Development of morphological and functional polarity in primary cultures of immature rat uterine epithelial cells

The present study describes a culture environment in which luminal epithelial cells isolated from immature rat uteri and cultured on a matrix-coated permeable surface, with separate apical and basal secretory compartments, proliferate to confluence. Subsequently the cells undergo a process of differentiation accompanied by progressive development of functional polarity. Ultrastructural and immunocytochemical evidence verifies the ability of these primary cultures to regain polar organization, separate membrane domains, and form functional tight junctions as demonstrated by the development of transepithelial resistance. The appearance of uvomorulin is restricted to the lateral cell surface. Coordinated indices of functional polarity that develop progressively in post-confluent cultures include the preferential uptake of [35S]methionine from the basal surface and a rise in uterine epithelial cell secretory activity characterized by a progressive preference for apical secretion. The time dependent development of polarity was characterized by differences in the protein profiles of the apical and basolateral secretory compartments. The maintenance of hormone responsiveness by the cultured cells was validated by the secretion of two proteins identified as secretory markers of estrogen response in the intact uterus. The technique of culturing the cells on a matrix-coated permeable surface with separate secretory compartments produces a uterine epithelial cell that morphologically and functionally resembles its in situ equivalent. The culture method and analytical approach used in this present study may be applied to primary cultures of a variety of natural epithelia, which have hitherto proven resistant to more conventional culture methodologies.     

Access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.     

Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture

Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFbeta) into the basolateral chamber ( approximately 70% > apical) and tumor necrosis factor-alpha (TNFalpha) into the apical compartment ( approximately 30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24-48 h increased epithelial cell TER ( approximately 70% higher than control) and decreased TNFalpha release into both the apical and basolateral chambers ( approximately 30%-50%). In contrast, TGFbeta release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFalpha release persisted for 5-7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNFalpha without effecting TGFbeta release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.     

Surface area of apical and basolateral plasmalemma of epithelial cells of the ductuli efferentes testis of the rat

Serial sectioning was used to determine the occurrence of ciliated cells, and a morphological technique was used to estimate the relative and absolute surface areas of apical and basolateral membrane of the epithelial cells lining the ductuli efferentes of the rat. It was found that the ciliated cells constitute 15% of the epithelial cells and occur as groups of mainly 1–3 cells which are distributed at random in the duct epithelium. For the non-ciliated cells it was estimated that the formation of microvilli by the apical membrane increased the surface area of that border by a factor of 37-fold. The average surface density of the basolateral membrane was 76% the surface density of the apical membrane. However, there was a 3-fold increase in surface density along the apicalbasal axis of the basolateral plasmalemma. In the Discussion, the ductuli efferentes are compared to their homologue, the proximal tubules of the kidney, in the rates of fluid transport and membrane adaptations of their epithelium.     

The immunocytochemical localization of a cat uterine protein that is estrogen dependent (CUPED)

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.     

Oxytocin and vasopressin involved in restraint water-immersion stress mediated by oxytocin receptor and vasopressin 1b receptor in rat brain

Aims

Vasopressin (AVP) and oxytocin (OT) are considered to be related to gastric functions and the regulation of stress response. The present study was to study the role of vasopressinergic and oxytocinergic neurons during the restraint water-immersion stress.

Methods

Ten male Wistar rats were divided into two groups, control and RWIS for 1h. The brain sections were treated with a dual immunohistochemistry of Fos and oxytocin (OT) or vasopressin (AVP) or OT receptor or AVP 1b receptor (V_1bR).

Results

(1) Fos-immunoreactive (Fos-IR) neurons dramatically increased in the hypothalamic paraventricular nucleus (PVN), the supraoptic nucleus (SON), the neucleus of solitary tract (NTS) and motor nucleus of the vagus (DMV) in the RWIS rats; (2) OT-immunoreactive (OT-IR) neurons were mainly observed in the medial magnocellular part of the PVN and the dorsal portion of the SON, while AVP-immunoreactive (AVP-IR) neurons mainly distributed in the magnocellular part of the PVN and the ventral portion of the SON. In the RWIS rats, Fos-IR neurons were indentified in 31% of OT-IR neurons and 40% of AVP-IR neurons in the PVN, while in the SON it represented 28%, 53% respectively; (3) V_1bR-IR and OTR-IR neurons occupied all portions of the NTS and DMV. In the RWIS rats, more than 10% of OTR-IR and V_1bR-IR neurons were activated in the DMV, while lower ratio in the NTS.

Conclusion

RWIS activates both oxytocinergic and vasopressinergic neurons in the PVN and SON, which may project to the NTS or DMV mediating the activity of the neurons by OTR and V_1bR.     

