Demonstration of uterine receptivity in vitro by co-culture of rat epithelial cells and blastocyst

Uterine receptivity is prerequisite for the attachment of the embryo to the uterine epithelium and involves a specialized polarity-dependent property of uterine epithelial (UE) cells. These UE cells, when polarized in culture, behave like cells in utero by exhibiting apico-basal polarity. In order to develop an implantation model in vitro, UE cells were polarized on extracellular matrix (ECM), and polarity was validated by response to estradiol-17β administered exogenously. UE cells of pregnant rats at day-3 and day-4 post-coitum (p.c.) and of non-pregnant rats were cultured on bare and extracellular-matrix-coated petri dishes until confluency. Hatched blastocysts were transferred to the cultures, and adhesion was monitored every 24 h. Although blastocysts attached to UE cells that were taken from non-pregnant rats and from rats of day-3 p.c. and cultured on bare plastic, they failed to attach to these cells polarized on ECM. However, blastocysts attached firmly to UE cells that had been taken from rats of day-4 p.c. and polarized on ECM. Receptivity of UE cells taken from non-pregnant and pregnant (day-4 p.c.) rats was quantitated by flow cytometric estimation of cellular levels of β3 integrin. The expression of β3 integrin in UE cells from rats of day-4 p.c. was highly significant (P<0.01) when compared with its expression in UE cells from non-pregnant rats. The expression of β3 integrin in UE cells of day-4 p.c. confirmed the receptivity of these cells to blastocyst implantation. Uterine receptivity was also validated in vivo by inducing the decidual cell reaction in rats ovariectomized on day-3 and day-4 p.c. Whereas remarkable deciduoma was noticed in the animals of day-4 p.c., it was absent in the animals of day-3 p.c., thereby indicating that the uterus was receptive on day-4 p.c. only. Thus, blastocysts do not attach to polarized UE cells that have been obtained from a non-receptive uterus. Attachment will occur only if the cells are obtained from a receptive uterus. UE cell receptivity is therefore essential for mimicking the process of implantation in vitro.The authors are grateful to the Ministry of Health and Family welfare, Government of India, for financial support     

Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization

This study was designed to determine the effect of co-culture with porcine oviductal epithelial cell (POEC) monolayers on in vitro fertilization of pig oocytes. The in vitro penetrability of mature (experiment 1) or immature (experiment 2) oocytes was studied in presence or absence of POEC during IVF with fresh semen. In experiment 3, boar and POEC effects were analyzed but in this case with frozen-thawed spermatozoa. In experiment 4, the spermatozoa were pre-incubated before IVF with or without POEC in order to assess their effect on IVF sperm-related parameters. In experiment 5, the effect of POEC was studied by co-culturing them with oocytes before IVF to determine if monospermy was improved. The results showed that high sperm concentration and POEC increase oocyte penetrability (P<0.01) and decrease monospermy rate (P<0.01), in both mature and immature oocytes (P<0.01) with fresh semen and a 18 h culture time. With frozen semen was detected a boar and POEC effect (P<0.01) on penetration rate. The sperm pre-culture 2 h with POEC also resulted in an increase of sperm penetration in terms of number of sperm per oocyte (P<0.01) and this treatment did not increase monospermy when contact time between gametes was limited to 6 h although monospermy was higher when POEC were present during IVF. Finally, exposure of oocytes to POEC for 4 h before IVF facilitated monospermic penetration to over 70% (P<0.01). In conclusion, the use of POEC in porcine IVF systems provides the possibility of working with low sperm concentrations and the effect of POEC on monospermy depends on sperm concentration, boar and contact time between gametes. Moreover, the exposure of oocytes to POEC before IVF improves the rate of monospermy.     

