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Secretory function of the Fallopian tube epithelial cells in vitro
Trophoblast Research 13:87-104, 1999
S E C R E T O R Y F U N C T I O N OF T H E F A L L O P I A N T U B E E P I T H E L I A L CELLS I N V I T R O - A Review Ovrang Djahanbakhch, Ertan Saridogan, M. Ertan Kervancioglu, Tariq M a h m o o d , Lin Li and Jurgis G. Grudzinskas Department of Obstetrics and Gynaecology St. Bartholomew's and The Royal London School of Medicine and Dentistry London, United Kingdom
INTRODUCTION The oviduct has m a n y roles in the process of natural conception: the major two being retrieval of the oocyte released by the ruptured ovarian follicle and facilitation of final maturation of the gametes. Both the sperm and oocyte are transported in the Fallopian tube to the site of fertilization and meet within a well-defined time limit. Fertilization takes place at the isthmic-ampullary junction, and then the pre-embryo is transported to the uterine cavity at the optimal time for nidation. Significantly, the sperm and pre-embryo, which differ antigenically from the mother, are not attacked by the immune system. The factors controlling gamete and pre-embryo movement, maturation and interactions with the maternal immune system are not fully understood. The physiological study of the Fallopian tube has been hampered by its relative inaccessibility. Our knowledge comes from several sources: animal studies, studies on h u m a n Fallopian tube specimens removed surgically, studies of the tubal fluid obtained by tubal cannulation, in vitro short term explants or epithelial cell cultures from the Fallopian tubes, and in vivo endoscopic, electrophysiological and telemetric studies on Fallopian tubes. All these methods have inherent advantages and disadvantages and when data from these studies are analyzed, limitations of the methodology used have to be considered e.g., conclusions drawn from animal studies cannot necessarily be extrapolated directly to h u m a n reproduction. The aim of this paper is to review studies on the cellular and metabolic aspects of Fallopian tube epithelial cells.
Histological Organization of Fallopian Tube Mucosa The mucosa of the Fallopian tube is convoluted into folds longitudinally around the lumen. The size and complexity of these folds increases progressively along the lumen towards the fimbriated end where there are many secondary and tertiary folds superimposed on the primary fold. Each fold consists of a single epithelial cell layer on the basement membrane. This is surrounded by a connective tissue layer, the lamina propria, which separates the epithelial layer from the muscle layer, the tunica muscularis. In the epithelial layer four types of cells have been identified, the most c o m m o n being are ciliated and secretory cells. Less c o m m o n l y seen are the smaller peg
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(intercalary) and basal (indifferent) cells. In the lamina propria there are plasma cells, the most c o m m o n of which are present throughout the length of the tube, and secrete lgA (Kutteh et al., 1988). Plasma cells of the IgG or IgM class are also present, but are less frequently seen. The ciliated cells are cuboid in shape and contain a fine granular cytoplasm. These cells are further characterized by their vesiculated endoplasmic reticulum, long mitochondria and cytoplasmic droplets. The nucleus is usually centrally located and is round or oval in shape. A perinuclear halo of clear cytoplasm is often seen. The cilia are attached to a refractile row of basal granules beneath the cell membrane. They are approximately 7 F-m long and consist of two central filaments surrounded by nine double lateral filaments. Ciliated cells are most commonly seen on the apical aspect of the mucosal folds. Along the length of the mucosal folds some workers have reported a uniform distribution, while others suggest a progressive decrease in their occurrence from the fimbriated end to the isthmus (Ferenczy et al., 1972; Patek et al., 1972a,b; Critoph and Dennis, 1977). Secretory cells are distributed throughout the length of the Fallopian tube, being most c o m m o n in the ampulla where they constitute approximately 50% of epithelial cells. Secretory cells have a more granular cytoplasm (secretory granules), a well-developed endoplasmic reticulum and Golgi bodies. The mitochondria are smaller than in ciiiated cells and the nucleus is dark and oval with its longitudinal axis parallel to that of the cell. As with the secretory epithelial cells of the endometrium, the position of the nucleus varies throughout the ovarian cycle. Intercalary cells appear as small cells with little cytoplasm and a long dark nucleus, compressed between adjacent cells as they are seen most often in the luteal phase of the ovarian cycle (Fredericks, 1986). The basal cells arc small, rounded and also have sparse cytoplasm surrounding a dark nucleus and are located adjacent to the basement membrane. The Effect of Ovarian Steroids on Tubal M o r p h o l o g y The general morphology of the Fallopian tube mucosa varies during the ovarian cycle, though the variations are much less pronounced than those observed in the endometrium. The changes are qualitatively similar in different parts of the tube, but there are quantitative differences. As mitosis is rare in the Fallopian tube epithelmm, there is little or no cyclic change in cell number (Jansen, 1984). The epithelial thickness is uniformly low during menstruation and increases during the follicular phase reaching its m a x i m u m height (30 ~tm) during the late follicular phase (Verhage et al., 1979). At this time both ciliated and non-ciliated cells are of equal height. In the luteal phase, the epithelium loses its height gradually to a m i n i m u m (10-15 lam) during menstruation (Fredericks, 1986). There are minor differences seen during the cyclic changes of the ciliated and non-ciliated cell in height. Patek et al. (1972b) reported ciliated cells of the h u m a n tubal mucosa to be reduced in height during the menstrual phase. The height is increased in the proliferative phase when the ciliated cells are covered by well-shaped, regularly distributed microvilli. The m a x i m u m height is reached in the periovulatory period, at this time both cell types are of equal height, with non-ciliated cell apices forming domes between the tufts of cilia. After ovulation the cilia become more prominent as the secretory cells shorten. Then the ciliated cells become broader and lower, while the nonciliated cells are left with pedunculated apices during the luteal phase (Jansen, 1984).