The distribution of dopamine-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

Summary An indirect gold-labeling method utilizing the lectin from Limax flavus was employed to characterize the subcellular distribution of sialic acid in glycoconjugages of the salamander olfactory mucosa. The highest density of lectin binding sites was in secretory vesicles of sustentacular cells. Significantly lower densities of lectin binding sites were found in secretory granules of acinar cells of both Bowman's and respiratory glands. Lectin binding in acinar cells of Bowman's glands was confined primarily to electron-lucent regions and membranes of secretory granules. In the olfactory mucus, the density of lectin binding sites was greater in the region of mucus closest to the nasal cavity than in that closest to the epithelial surface. At the epithelial surface, the density of lectin binding sites associated with olfactory cilia was 2.4-fold greater than that associated with microvilli of sustentacular cells or non-ciliary plasma membranes of olfactory receptor neurons, and 7.9-fold greater than non-microvillar sustentacular cell plasma membranes. Lectin binding sites were primarily associated with the glycocalyx of olfactory receptor cilia. The cilia on cells in the respiratory epithelium contained few lectin binding sites. Thus, sialylated glycoconjugates secreted by sustentacular cells are preferentially localized in the glycocalyx of the cilia of olfactory receptor neurons.     

Actin binding protein expression is altered in uterine luminal epithelium by clomiphene citrate, a synthetic estrogen receptor modulator

Clomiphene citrate (CC), a synthetic oestrogen, is often prescribed as a superovulator in treating infertility. Although CC works efficiently, pregnancy rates following CC treatment are approximately 10 times lower than "natural" rates. This study investigates how a dose of 1.25 mg CC given to ovariectomized rats before the implantation priming hormones (a single dose of progesterone for 3 days and a dose of estradiol-17beta on d3, P-P-PE), alters the expression and distribution of alpha-actinin, gelsolin and vinculin. Actin binding proteins show a specific distribution within the uterine epithelium during implantation, linking the actin cytoskeleton to integrin expression on the uterine surface and in this way aiding "adhesiveness" for blastocyst apposition to the uterine epithelium. In this study, immunocytochemistry on frozen uterine sections using mouse monoclonal antibodies against alpha-actinin, gelsolin and vinculin and peroxidase-conjugated secondary antibodies, show that CC, administered before the P-P-PE regimen, down-regulates the expression of vinculin, does not alter the expression of gelsolin and up-regulates alpha-actinin on the uterine apical surface, when compared to P-P-PE treated animals. All three proteins are down-regulated on the apical surface of the luminal epithelium and glands in all groups when compared to pregnant controls. Vinculin was only localized in the basolateral compartment of the uterine epithelial cells in the CC treated groups. By down-regulating these proteins on the uterine surface and up-regulating vinculin on the basolateral membrane of the epithelium, CC may impede adhesion and invasion of blastocysts at implantation. These results may aid the exogenous manipulation of uterine tissue to control fertility and improve assisted reproductive out-comes.     

Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

The proamphiregulin cytoplasmic domain is required for basolateral sorting, but is not essential for constitutive or stimulus-induced processing in polarized Madin-Darby canine kidney cells

In this study, the role of the amphiregulin precursor (pro-AR) cytoplasmic domain in the basolateral sorting and cell-surface processing of pro-AR in polarized epithelial cells has been investigated using Madin-Darby canine kidney cells stably expressing various human pro-AR forms. Our results demonstrate that newly synthesized wild-type pro-AR (50 kDa) is delivered directly to the basolateral membrane domain with >95% efficiency, where it is sequentially cleaved within the ectodomain to release several soluble amphiregulin (AR) forms. Analyses of a pro-AR cytoplasmic domain truncation mutant (ARTL27) and two pro-AR secretory mutants (ARsec184 and ARsec190) indicated that the pro-AR cytoplasmic domain is not required for efficient delivery to the plasma membrane, but does contain essential basolateral sorting information. We show that the pro-AR cytoplasmic domain truncation mutant (ARTL27) is not sorted in polarized Madin-Darby canine kidney cells, with approximately 65% of the newly synthesized protein delivered to the apical cell surface. Under base-line conditions, ARTL27 was preferentially cleaved from the basolateral surface with 4-fold greater efficiency compared with cleavage from the apical membrane domain. However, ARTL27 ectodomain cleavage could be stimulated equivalently from either membrane domain by a variety of different stimuli. The metalloprotease inhibitor BB-94 could inhibit both base-line and stimulus-induced ectodomain cleavage of wild-type pro-AR and ARTL27. These results indicate that the pro-AR cytoplasmic domain is required for basolateral sorting, but is not essential for ectodomain processing. Preferential constitutive cleavage of ARTL27 from the basolateral cell surface also suggests that the metalloprotease activity involved in base-line and stimulus-induced ARTL27 ectodomain cleavage may be regulated differently in the apical and basolateral membrane domains of polarized epithelial cells.     

Apical Polarity of N-CAM and EMMPRIN in Retinal Pigment Epithelium Resulting from Suppression of Basolateral Signal Recognition

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM''s basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.     