Alterations in microvilli of uterine epithelial cells after colchicine treatment

Colchicine has been injected intra-peritoneally to pregnant rats to determine the effect of this drug on the microvilli of uterine luminal epithelial cells. Microvilli in control animals (saline injected) were 0.51 +/- 0.07 micron tall, whereas in colchicine treated they were only 0.24 +/- 0.02 micron tall. We conclude that colchicine reduces microvillar height in these cells via interaction with microtubules, and suggest that modulations of microtubules probably contribute to the alterations in the microvilli of these cells seen during normal pregnancy.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.     

Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells

To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep. After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4.5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable. We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of a mouse primary co-culture of endometrial epithelial cells and peripheral blood leukocytes: effect on epithelial barrier function and leukocyte survival

This study aimed to establish an in vitro co-culture model that would allow us to study the interaction between endometrial epithelial cells and immune cells. Flow cytometry analysis and cell surface marker staining were used to identify suitable immune leukocytes from a range of sources, such as intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes. Optimizing culture conditions such as cell viabilities, cell seeding ratios and densities and co-culture methods were examined and determined. Results showed that co-culture of mouse endometrial epithelial cells (EEC) with peripheral blood leukocytes (PBL) at seeding densities of 3.0 x 10(6) and 1.0 x 10(6)cells/ml, respectively, appeared to affect both the survival of leukocytes and epithelial barrier function. Cell viability counts of immune cells showed 95% and 72.5% cell survival after isolation and after 4 days in co-culture with EEC, respectively, but only 11% cell survival when cultured alone for 4 days without EEC. Short-circuit current (I(sc)) results also showed that EEC and PBL co-culture exhibited a four-fold increase in the transepithelial resistance (TER) as compared to EEC culture alone, indicating enhanced protective barrier function. Taken together, the currently established in vitro co-culture model of endometrial epithelial cells and immune cells may provide a means to investigate local cellular immune responses upon uterine infections.     

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.     

Indirect co-culture with tendons or tenocytes can program amniotic epithelial cells towards stepwise tenogenic differentiation

Background

Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue.

Methodology/Principal Findings

Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43).

Conclusions/Significance

The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or explants. The co-cultural system can be proposed as a low cost and easy technique to engineer tendon for biological study and cell therapy approach.     

Colloidal iron hydroxide staining of surface carbohydrates after glycerol treatment of uterine epithelial cells

The structural relationships between intramembraneous particles (IMPs) and surface carbohydrates has been studied in uterine epithelial cells with a colloidal iron hydroxide (CIH) technique. To aggregate IMPs, glycerol treatment of unfixed cells was used and this treatment also caused some patching of CIH deposits on the cell surface. We conclude that some of the CIH receptors may be the surface expression of the IMPs.     

Metabolic interactions of parenchymal hepatocytes and dividing epithelial cells in co-culture.

Previous work has led to the identification of a novel class of effector molecules [DIFs (differentiation-inducing factors) 1-3] released from the slime mould Dictyostelium discoideum. These substances induce stalk-cell differentiation in Dictyostelium discoideum and are thought to act as morphogens in the generation of the prestalk/prespore pattern during development. The DIFs are phenylalkan-1-ones, with chloro, hydroxy and methoxy substitution on the benzene ring. DIFs 1-3 and a number of their analogues have been synthesized by using a simple two-step procedure, and each analogue has been characterized by m.s., u.v. and n.m.r. spectroscopy. The crystal structure of synthetic DIF-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one, was investigated. The specific biological activity of each analogue was determined in a bioassay, where isolated Dictyostelium amoebae are induced to differentiate into stalk cells. The major biologically active substance, DIF-1, caused 50% stalk-cell differentiation at 1.8 x 10(-10) M; the C4 alkyl homologue (DIF-2) and C6 homologue possessed 40 and 16% of the activity of DIF-1 respectively. Further increase or decrease in the alkyl chain length resulted in a marked decrease in specific activity. The pattern of substitution on the benzene ring is a major determinant of bioactivity, since the specific activities of the 2,4-dihydroxy-6-methoxy and trihydroxy analogues were less than 1% of that of DIF-1. Substitution of bromine in DIF-1 had little effect on bioactivity; in contrast the activity of monochloro-DIF-1 (DIF-3) was diminished. There was no evidence for antagonism or synergy between DIF-1 and any of its analogues. This series of analogues will facilitate further studies in the biological effects and mode of action of DIF-1.     

