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.     

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.     

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo.     

A morphological,morphometric and histochemical study of the oviduct in pregnant and non‐pregnant females of the plains viscacha (Lagostomus maximus)

The oviduct is a very thin organ with a very tortuous appearance. It is divided into three segments: the infundibulum, the ampulla and the isthmus. Particularly, the oviduct of the viscacha lacks the intramural portion described in other species. The mucosa shows longitudinal pleats. The free edge of the infundibulum ends as small fimbriae that are of variable length and do not completely cover the ovary. The proportion of ciliated and secretory epithelial cells varied both in relation to the segments of the oviduct analysed and to the physiological state (anoestrus, follicular phase, early pregnancy and late pregnancy). The glycocalix and the apical region of the superficial epithelial cells are PAS and alcian‐blue positive. The muscular layers vary in thickness in different regions. Some lectins such as UEA‐1 and DBA showed variations in the binding pattern during the different physiological stages analysed whereas RCA‐1and WGA had a very stable pattern.     

Ultrastructure of mucous cells from the oviduct epithelium of the rabbit (Lepus cuniculus). Preliminary study]

Authors studied the ultrastructural features of the mucous cells present in the three segments of the rabbit oviduct in anoestrous. Results showed that only one kind of mucous cell was present in the isthmus while two different kinds of mucous cells were found in the ampulla and infundibulum. The ultrastructural features observed in the isthmic cells correlated well with the histochemical data already described in that segment. However such correlations could not be made between the ampulla and infundibulum. Authors suggest that the ampulla can be considered a transitional segment between isthmus and infundibulum.     

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   

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.     

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.     

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.

     

Nuclear estrogen and progesterone receptors in the oviduct of heifers under natural and superovulated estrous cycles

Oviducts from 22 crossbred heifers were examined for the presence of nuclear estrogen (ERalpha) and progesterone (PR) receptors at different phases (estrus, metaestrus and diestrus) of naturally occurring estrous cycles and estrous cycles during which superovulation was induced. Receptors were detected by immunohistochemistry in the epithelial cells, connective tissue and muscular layer of oviductal infundibulum, ampulla, ampullary/isthmic transition and isthmus. Epithelial ERalpha was found along the entire oviduct regardless of the cycle phase and of the circulating concentrations of 17beta-estradiol (E(2)) and progesterone (P(4)). Epithelial PR was found mainly at the ampullary-isthmic transition and isthmus and more intensely at the estrus phase but their amount was not correlated with P(4) concentrations. ERalpha in the connective tissue was more abundant at the infundibulum and ampulla, regardless of the phase of the estrous cycle and of E(2) and P(4) circulating concentrations. PR in the connective tissue was found mostly at the ampulla, regardless of the estrous cycle phase but no correlations were found between amount and P(4) concentrations. ERalpha staining intensity in the muscular layer was similar at all phases of the estrous cycle and at all anatomical segments of the oviducts. However, PR staining was more intense at the isthmus during the metaestrus phase and it was negatively correlated with P(4) concentrations. In general, data from the present research suggest that P(4) exerts an inhibitory role upon ERalpha and PR. Also, no differences were found between animals subjected or not to superovulation.     

Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination

A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.     

Histochemical features of the mucus of the surface epithelium of the rabbit oviduct]

Some cytochemical observations were made on the secretion of the mucocytes in the surface epithelium of the different regions of the rabbit oviduct in anoestrous. It was concluded that the mucocytes of the oviduct contained mucin which was neutral or acid in nature. The three regions of the rabbit oviduct contained carboxylated acid mucopolysaccharide, but sialomucin was found in the infundibulum and sulphomucin was demonstrated in the isthmus and ampulla.     

Origin of oestrus-associated glycoproteins in bovine oviductal fluid.

The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi [35S]methionine in serum-free medium for 6 h at 37 degrees C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.     

Identification,characterization and localization of three proteins expressed by the porcine oviduct

At estrus, the oviduct undergoes endocrine-induced changes which provide an essential microenvironment for maturation of gametes, fertilization and embryonic development. Several oviduct expressed proteins which interact with gametes or embryos, including the oviduct-specific, estrogen-dependent glycoprotein (OGP), have been identified and characterized. The objective of the present study was to identify, characterize and localize other proteins expressed by the porcine oviduct during estrus that may function in an autocrine or paracrine manner to enhance fertilization and embryonic development. Oviducts were collected during the estrous cycle or early pregnancy, flushed and divided into functional segments, and portions of the infundibulum, ampulla and isthmus were fixed for immunocytochemical analysis or cultured. Culture media was semi-purified by heparin-agarose affinity chromatography, proteins were transferred to polyvinylidene fluoride (PVDF) membrane after two-dimensional (2D)-SDS-PAGE and three different proteins were identified, excised and subjected to N-terminal amino acid analysis. These proteins were identified as complement component C3b, the carboxy-terminal propeptide of alpha 1 (III) procollagen (PIIICP), and the heavy chain variable region of IgA. Electrophoresis and fluorography of media from Days 0 to 12 of early pregnancy or the estrous cycle revealed both spatial and temporal expression of C3b and IgA heavy chain but not PIIICP by the oviduct. Further, all three proteins were identified in oviduct fluid by electrophoresis, immunoblot or immunoprecipitation analysis. Complement component C3b and IgA heavy chain were immunolocalized in all three oviduct segments on all days; however, temporal and spatial differences were demonstrated. Staining was greater in the infundibulum and during estrus for all three identified proteins. In summary, three proteins expressed by the oviduct at estrus and during early pregnancy were identified; characterization and localization suggest they may play a critical role in protecting the luminal environment, participating in ECM remodeling and gamete interactions.     

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle‐stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre‐ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct‐specific glycoprotein 1, heat‐shock protein family A member 5, α‐l ‐fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.     

Immunohistochemical demonstration of S-100 protein in epithelial cells of bovine oviduct

The present study deals with an immunohistochemical localization of S-100 protein in the bovine oviduct. The epithelium of the infundibulum, ampulla and isthmus showed a positive staining for S-100 protein. The immunoreactivity for S-100 was observed both in the ciliated and nonciliated (secretory) cells of the oviductal epithelium at any stages of the estrous cycle. The immunoreactivity was also found in nervous elements and endothelial cells of blood vessels. No cell outside these cells showed any immunoreactivity for S-100. Although the functional significance of S-100 protein in the oviductal epithelium remains to be elucidated, the present results introduce new perspectives into the investigation of function and localization of S-100 protein.