Biosynthesis and immunocytochemical localization of an estrogen-dependent glycoprotein and associated morphological alterations in the sheep ampulla oviduct.

Published reports have shown that an M(r) 90,000-92,000 protein is released into the oviductal lumen of the sheep, during estrus at a time corresponding to ovulation and fertilization, where it associates with the embryo. The objectives of this study were (a) to determine whether estradiol-17 beta (E) alone or in combination with progesterone (P) induces the synthesis of the M(r) 90,000-92,000 protein from the ampulla and/or isthmus oviduct; (b) to monitor structural alterations in oviductal epithelial cells associated with the synthesis of this protein; and (c) to generate a polyclonal antiserum to the protein and use the antiserum to verify its cellular location and tissue specificity. Oviductal flushings and explant culture media were obtained from ovariectomized animals treated with E alone or with E plus P. The M(r) 90,000-92,000 protein was present in 3H-leucine- and 3H-glucosamine-labeled culture media of the ampulla (not isthmus) oviduct in animals treated with E alone or with E plus P. The glycoprotein was detected in gels of oviductal flushings obtained from animals treated only with E. A specific polyclonal antiserum to the protein was made and cross-reacted on Western blots of oviductal flushings from E-treated animals and ampulla (not isthmus) oviduct culture media from animals treated with E alone or with E plus P. The secretory apparatus of the epithelial cells of the ampulla oviduct matured and differentiated in response to E. Light and electron microscopic immunocytochemistry localized the M(r) 90,000-92,000 glycoprotein to secretory granules in the nonciliated cells of the ampulla oviduct. Immunoperoxidase reaction product was absent in tissue sections and Western blots of other reproductive and nonreproductive tract tissues obtained from steroid-treated animals. Therefore, the secretory cells of the ampulla oviduct of the sheep synthesize and release an E-induced, oviduct-derived M(r) 90,000-92,000 glycoprotein.     

Long-term culture of feline oviduct epithelial cells on permeable filter supports

Basic knowledge about cellular and molecular mechanisms underlying feline reproduction is required to improve reproductive biotechnologies in endangered felids. Commonly, the domestic cat (Felis catus) is used as a model species, but many of the fine-tuned, dynamic reproductive processes can hardly be observed in vivo. This necessitates the development of in vitro models. The oviduct is a central reproductive organ hosting fertilization in the ampulla and early embryonic development in the isthmus part, which also functions as a sperm reservoir before fertilization. In other species, culturing oviduct epithelial cells in compartmentalized culture systems has proven useful to maintain oviduct epithelium polarization and functionality. Therefore, we made the first attempt to establish a compartmentalized long-term culture system of feline oviduct epithelial cells from both ampulla and isthmus. Cells were isolated from tissue samples (n?=?33 animals) after routine gonadectomy, seeded on permeable filter supports and cultured at the liquid–liquid or air–liquid interface. Cultures were harvested after 21 days and microscopically evaluated for epithelial differentiation (monolayer formation with basal–apical polarization) and protein expression of marker genes (oviduct-specific glycoprotein, acetylated tubulin). Due to the heterogeneous and undefined native tissue material available for this study, the applied cell culture approach was only successful in a limited number of cases (five differentiated cultures). Even though the protocol needs optimization, our study showed that the compartmentalized culture approach is suitable for maintaining differentiated epithelial cells from both isthmus and ampulla of the feline oviduct.

     

GRP78 expression and immunohistochemical localization in the female reproductive tract of mice

The 78-kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone, with multiple functional roles in protein processing and provision of cellular protection. However, the physiological role of GRP78 in embryo development is not clear. Localization of GRP78 and expression of its mRNA in the reproductive organs throughout the estrous cycle in mice were investigated by immunohistochemistry and real-time polymerase chain reaction, respectively. Whereas there was intense staining for GRP78 in the oviduct at estrus, the ciliated cells of isthmus had better staining than those of infundibulum and ampulla at all phases of the cycle. Furthermore, GRP78 was located in the uterine luminal and glandular epithelial cells throughout the estrous cycle, particularly during the estrus phase. However, levels of GRP78 mRNA in the oviduct and uterus varied during the cycle, with peaks at estrus. In conclusion, GRP78 expression varied with the phase of the murine estrous cycle; this might be related to gamete transport, fertilization and early development of the zygote/embryo.     