Colloidal iron hydroxide staining of surface carbohydrates after glycerol treatment of uterine epithelial cells

Summary The structural relationships between intramembranous particles (IMPs) and surface carbohydrates has been studied in uterine epithelial cells with a colloidal iron hydroxide (CIH) technique. To aggregate IMPs, glycerol treatment of unfixed cells was used and this treatment also caused some patching of CIH deposits on the cell surface. We conclude that some of the CIH receptors may be the surface expresion of the IMPs.     

Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture

The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.     

Rabbit sperm cryopreservation: a review

Sperm cryopreservation is a great challenge, since many sperm are irreversibly damaged or present altered functionality after the whole process. Although components of extenders for sperm cryopreservation are quite similar between species, sperm from each of the species present peculiarities that force researchers to optimize the extenders and protocols for each particular species. In this review, information related to rabbit sperm cryopreservation is compiled. The topics discussed include the extenders and protocols developed for rabbit sperm cryopreservation, as well as fertility data obtained after artificial insemination with cryopreserved sperm and factors that may have an impact on the results obtained. In addition, suggestions for improving the results after cryopreservation of rabbit sperm are also proposed.     

Survival of early preimplantation porcine embryos after co-culture with cells producing an avian retrovirus

To determine the relative survival of porcine embryos after co-culture with cells producing an avian retrovirus, four-cell stage embryos were obtained from sows following synchronization with altrenogest and superovulation with gonadotropins. These embryos were randomly assigned to the following treatments: no manipulation (zona-intact); zona removed with acidified Tyrode's solution (zona-free); and zona removed followed by co-culture with D-17 canine cells producing an avian retrovirus vector derived from spleen necrosis virus (zona-free + co-culture). The survival rates of four-cell stage embryos to morulae or early blastocysts during a 48-h culture period were 93.3, 80.0 and 57.7% in zona-intact, zona-free and zona-free + co-culture groups, respectively. Following embryo transfer, the development of embryos to fetuses at six weeks of gestation was 37.5, 30.0 and 11.7% in zona-intact, zona-free and zona-free + co-culture groups. These results indicate that early preimplantation porcine embryos can develop to apparently normal fetuses following co-culture with cells producing a retrovirus, and the feasibility of this method for retrovirus-mediated gene transfer in pigs was demonstrated.     

Progesterone receptor repression by estrogens in rat uterine epithelial cells

Measurements performed using cell lines or animal tissues have shown that the progesterone receptor (PR) can be induced by estrogens. By use of immunohistochemistry we studied the effects of estrogens on the PR levels in the individual cell types of the target organs uterus and breast. In the uteri of rats, ovariectomy induced a decrease in PR immunoreactivity within the myometrium and outer stromal cell layers. In contrast, in the uterine luminal and glandular epithelium and surrounding stromal cell layers the PR immunoreactivity was significantly enhanced. The same picture emerged when intact rats were treated with the pure estrogen receptor antagonist, ZM 182780 (10 mg/kg/d). Treatment of ovariectomized rats with estradiol resulted in high PR levels in the myometrium and stroma cells but low PR immunoreactivity in the epithelial cells. The ER-mediated repression of the PR immunoreactivity was evidently restricted to the uterine epithelium, as we found that in the epithelial cells of the mammary gland and in cells of N-nitrosomethylurea-induced mammary carcinomas the PR expression was induced by estrogens and was blocked by the pure antiestrogen ZM 182780. These results clearly show that in the rat the activated ER induces diverging effects on PR expression in different cell types even within the same organ.     

Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells

We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate- containing molecules (HS[PG]) were the major sulfated products synthesized and secreted by these cells. The ability of epithelial cells to secrete KSPG greatly increased in parallel with the development of cell polarity. Furthermore, KSPG secretion occurred preferentially to the apical medium in highly polarized cultures. In contrast, HS(PG) secretion did not increase along with development of polarity, although most HS(PG) (85%) were secreted apically as well. Pulse-chase studies indicated that highly polarized cultures secreted 80-90% of the sulfated macromolecules they synthesized, predominantly to the apical secretory compartment. The half-lives for KSPG and HS(PG) secretion were approximately 3 and 4 h, respectively. Parallel studies of cells cultured on tissue culture plastic-coated with biomatrix indicated that neither the state of confluency nor the biomatrix was primarily responsible for inducing the KSPG secretion observed in polarizing cultures. Experiments with uterine strips indicated that the steroid hormone, 17-beta-estradiol, markedly stimulated synthesis and secretion of sulfated macromolecules, but had no preferential effect on KSPG production. The ratio of KSPG to HS(PG) secretion from uterine strips was similar to that found in the apical medium of highly polarized cell cultures. Thus, the pattern of proteoglycan secretion observed in polarized cell cultures mimicked that observed for uterine cells, although the preferential increase in KSPG production by polarized cells could not be attributed to an estrogen response. Collectively, these studies describe the major sulfated molecules secreted by rat uterine epithelial cells under varying conditions and provide evidence for a novel influence of cell polarity on the cell's ability to secrete sulfated glycoconjugates.     

微流控芯片技术用于精子与上皮细胞分离的研究

为研究建立使用微流控芯片技术分离精子与阴道上皮细胞的方法,选用制作工艺简单的玻璃-PDMS芯片,对混合样本进行分离。加样前分别在进、出口池中加入7此和lO此缓冲液,然后在进样口加入2此混合样本。至少静置8nlin后,从出口池中取出3此形成重力驱动的微流体后开始分离,每隔5min在进样口补加1此缓冲液。达到理想分离效果时,用移液器从出口池取出分离出的精子,核酸酶去除游离DNA,经过提取、扩增和电泳分离脸测等步骤得到精子的分型结果。结果显示,使用基于重力驱动微流体原理的微流控芯片可在30min内分离出精子,不会有上皮细胞进入分离通道;通过核酸酶对分离出的精子液的去游DNA处理,可得到单一、完整的精子分型。与传统的差异裂解法相比,这种方法在很大程度上节省了检验时间,在性侵案件中具有一定的法医物证分析价值。     

Enhanced neural differentiation of neural stem cells and neurite growth by amniotic epithelial cell co-culture

Amniotic epithelial cells (AECs) were reported to show a neuroprotective effect on neurons, but there was no direct evidence for a functional relationship between neural stem cells (NSCs) and AECs. The aim of this study was to determine whether AECs could stimulate differentiation and expand neurogenesis of NSCs, and whether the roles were due to a diffusible factor or required direct cell-cell contact. AECs were isolated from rat amnion on E14-16 and NSCs were isolated from neocortical tissue. The growth and differentiation of NSCs were compared under different conditions. The results showed that NSCs cultured with FGF-2 proliferated and formed floating neurospheres while those grown in B27 without FGF-2 failed to thrive. Those grown either with AEC conditioned medium or in transwells showed significantly improved survival. Moreover, the neural differentiation and length of neurite were greater in exogenous FGF groups when NSCs were allowed direct contact with AECs. Western blotting, immunocytochemistry and RT-PCR indicated that rat AECs could secrete NT-3 and BDNF. Furthermore, the presence of FGF-2 enhanced the function of AECs. These findings identified that AECs may be regarded as a critical component of NSCs niche and suggested that direct cell-to-cell contact may provide additional and independent support. Such information would circumvent the need for AECs-NSCs co-culture and could potentially facilitate the production of neurons for future clinical applications.