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There is disagreement about the occurrence of specific ultrastructural changes in ciliated cells during the menstrual cycle. However, there may be an increase in the number and size of cytoplasmic granules and mitochondria, as the cycle progresses (Fredericks, 1986). In the human oviduct, there is never complete shedding and regeneration of cilia unlike the infundibulum of the rhesus monkey oviduct (Brecmer, 1969). Verhage et al., (1979) reported cyclic changes in the cilia of ampullary and fimbrial cells of the human Fallopian tube. They described deciliation of 10-12% of cells during the luteal phase, with regeneration in the early follicular phase of the next cycle. During the menstrual phase the cilia appeared "droopy". In the follicular phase the cilia became erect and vigorous. The rate of beat of the cilia was seen to be greatest at, or just after, ovulation, suggesting that ciliary activity is related to the circulating level of ovarian steroids (Critoph and Dennis, 1977). Using a method that detects variation in light intensity we studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have observed that incubation with progesterone (10 gM) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This progesterone induced reduction in ciliary beat frequency was prevented by the progesterone receptor antagonist mifepristone. Estradiol alone had no effect on ciliary beat frequency but did prevent the reduction in ciliary beat frequency seen with progesterone when the tissue was incubated with the two steroids together (Mahmood et at., 1998). In humans, secretory cells of the tubal epithelium are generally low in height during the menstrual phase. However, some appear in clusters, are round or sphere shaped, and bulge into the tubal lumen (Patek et al., 1972b), these features being retained from the late luteal phase of the previous menstrual cycle During the follicular phase, secretory cells increase in height and are covered with microvilli. There are cytoplasmic apical projections or domes at the luminal surface, these finger-like projections are prominent in the follicular phase and in the imcnediate preovulatory phase this surface activity reaches a peak, with very irregular protrusions, some of which are higher than the adjacent cilia (Crow et al., 1994). The apical projections become wasted and even completely separated from the underlying cells. This form of apocrine or decapitation secretion results in release of secretory granules with large cell fragments, sometimes including the nucleus and a number of whole cells. Secretory material in the lumen covers the cell surface and obscures the cilia. This secretory activity is particularly pronounced in the isthmus (Jansen, 1980). In the periovulatory period the microvilli become swollen and adhere to each other forming tuft-like structures. After ovulation a decrease in secretion becomes apparent, and cilia start regaining their prominence By days 18 and 19, the luminal secretion disappears, the secretory cell dominance is lost and the cilia are prominent and erect (Jansen, 1980)~ These changes are associated with ultrastructural alterations which reflect changing secretory activity (Ludwig and Metzer, 1976; Pauerstein and Eddy, 1979). At the onset of the proliferative phase the Golgi apparatus is compact, the endoplasmic reticulum limited and the mitochondria diminished. These structures become more prominent during the follicular phase and about cycle day 10 granules of ribonucleoprotein (Palade granules) become numerous. By the end of the follicular phase, secretory granules appear beneath the cell membrane adjacent to the lumen. In the luteal phase the endoplasmic reticulum dilates and secretory droplets and granules appear, while the Golgi apparatus expands the mitochondria decrease in number. Some secretory cells rupture and release their contents during the midluteal phase. Liposomes
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appear in increased numbers in the luteal phase. Secretory activity during the menstrual cycle is more prominent in the isthmus than in the ampulla (Ludwig and Metzer, 1976; Pauerstein and Eddy, 1979).
