJ) than in the other segments. This is the first time CD44 has been detected in the epithelium of the tubal sperm reservoir in cattle, suggesting a pathway for the action of hyaluronan in this segment.