Catecholamines within the rabbit oviduct at fertilization time

Dopamine, norepinephrine and epinephrine levels of rabbit oviductalfluids were measured in order to establish whether the ampullarysegment contained higher concentrations correlating with thesperm hyperactivity observed in the ampulla of different mammals.In this study, we found no statistically significant differencesbetween flushings from the ampulla or the isthmus at oestrus,ovulation or 72 h postcoitum (p.c). However, after correctingfor the volume secreted, the concentrations of catecholaminesare always lower in the ampulla than in the isthmus. In allcases the levels of biogenk amines were highest at oestrus thenfell at ovulation. Individual variation of these levels at thetime of ovulation was observed only in the ampulla, probablydue to some contribution from follkular fluid. During the earlyluteal phase (72 h p.c.) the catecholamine levels again increaseslightly, especially in the isthmus. We conclude that whiplashmotility cannot be explained by a higher level of catecholaminesin the ampulla.     

Andrology: Hypotaurine in spermatozoa and genital secretions and its production by oviduct epithelial cells in vitro

Taurine and/or hypotaurine are necessary compounds for spermcapacitation, fertilization and embryo development. Hypotaurinehas a protective role against peroxidative damage. The objectof this work was, on the one hand, to determine the preciseamounts of hypotaurine and taurine in the sperm environmentat the moment of fertilization, and on the other hand to evaluatethe production of hypotaurine and taurine by oviduct epithelialcells. Hypotaurine and taurine were quantified in spermatozoaand seminal and tubal fluid of various species, and in secretionsby oviduct epithelial cell layers in vitro. Significant amountsof taurine and hypotaurine were identified. Both compounds werequantified in pre-ovulatory follicular fluid, i.e. in one ofthe fluids present at the site of fertilization. We also observedthat hypotaurine and taurine are synthesized and secreted invitro by oviduct epithelial cells. We were able to demonstratethat hypotaurine is stable when added to an in-vitro fertilization(IVF) culture medium. The effects of this compound should bemore carefully studied in human IVF.     

小鼠植入前胚胎对输卵管上皮细胞DNA甲基转移酶1表达的影响

目的 探讨小鼠植入前胚胎对输卵管上皮细胞DNA甲基转移酶1（DNA methyltransferase 1，Dnmt1）表达的影响。方法 应用免疫组织化学方法确定Dnmt1蛋白在小鼠输卵管的组织学定位；选取植入前胚胎的2细胞期、4细胞期和8细胞期作为3个观察点，采用实时逆转录-聚合酶链反应和Western印迹分别检测正常妊娠和假孕小鼠输卵管上皮Dnmt1 mRNA和蛋白表达水平的变化。结果 Dnmt1蛋白主要表达于上皮层；妊娠组小鼠各期Dnmt1 mRNA表达水平均显著性低于同期假孕组（P〈0．05），妊娠组4细胞期Dnmt1蛋白表达水平显著性低于同期假孕组（P〈0．05）。结论 小鼠植入前期的胚胎对母体输卵管上皮Dnmt1 mRNA和蛋白表达存在调节作用，提示胚胎可能通过DNA甲基化间接调控输卵管上皮某些基因的表达，Dnmt1可能参与胚胎-母体交流的部分机制。     

Culture of bovine oviduct epithelial cells (BOEC)