Fallopian Tube Epithelial Secretory Proteins The oviduct synthesizes and secretes both specific proteins and proteins of general Mullerian origin, some of which vary according to anatomical site, stage of the estrus or ovarian cycle, and during pregnancy. There are as yet few systematic, controlled studies of specific protein synthesis and secretion in normal human Fallopian tube and there are no simple assays to measure specific tubal epithelial proteins to evaluate these substances as potential markers of Fallopian tube function. For clinicians these phenomena will be important both for the possible development of new methods of contraception and in the assessment of sub-fertile women who do not have anatomical tubal factors as a cause of their infertility. Fallopian tube mucosal explants or epithelial cells are used to study the in vitro synthesis and secretion of proteins. In these systems a radiolabeled amino acid or glucosamine is added to the culture medium which is incorporated into newly synthesized proteins. Using this technique, estrogen-dependent high molecular weight oviduct secretory glycoproteins have been described in baboons (Fazleabas and Verhage, 1986; Verhage and Fazleabas, 1988), humans (Buhi et al., 1989a; Verhage et al., 1988), sheep (Murray, 1992, 1993), pigs (Buhi et al., 1989b, 1992, 1993) and cows (Boice et al., 1990). This glycoprotein has later been characterized and called 'oviductin'. In addition, a 66 kDa oviduct specific protein was demonstrated in rabbits (Hyde and Black, 1986), 16 and 88 kDa proteins in baboons (Fazleabas and Verhage, 1986), 30, 55, 85-97 and 97 kDa proteins in cows (Malayer et al., 1988), 20 and 60 kDa proteins in pigs (Buhi et al., 1989b). In humans we have reported in vitro de novo synthesis of immunoglobulin and previously undescribed 17 and 25 kDa proteins (Maguiness et al., 1993b). These 25 and 17 kDa proteins (designated as tubal epithelial protein-I, TEP-1 and TEP-2, respectively) were not secreted by the endometrium. Furthermore, TEP-2 was not secreted by the Fallopian tube from postmenopausal women, but only by premenopausal women, suggesting dependence on ovarian function. Using immunohistochemical localization and radioimmunoassay, Pregnancy Associated Placental Protein-A, PAPP-A (Sjoberg et al., 1986), and Placental Protein 5, PP 5 (Butzow, 1989) have been demonstrated in the human Fallopian tube. This methodology, refined by the use of gel filtration, has also identified the presence of Placental Protein 10, PP 10 (Tiitinen et al., 1986) and Placental Protein 14, PP 14 (Julkunen et al., 1986). Placental protein 14 is one of the major secretory proteins of the endometrium and is also known as progesterone-dependent endometrial protein (Seppala et al., 1992). Recent publications refer to this protein as glycodelin A due to its glyeoprotein structure (Dell et al., 1995) and it is present in hemopoietic cells and the ovarian tissue (Kamarainen et al., 1994; 1996). It has been reported that PP 14 has immunosuppressive effects (Olajide and Chard, 1992) and also inhibits sperm binding to zona pellucida in humans (Oehninger et al., 1995). Recently, it has been hypothesized that glycodelin A is involved in the human feto-embryonic defense system by providing an immunosuppressive environment which protects the embryo from immediate attack by
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e n d o m e t r i a l natural killer cells (Clark et al., 1996). In vitro de novo synthesis and the release of PP 14 by the Fallopian tube mucosal explants has been observed d u r i n g the ovarian cycle (Verhage et al., 1988; Maguiness et al., 1993a). Verhage et al. (1988) reported that in vitro PP14 secretion was detected only in the late luteal phase in the Fallopian tube and similarly, it was shown that PP 14 secretion b y the e n d o m e t r i a l cells in vitro increased almost 10 times in the late luteal phase (Laird et al., 1993). However, we have d e m o n s t r a t e d that PP 14 synthesis and secretion by the Fallopian tube mucosa occurred t h r o u g h o u t the ovarian cycle i n d e p e n d e n t of any cyclical changes (Maguiness et al., 1993a).