Polarized distribution of NHE1 and NHE2 in the rat epididymis

Previous studies from our laboratory have provided evidence that the rat epididymis utilizes the Na(+)/H(+) exchanger to transport acid and base. The present study was undertaken to use immunohistochemistry for investigating the localization (apical versus basolateral) and distribution of NHE1 and NHE2 proteins along intact rat epididymis. Both proteins were found to be exclusively localized within the epithelium. Immunoreactivity for NHE1 was detected on the basolateral surface, whereas NHE2 immunoreactivity was detected on the apical side of the epithelium. Interestingly, NHE1 was found along the entire length of the epididymal tubule whereas NHE2 was absent in the initial segment but present in the caput, corpus, and cauda regions. These results, when interpreted along with those of previous functional studies, may suggest that the apical NHE2 is involved in Na(+) reabsorption and the basolateral NHE1 in HCO(3)(-) secretion in the rat epididymis.     

Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA

We expressed cDNA for the rabbit polymeric immunoglobulin receptor in polarized Madin-Darby Canine Kidney epithelial cells, which normally do not produce this receptor. The receptor appeared to function as in vivo; dimeric IgA was transported from the basolateral to the apical surface and released into the apical medium, together with the cleaved fragment of the receptor, known as secretory component. This system enabled us, for the first time, to study quantitatively IgA transcytosis in vitro and thus make the following observations. First, greater than 90% of the newly made receptor that is ultimately cleaved to secretory component and released into the apical medium goes first to the basolateral surface. Second, transport of the receptor does not depend on ligand binding. Third, transcytosis of bound ligand has a t 1/2 of 30 min.     

Human coronavirus 229E infects polarized airway epithelia from the apical surface

Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.     

Apical and basolateral EGF receptors regulate gastric mucosal paracellular permeability

Previous studies found that monolayers formed from canine oxyntic epithelial cells in primary culture displayed remarkable resistance to apical acidification and both mitogenic and migratory responses to epidermal growth factor (EGF) treatment. In our present studies, we found that EGF increased transepithelial resistance (TER) but not short-circuit current in these monolayers. Parallel effects of EGF on decreasing mannitol flux and increasing TER implicate direct regulation of paracellular permeability. EGF acting at either apical and basolateral receptors rapidly increased TER, but the apical response was sustained whereas the basolateral response was transient. (125)I-labeled EGF binding revealed specific apical binding, but receptor numbers were 25-fold lower than on the basolateral surface. Both apical and basolateral EGF activated tyrosine phosphorylation of EGF receptors (EGFR), beta-catenin, and cellular substrate as evident on confocal microscopy. Although apical EGF activated a lesser degree of receptor autophosphorylation than basolateral EGF, phosphorylation of beta-catenin was equally prominent with apical and basolateral receptor activation. Together, these findings indicate that functional apical and basolateral EGFR exist on primary canine gastric epithelial cells and that these receptors regulate paracellular permeability. The sustained effect of apical EGFR activation and prominent phosphorylation of beta-catenin suggest that apical EGFR may play a key role in this regulation.     

PI3K/Akt responses to oxytocin stimulation in Caco2BB gut cells

Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5 nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8 nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5 nM OT for 30 min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms.     

Possible involvement of oxytocin and its receptor in the local regulation of prostaglandin secretion in the cat endometrium

Ovarian originated oxytocin (OT) is involved in several reproductive process, amongst them its role in the regulation/modulation of the estrous cycle in several species has been demonstrated. Although the systemic role of endometrial originated prostaglandins (PGs), especially prostaglandin F(2α) (PGF(2α)), is equivocal in cats, their possible involvement in the local regulation of uterine events during the estrous cycle is uncertain. We examined the spontaneous and LH-stimulated OT production in cultured luteal cells, the spatial and temporal arrangement of OT receptors (OTR) in a cat endometrium and, finally the effects of OT on PG secretion and prostaglandin-endoperoxide synthase (PTGS2) expression in the feline cultured endometrial cells. Uteri together with ovaries were collected from adult domestic cats (n=27) at different stages of the estrous cycle, after routine ovariohysterectomy procedures. The endometrial and luteal cells were separated enzymatically. Luteinizing hormone (LH) augmented OT secretion in cultured luteal cells 2-fold compared with control (P<0.05). Oxytocin receptor was abundantly expressed in different ovarian structure, as well as in uterine tissues collected at early/developing and mid-luteal phase. The secretion of PGF(2α) by endometrial epithelial cells was increased by OT at a dose 10(-7)M (P<0.001). Atosiban (specific OTR blocker) alone did not affect PG secretion but atosiban in combination with OT abolished the stimulating effect of OT on PGF(2α) secretion. Oxytocin augmented PGE(2) secretion at a dose 10(-7)M and 10(-6)M in the endometrial stromal cells (P<0.001). The treatment with atosiban did not abrogated positive effect of OT on PGE(2) production in the stromal cells. Effect of OT on PTGS2 mRNA expression, the rate-limiting enzyme in PG production, was examined by Real Time-PCR and PTGS2 mRNA expression was significantly affected by OT in both epithelial and stromal cell cultures (P<0.01). The present observations have shown that OT is locally produced by the early/developing corpora lutea and that corpora lutea delivered OT may regulate PG secretion in a cat endometrium especially at early- and mid-diestrus, by affecting PTGS2 mRNA expression.