Background: Bovine oviduct epithelial cells are widely used in co-culture experiments to improve early embryonic development and in vitro fertilization in embryo transfer programmes for domestic animals. Methods: The present study compares different methods for harvesting and culture of bovine oviduct epithelial cells in order to optimize handling. Bovine oviduct epithelial cells were mechanically or enzymatically isolated and cultured on glass, on permeable membranes, or in suspension. Growth of the cells and their state of differentiation was examined by means of classical staining methods, immunohistochemistry and electron microscopy. Results: Initial cell suspensions contained sheets of ciliated and nonciliated (secretory) cells; 24 h after seeding, free floating epithelial cells formed vesicles with cilia on their external surface. First adhesion of cells was seen 72 h after seeding. Later on, cells grew continuously and confluent monolayers were formed after 7 days. Results were identical after mechanical or enzymatical cell harvesting and were identical on both substrata tested, i.e., on glass and on permeable membranes. Light and electron microscopy proved the monolayers to resemble a polarized, simple, cuboidal to columnar epithelial membrane with intact junctional complexes and numerous apical microvilli. Their epithelial nature was established by immunostaining for cytokeratins. Cilia were missing and secretory granules were scarce. A layer of acidic glycoprotein material was demonstrated on the apical surface. Monolayers of bovine oviduct epithelial cells stored lipid droplets and large quantities of glycogen. About 50% of the seeded cells did not adhere but survived in the culture medium as free floating cells. These suspended cells maintained morphological criteria of differentiation (cilia and secretory granules) until day 12 of culture. Proliferation rates of cultivated cells were determined by counting mitoses and by immunostaining with MIB1 antibody. Results showed coincidence of rapid proliferation and morphological dedifferentiation of monolayers. Suspended cells, by contrast, did not proliferate but retained cellular differentiation under identical culture conditions. Conclusions: The results strongly suggest that monolayers of bovine oviduct epithelial cells will not fully substitute for original oviduct epithelium when used in co-culture experiments after in vitro fertilization. © 1995 Wiley-Liss, Inc.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Fetal bovine oviduct epithelial cell monolayers: Method of culture and identification

Summary Bovine epithelial cell monolayers were obtained for culture from fetal oviduct after in situ trypsinization. Isolated ciliated and secretory cells obtained in high yield with good viability were suspended in B2-MENEZO'S medium supplemented with 7.5% fetal bovine serum FBS. The plated primary cultures reached confluency 2 days after initial seeding, producing a monolayer of cohesive polygonal cells with viability of 85 to 95%. Associated with this large epithelial cell population, ciliated cells as well as a few elongated spindle cells were observed. After the first subculture the ciliated cells disappeared and the epithelial cells in the monolayer grew more rapidly to confluence than adult-derived cultures. In addition, frozen-thawed oviduct epithelial cells also maintained a level of 75 to 85% viability during postthaw subculture. The epithelial cells maintained their secretory activity in culture as indicated by electron microscopy and immunocytochemistry. The cell culture monolayers contained keratin, a specific cytoskeletal component of epithelial cells. This culture system may offer benefits for in vitro culture of mammalian embryos. This research was supported by the Mérieux Foundation.     

In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells.

Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fertilization and early embryology: Comparison of pyruvate uptake by embryos derived from conception and non-conception natural cycles

The uptake of pyruvate by human embryos derived from naturalcycles in the first 24 h following fertilization was examined.Since only one egg was obtained and therefore only one embryotransferred to the woman, it was possible to relate pyruvateconsumption by a particular embryo to the outcome of that cycle(pregnancy or no pregnancy). The results showed that embryoshave a wide range of pyruvate uptake values (2–53 pmol/embryo/h)but that this variation was reduced significantly to an intermediaterange of values in those embryos that were able to implant (10–30pmol/embryo/h). An association was found between embryo morphologyand pyruvate consumption. Morphologically good embryos weremore likely to implant if they demonstrated an intermediatepyruvate uptake. However, poor embryos did not implant evenif they had a pyruvate uptake of 10–30 pmol/embryo/h.No relationship was found between the type of infertility andpyruvate consumption of individual embryos. It is suggestedthat the ability of an embryo to implant is multifactorial andthat both morphology and pyruvate uptake may be factors.     

Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells

A study was undertaken to evaluate embryonic development andestablish pregnancies with human embryos after in-vitro culturein two different systems. Treatment A consisted of culturingzygotes in serum-supplemented human tubal fluid culture medium(HTF). Treatment B consisted of culturing zygotes on a monolayerof bovine oviductal epithelial cells with HTF. At the time ofembryo replacement, embryos in treatment B had 4.11 blastomerespresent, which was greater (P < 0.05) than the 3.81 presentfor embryos in treatment A. In addition, the cellular fragmentationrate for treatment A embryos was 1.10, which was greater (P< 0.05) than the fragmentation rate of 0.38 for embryos withintreatment B. The incidence of ongoing pregnancy was higher afterreplacement of co-cultured embryos (treatment B) (43%) thanreplacement of conventionally cultured embryos (treatment A)(29%). The implantation rate per embryo increased (P < 0.05)from 11.5 to 18.4% after co-culture. In treatment B the proportionof ‘spare’ embryos developing to expanded blastocystswas 58.5%, which was greater (P < 0.05) than the blastocystdevelopment rate of 29.3% observed for embryos within treatmentA.     