Electrophoretic characterization of proteins in oviduct fluid of cows during the estrous cycle

This study was undertaken to examine the in vivo protein composition of the bovine oviduct during the estrous cycle. Oviduct fluid was collected daily from four oviduct-cannulated dairy cows for a total of four complete estrous cycles. Fluid secretion followed a definite cyclic pattern, with maximum secretion occurring at estrus in all cycles. Protein concentration fluctuated during the cycle and varied among animals. In general, protein concentration was lower at the time of estrus. Total protein in oviduct fluid, however, was higher around estrus, indicating increased transudation or secretion by the oviduct. One-dimensional SDS-PAGE separation revealed the protein pattern of oviduct fluid to be generally similar to that of blood serum and follicular fluid. Two proteins appeared to be oviduct-specific. The first, a protein of approximately 47 kDa, was evident in oviduct fluid throughout the estrous cycle. The second protein, evident as a broad diffuse staining band above albumin, appeared for only 3-4 days at or near ovulation. This protein had a molecular weight of 80-95 kDa and stained positive for carbohydrate with periodic acid-Schiff reagent. These studies indicate that the in vivo protein composition of oviduct fluid varies with the estrous cycle, and that around estrus, an oviduct-specific glycoprotein is present.     

Oviduct-specific expression of tissue plasminogen activator in laying hens

Egg-laying hens are important candidate bioreactors for pharmaceutical protein production because of the amenability of their eggs for protein expression. In this study, we constructed an oviduct-specific vector containing tissue plasminogen activator (tPA) protein and green fluorescent protein (pL-2.8OVtPAGFP) and assessed its expression in vitro and in vivo. Oviduct epithelial and 3T3 cells were cultured and transfected with pL-2.8OVtPAGFP and pEGP-N1 (control vector), respectively. The pL-2.8OVtPAGFP vector was administered to laying hens via a wing vein and their eggs and tissues were examined for tPA expression. The oviduct-specific vector pL-2.8OVtPAGFP was expressed only in oviduct epithelial cells whereas pEGP-N1 was detected in oviduct epithelial and 3T3 cells. Western blotting detected a 89 kDa band corresponding to tPA in egg white and oviduct epithelial cells, thus confirming expression of the protein. The amount of tPAGFP in eggs ranged 9 to 41 ng/mL on the third day after vector injection. The tPA expressed in egg white and oviduct epithelial cells showed fibrinolytic activity, indicating that the protein was expressed in active form. GFP was observed only in oviducts, with no detection in heart, muscle, liver and intestine. This is the first study to report the expression of tPA in egg white and oviduct epithelial cells using an oviduct-specific vector.     

Oviductal epithelium of the baboon: hormonal control and the immuno-gold localization of oviduct-specific glycoproteins

Oviducts were obtained from a series of cycling and ovariectomized steroid-treated baboons. The lining epithelium of the ampulla and isthmus was analyzed by light and electron microscopy. Both morphological and cytomorphometric analyses revealed that the morphological and functional state of the oviductal epithelium in the baboon is controlled by the ovarian steroids. Additionally, a clear cephalocaudal steroid-responsive gradient was observed when the data from the ampulla and isthmus of the same animal were compared. Within the ampulla, estradiol induced hypertrophy, hyperplasia, ciliogenesis, and secretory activity, whereas adding progesterone to the treatment regimen (+/- estradiol) resulted in atrophy, deciliation, apoptosis, and loss of the secretory activity. These cyclic processes were less evident in the isthmus. We also used an indirect electron microscopic immunogold technique and a previously characterized polyclonal antibody to determine the localization of oviduct-specific glycoproteins. These glycoproteins were present in every secretory granule observed, regardless of oviduct region, electron density, or size of the secretory granule. In summary, our data show that 1) estradiol induces and maintains the mature epithelium of the baboon oviduct, 2) steroid withdrawal or the administration of progesterone causes regression of the epithelium, and 3) the previously identified estradiol-dependent oviduct-specific glycoproteins are synthesized within and released from the secretory epithelial cells.     