Retinoids during the in vitro transition from bovine morula to blastocyst

BACKGROUND: The conversion of retinol (ROH) to retinoic acid (RA) is crucial during development but has been not studied during blastocyst formation. METHODS AND RESULTS: In vitro-produced bovine morulae were treated for 24 h with citral (which inhibits the synthesis of RA from ROH), citral + all trans retinoic acid (ATRA), ATRA or no additives. Citral interfered with blastocyst development, whereas exogenous RA had no effect. RA, however, reversed the effect of citral on development and stimulated cell proliferation. Neither citral nor RA changed the apoptotic index, but RA triggered an increase in the apoptotic frequency of the inner cell mass. Citral and RA reduced the necrotic index. Na/K-ATPase alpha1-subunit mRNA concentrations (analysed by real-time PCR) increased after hatching and showed dependence on retinoid activity, but no evidence was found of any retinoid effect on p53 expression. Nevertheless, the p53 mRNA concentration increased in response to proliferation in hatched blastocysts. CONCLUSION: The preimplantation bovine embryo metabolizes endogenous ROH to RA, which participates in important cell processes. The true extent of the influence of RA is unknown, although the modulation of retinoid metabolism seems to be a means of increasing cell proliferation. This knowledge might be used to improve embryo quality and the efficiency of stem cell derivation.     

Physiology: Morphological characteristics of human endometrial epithelial cells cultured on rat-tail collagen matrix

Epithelial cells were isolated from normal human endometriumand cultured on reconstituted matrices of rat-tail collagengels. Cells attached within 24 h after plating. Initially, epithelialcells were not structurally polarized, had projections fromboth apical and basal domains and were generally flat to cuboidalin shape. When the gels were made free floating 36–48h after initiation of the cultures, the epithelial cells becamecolumnar in shape as the gels underwent contraction. Althougha maintained growth profile was observed during the 10 daysof culture, there was a linear decrease in gel matrix diameteralong with increasing loss of transparency. Electron microscopyrevealed the presence of apical microvilli, junctional complexes,lipid droplets and endoplasmic reticulum in cultured epithelialcells. The basolateral dilatations and lateral membrane plicationswere seen in these cells by 6–8 days in culture. Withgel contraction, rearrangement of matrix material was observed.Occasionally there were basal lamina-like structures adjacentto the flattened basal surface and the formation of gland-likestructures within the matrix. The three-dimensional primaryculture of human epithelial cells on collagenous biomatrix appearsto be a potential experimental tool for the study of cell-celland cell-matrix interactions, and of the study of the effectsof endocrine and paracrine factors on these cells in vitro.     

Establishment of a ciliated epithelial cell line from human Fallopian tube

Human tubal epithelial cells in primary culture were transfected with simian virus 40 (SV40) large T antigen plasmid, and an immortalized ciliated cell line, named as NT/T-S, was established without crisis. Transmission electron microscopy proved that NT/T-S cells had cilia, microvilli, junctional complexes, rough endoplasmic reticula, free ribosomes and microtubules. NT/T-S cells were evaluated preliminarily on the basis of co-culture study using surplus embryos at the 4- to 8-cell stage in our IVF and embryo transfer programme. All of the 133 embryos had >/=10% fragments (based on the surface area) and were unworthy of cryopreservation. Up to 57% (16/28) of the embryos with 10-30% fragments reached the blastocyst stage by co-culture. In contrast, blastocyst formation was observed in <10% of the control embryos, some of which were co-cultured with NFL/T cells (the immortalized human fetal liver epithelial cells) (1/16), and the others were incubated with the co-culture medium alone (1/18). Various cytokines/growth factors such as leukaemia inhibitory factor (LIF), interleukin (IL)-6, IL-8 and basic fibroblast growth factor were secreted by NT/T-S cells as well as by the tubal epithelial cells in primary culture. The establishment of a ciliated cell line will provide a valuable resource for the further studies of the Fallopian tube in the early events of pregnancy.     

Effects of stage of the bovine oestrous cycle on in-vitro characteristics of uterine and oviductal epithelial cells.

The objective of this experiment was to evaluate the effects of stage of the bovine oestrous cycle on in-vitro morphology, growth and monolayer foundation of uterine and oviductal epithelial cells. Epithelial cells were isolated from the uterus and oviducts collected from cyclic cattle on the day of oestrus (Treatment A), and between days 4 to 6 (Treatment B), days 8 to 10 (Treatment C) and days 14 to 16 (Treatment D) of the oestrous cycle. The morphological development, per cent cell viability and cell attachment were evaluated during primary culture and after the first and third subpassages. The highest per cent cell viability and cell attachment during primary culture, respectively, were noted in Treatment B for both uterine (87.7 and 87.5%) and oviductal (88.4 and 87.2%) cell populations. Uterine epithelial cell populations in Treatments C and D, respectively, had the lowest viability (76.5 and 68.8%) and attachment (10.8 and 10.5%) during primary culture. There were marked improvements in cell viability and cell attachment following the first subpassage (P less than 0.001) compared with primary cultures for both uterine and oviductal cells. These results indicate that the stage of the oestrous cycle has dramatic effects on uterine and oviductal epithelial cell morphology and developmental patterns during primary in-vitro cultures. The stage of the oestrous cycle when cells are collected may be more important than was once realized when culturing early stage embryos in vitro.     

Uterus and endometrium: Study of the intramural oviduct response to tubal and uterine distension: identification of tubo-uterine sphincter and reflex

The effect of tubal and uterine distension on the intramuralportion of the oviduct (IMO) was studied in 11 women (mean age31.1 years). The IMO length was determined and the pressureresponse of the IMO to distension of the anaesthetized oviductand uterus was examined. A 1.2 cm long high pressure zone (32.2± 6.9 cm H₂O) was identified in the IMO. An IMO pressuredrop occurred upon oviduct distension with 2 ml of CO₂ (P <0.01); distension with greater volumes induced the same pressuredrop (not significant). The IMO pressure increased with 10 mluterine distension (P < 0.05); distension with greater volumesdid not induce further pressure rises (not significant). Distensionof each of the anaesthetized oviduct, uterus and IMO separatelyeffected no IMO pressure response. A ‘tubo-uterine’physiological sphincter is postulated at the high pressure zoneof the IMO. It relaxes or contracts upon tubal or uterine distensionrespectively. The sphincter response to tubal or uterine distensionis suggested to occur by a reflex action through a ‘tubo-uterine’reflex, as shown by reproducible results and sphincteric non-responseupon anaesthetization of the two arms of the reflex.     

Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos

BACKGROUND: Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish. METHODS: During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters. RESULTS: Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability. CONCLUSIONS: The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop.     

Fertilization and early embryology: Differential effect of epithelial cell-conditioned medium fractions on preimplantation mouse embryo development

Coculture studies using preimplantation embryos have led toa number of conflicting studies. In the human, ethical considerationshave led to the preferential use of epithelial cell lines asdistinct from human Fallopian tube cells. In an attempt to isolatefactors influencing embryo development we have cultured 2-cellOF1 mouse embryos in media [Ménézo's B2 and Whittingham'sT6 supplemented with vitamins and amino acids (T6VA)] conditionedon two types of kidney epithelial cells (MBDK and Vero). Differentmolecular weight fractions of conditioned medium were used toshow the absence or presence of specific embryotrophic factors.With MDBK cells, B2 conditioned medium enhanced embryo developmentup to the blastocyst stage, while no blastocysts developed inB2 alone. When using T6VA medium, both the control and conditionedmedia showed a high percentage of blastocyst formation (57.0and 54.0% respectively), while the different molecular weightfractions showed no added improvement. With Vero cells, B2 alone,B2 conditioned medium and fractions were all detrimental toembryo development. A high percentage of blastocyst formation(between 64.7 and 75.8%) was observed in T6VA alone, T6VA conditionedmedium and fractions. Low blastocyst formation in a controlmedium can show strong positive results when medium is conditionedby cells. In contrast, a good base medium, such as T6VA, canequal the results using conditioned medium. Different cellsin contact with different types of medium show variability inthe pattern of responses, highlighting the presence of falsepositives in coculture studies.     