Histology of the adult mouse oviduct and endometrium following a single prenatal exposure to diethylstilbestrol

Light microscopy was used to examine the oviduct and endometrium of offspring from mice administered DES (10 micrograms/kg in 0.1 cc of corn oil, subcutaneously) or corn oil alone on Day 15 of gestation. Offspring were sacrificed at 5, 7 and 9 months of age. Oviduct changes in DES exposed offspring included numerous abnormal secretory cells which lined the mucosal folds of the isthmus. These cells contained a distinct granular cytoplasm which was eosinophilic and a nucleus displaced towards the apical surface. In addition both the ampulla and isthmus had mucosal folds which extended to the serosal surface and an accumulation of subepithelial fibrinoid material. Endometrial changes included squamous metaplasia of both the surface and glandular epithelial layer as well as extensive cystic glandular hyperplasia. In addition the endometrial connective tissue stroma exhibited fibrinoid accumulation. These changes may reflect an altered endocrine environment resulting from ovarian abnormalities during adulthood.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

The concentrations of catecholamines and oxytocin receptors in the oviduct and its contractile activity in cows during the estrous cycle

In vitro experiments on oviducts of cyclic cows were undertaken to study: (1) the content of dopamine (DA), noradrenaline (NA) and adrenaline (A) in infundibulum, ampulla and isthmus, (2) the concentration of oxytocin receptors (OTR) in oviductal tissues and (3) the motility of ampulla and isthmus. Changes of DA content were observed in the infundibulum and the ampulla with maximal values occurring on Days 6-10 of the estrous cycle. The mean NA content was greatest in infundibulum相似文献   

Identification and localization of plasminogen activator inhibitor-1 within the porcine oviduct

The porcine oviduct synthesizes de novo and secretes a number of proteins into culture medium, many of which are unidentified. The objectives of the present study were to 1) semipurify and identify a M(r) 45 000 secreted protein of the oviduct, 2) examine its synthesis within the three functional segments (infundibulum, ampulla, and isthmus), and 3) evaluate its distribution throughout the oviduct. Oviductal tissue was collected during early pregnancy, divided into functional segments, and subsequently cultured. Medium was collected, and the M(r) 45 000 protein was concentrated by gel-filtration chromatography. The semipurified protein was transferred onto a polyvinylidene fluoride membrane and subjected to N-terminal amino acid analysis. The 26-amino acid sequence was 96% identical to that of pig plasminogen activator inhibitor (PAI)-1. Analysis by 1-dimensional SDS-PAGE and fluorography of rabbit anti-human PAI-1-immunoprecipitated product confirmed PAI-1. Subsequent 2-dimensional SDS-PAGE and fluorographic analyses of media revealed greater PAI-1 synthesis by the isthmus than by the ampulla or infundibulum. PAI-1 was immunolocalized throughout the oviduct and was heavily concentrated in the apical region of epithelial cells. Immunogold electron microscopy localized PAI-1 within putative secretory granules in the epithelial apical region and also associated with cilia in the isthmus. Isthmic PAI expression suggests a crucial role in protecting the preimplantation embryo from proteolytic degradation as well as in regulation of extracellular matrix turnover and remodeling.     

Ultrastructural changes in the oviductal epithelium of Merino ewes during the estrous cycle

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days -1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron-lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.     

Morphometric and ultrastructural features of the mare oviduct epithelium during oestrus

Morphometric, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations have displayed regional differences in the mare oviductal epithelium. The entire mucosa of the oviduct was lined with a pseudostratified epithelium, which consisted of two distinct cell types, ciliated and non-ciliated. Ciliated cells were predominant in the three different segments of the oviduct and their percentage increased from fimbriae to ampulla and significantly decreased in the isthmus. SEM revealed in the infundibulum finger-like mucosal folds, some of them interconnected, in the ampulla numerous and elaborated branched folds of the mucosa, whereas the isthmus displayed a narrow lumen, short and non-branched mucosal folds. In the ampulla and isthmus the majority of non-ciliated cells showed apical blebs provided or not of short microvilli. TEM displayed different ultrastructural features of ciliated and non-ciliated cells along the oviduct. Isthmus ciliated cells presented a more electron-dense cytoplasm than in infundibulum and ampulla cells and its cilia were enclosed in an amorphous matrix. The non-ciliated cells of infundibulum did not contain secretory granules but some apical endocytic vesicles and microvilli coated by a well developed glycocalyx. Non-ciliated cells of ampulla and isthmus contained secretory granules. Apical protrusions of ampulla displayed two types of secretory granules as well as occasional electron-lucent vesicles. Isthmus non-ciliated cells showed either electron-lucent or electron-dense cytoplasm and not all contained apical protrusions. The electron-dense non-ciliated cells displayed microvilli coated with a well developed glycocalyx. Three types of granules were observed in the isthmus non-ciliated cells. The regional differences observed along the epithelium lining the mare oviduct suggest that the epithelium of the each segment is involved in the production of a distinctive microenvironment with a unique biochemical milieu related to its functional role.     

Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus)

Oviductins belong to a family of oviduct-specific glycoproteins believed to play an important role in fertilization and/or early embryonic development. Oviductin cDNA between species is highly conserved and shares 58% to 98% similarity in the deduced amino acid sequences. Our objective in this study was to sequence the full open reading frame of the feline oviductin and to examine its expression during the estrous cycle on both mRNA and protein level. The obtained cDNA containing the full open reading frame was determined to be 1677 nucleotides coding for a deduced protein of 558 amino acids. Identities between species range from 74% (mouse) to 80% (human, baboon, and rhesus) within the N-terminal protein region. Major differences were localized in the carboxy terminal region, which corresponds to exon 11 of the gene. Feline oviductin contained one putative N-linked glycosylation site, six O-linked glycosylation sites, a potential heparin binding site, and two cholesterol recognition and/or interaction amino acid consensus (CRAC) domains. Oviductin expression was analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Both approaches revealed an estrous cycle-dependent expression in the ampulla and isthmus. Quantitative PCR showed highest oviductin mRNA copy numbers in the early and late follicular stage and reduced mRNA expression during all other stages. With the exception of the early follicular stage, feline oviductin mRNA abundance was not significantly different in the oviductal segments ampulla and isthmus. A prominent immunolabeling was seen in the early and late follicular stage which disappeared after ovulation, indicating a function of the protein during sperm storage and fertilization.     

Expression of prostaglandin E synthases in the bovine oviduct

The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E₂ is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH_2. The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH₂ to PGE₂, the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE₂ production were present in the Bos taurus oviduct.     

Isolation,culture, and characterization of equine oviduct epithelial cells in vitro

Oviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol-17β or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers. © 1995 Wiley-Liss, Inc.     

Distribution of NADPH-diaphorase and nitric oxide synthase (NOS) in different regions of porcine oviduct during the estrous cycle.

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo- and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units +/- SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 +/- 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 +/- 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 +/- 2.5; ampulla 35.6 +/- 4.2; infundibulum 24.7 +/- 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function.     

Expression of dihydrotestosterone synthases and androgen receptor in sheep oviduct ampulla and its regulation by estradiol and progesterone

Oviduct ampulla plays an important role in steroid hormone-regulated sperm-oocyte binding in female animals. Although studies have shown that androgen receptor are expressed in many species oviduct, the interaction among androgen receptor (AR), estradiol (E2) and progesterone (P4) in the sheep oviduct have rarely been reported. In this study, we evaluated the localization of two isoforms of dihydrotestosterone (DHT) sythetase enzymes 5α-reductase (5α‐red1, 5α‐red2) and AR in sheep oviduct ampulla by immunohistochemistry and immunofluorescence. Results showed that they were all distributed in oviduct epithelium layer. In epithelial cells, 5α‐red1, 5α‐red2 were expressed in cytoplast and nuclear, but AR were stained in nuclear. We also investigated their expression pattern in the sheep oviduct ampulla at different development stages of follicles (Large follicles stage; hemorrhagium, luteum and albicans of corpus stage) by molecular experiments. We found that 5α‐red1, 5α‐red2 and AR mRNA abundance and protein were expressed highest in corpus albicans stage and lowest in corpus hemorrhagium stage. In vitro, when sheep oviduct ampulla epithelial cells (SOAECs) were cultured and treated with different concentrations of E2/P4 (10^?9–10^?6 M), we found that E2 inhibited the expression of AR mRNA and protein, while P4 promoted this expression. In addition, when the SOAECs were treated with E2 (10^?8 M) and/or its non-selective inhibitor ICI182780 (10^?7 M) as well as with P4 (10^?6 M) and/or its non-specific inhibitor RU486 (10^?5 M), we found that E2 and P4 inhibited and promoted the expression of AR mRNA and proteins, respectively, via their nuclear receptor pathways. This study provides a basic insight for the further research of oviduct epithelium physiological function closely related to androgen.