Composition and morphology of lipid droplets from oviduct epithelial cells

This study was undertaken to determine the composition and morphology of lipid droplets in situ and isolated from oviductal epithelial cells and oviductal fluid. Oviductal epithelial cells were harvested enzymatically from oviducts of cows in either the luteal or the follicular stages of the ovarian cycle. Lipid droplets were isolated from cellular homogenates and characterized biochemically using thin layer chromatography. The morphology of lipid droplets in oviductal epithelial cells and in fractions isolated by ultracentrifugation from cellular homogenates was examined by electron microscopy. Lipid droplets isolated from oviduct epithelial cells varied in composition with the ovarian cycle and the oviductal region. There was more total lipid in droplets isolated from cows in the luteal than follicular phase of the ovarian cycle. Most of this difference was due to large amounts of esterified cholesterol present in the samples from luteal-stage animals. The most esterified cholesterol was found in droplets isolated from the oviductal isthmus of luteal cows. Droplets similar in lipid composition to those isolated from epithelial cells were found in oviductal fluid. Four distinct types of lipid inclusions were evident in electron micrographs of oviductal epithelia and characterized as osmiophilic droplets, lipofuscin-like clusters, lamellar structures, and composite bodies. All of the lipid inclusions were found in droplet isolates except for the extracted lipid portion of the composite body. The presence and diversity of oviduct epithelial lipid inclusions suggest that the oviductal epithelium may be very active in lipid metabolism, particularly cholesterol dynamics. © 1993 Wiley-Liss, Inc.     

Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production

The effects of water-soluble vitamins, singly or in combinations, on development of hamster 1-cell embryos were examined in a protein-free, chemically defined culture medium, HECM-6. Pantothenate significantly stimulated blastocyst development compared to the vitamin-free control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. When combinations of vitamins were tested, embryo development was either unchanged or significantly greater than in the control, but never significantly greater than development with pantothenate alone. A dose response to pantothenate indicated that 3 micromol/l was the optimum concentration. After embryo transfer, the percentage of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate (now designated HECM-9) was 24%, significantly higher than the 11% recovered from 100 1-cell embryos cultured in HECM-6 alone. This is the first report to show a stimulatory effect of a single vitamin on in-vitro development of preimplantation embryos in any mammalian species.     

Embryonic platelet-activating factor: an indicator of embryo viability

BACKGROUND: A definitive need exists to identify a biomarker of embryonic viability. Platelet-activating factor (PAF) production by human embryos is related to pregnancy potential. METHODS: Conditioned embryo culture media were obtained following conventional IVF on day 3, with PAF levels and pregnancy outcomes correlated. RESULTS: Overall pregnancy rate was 68% (17/25) with a mean of 84.1 (+/- 8.5) pmol/l/embryo PAF level. PAF levels ranged from a 216.4 pmol/l/embryo (pregnant) to a 3.7 pmol/l/embryo (not pregnant). There was a significant difference (P < 0.05) in PAF content between pregnant (92.1 +/- 9.5 pmol/l/embryo) and non-pregnant groups (52.5 +/- 16.6 pmol/l/embryo). Patients were categorized into three groups based upon PAF levels: low (< or= 5 pmol/l/embryo); medium (51-100 pmol/l/embryo) and high (>100 pmol/l/embryo). The low (60%) group had a significantly (P < 0.05) lower pregnancy rate than either the medium (85%) or high (89%) groups. A receiver-operator characteristic curve predicted a cut-off limit of 45 pmol/l/embryo for PAF content in human embryo conditioned culture media. CONCLUSIONS: The data demonstrate a correlation between PAF levels in human embryo conditioned culture media and pregnancy outcome. Additionally, as embryonic PAF levels increase so does the corresponding pregnancy rate. Therefore, PAF may be used as an indicator of embryo viability and for predicting pregnancy outcome.