Oviduct Epithelial Cell Culture Cell culture p r o v i d e s a useful means of s t u d y i n g various aspects of the function of epithelial cells of the Fallopian tube such as metabolism, protein synthesis, and response to d r u g s and infection. The effect of different physicochemical conditions and physiological agents such as h o r m o n e s and other regulatory substances on the Fallopian tube can be studied in cells obtained from different anatomical sites at each stage of the ovarian cycle, in cell culture the characterization and h o m o g e n e i t y of the sample can be tightly controlled. Single cell type can be i n d i v i d u a l l y assessed or the interaction of two different cell types can be o b s e r v e d (Byres et al., 1986). The use of p o l a r i z e d cell cultures allows the direction of substrate uptake and protein secretion to be studied (Carson et al., 1988). The translocation of h o r m o n e receptor complexes, fluctuations in cellular metabolic pools and cell-to-cell interactions can be e x a m i n e d (Freshney, 1987). The d i s a d v a n t a g e s of cell culture systems are that the original histology is lost, together with any paracrine influences. Cells cultured on an u n c o a t e d plastic surface lose the polarization n o r m a l l y present in intact tissues. P r e m a t u r e senescence and dedifferentiation m a y occur in the cells cultured on either glass or plastic surfaces ( G o s p o d a r o w i c z et al., 1980). It has been shown that extracellular matrices (ECM) stimulate proliferation a n d prevent de-differentiation ( H a d l e y et al., 1985). We currently use a commercially available ECM on a permeable filter for culture of epithelial cells (Kervancioglu et al., 1994b). I S O L A T I O N A N D CULTURE OF F A L L O P I A N TUBE EPITHELIAL CELLS The Fallopian tube epithelial cells can be isolated by several methods. The m e t h o d we have used to isolate epithelial cells from the Fallopian tube involves dissecting off the tubal mucosa and cutting it into small pieces a p p r o x i m a t e l y 1 m m ~ with fine scissors. The tissue pieces are w a s h e d to remove any traces of blood then, placed in culture m e d i u m s u p p l e m e n t e d with fetal bovine s e r u m and incubated at 37~ in an a t m o s p h e r e of 5% C O , / 9 5 % air and 95% h u m i d i t y ( p r i m a r y culture 1). Between d a y 7 and 10 of the p r i m a r y culture the tissue explants are r e m o v e d and the p r i m a r y culture p r o c e d u r e r e p e a t e d ( p r i m a r y culture 2). The cells which have become adherent to the p r i m a r y culture dishes are w a s h e d with calcium and m a g n e s i u m free H a n k s ' balanced salt solution, then trypsinized and subcultured into fresh culture m e d i u m . Subsequent cultures are p a s s a g e d at confluence (4-5 days) by transferring half of the cell population, using the same trypsinization technique, into two identical culture flasks containing subculture m e d i u m (Kervancioglu et al., 1994b).
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Mechanical isolation (Bongso et al., 1989) involves dislodging epithelial cells by using a needle and syringe to flush the lumen of the Fallopian tube with Hank's Balanced Salt Solution (HBSS). If the cell numbers obtained using this method were inadequate the authors resorted to gently scraping the epithelial laver. Enzymatic disaggregation of the epithelial layer can be achieved using trypsin (Ouhibi et al., 1989), collagenase (Takeuchi et al., 1991), or a combination of the two (Jung-Testas et al., 1986). Care needs to be taken with these enzymes as trypsin has been shown to have a deleterious effect oil cells if the reaction is performed at 37~ (Freshney, 1987). At lower temperatures (4~ a higher yield of viable cells can be obtained after 18 hours. If serum free m e d i u m is used the effect of trypsin has to be neutralized with an inhibitor, e.g., soyabean trypsin inhibitor. Polarized cell culture can be achieved by seeding isolated cells onto porous filters in inserts such as Millicell PC coated with an extra-cellular matrix (ECM) such as matrigel or collagen. The coated inserts are then placed into multiwell dishes and culture medium added to the upper and lower compartments (Kervancioglu et al., 1994b). MEDIA REQUIREMENTS A N D M A I N T E N A N C E
Various media have been used for the culture of h u m a n Fallopian tube epithelial cells: Chang, B2, MEM, M199, RPMI 1640. With the exception of Cbang's m e d i u m (Bongso et al., 1989) all other media require the addition of 7.5% to 20% serum. When cells become confluent they are detached from their substrate with trypsin/EDTA and transferred to fresh media in new culture dishes to low further cell proliferation in subculture. Without serum supplementation, primary culture has been carried on for up to 56 days in the presence of albumin, insulin and transferrin (Henriksen et al., 1990). However, the cell n u m b e r did not increase after day 10 and subculture was not successful. Supplements used by others include lactalbumin, hydrolyzed epidermal growth factor and serotonin (Takeuchi et al., 1991). We have successfully cultured and subcultured tubal epithelial cells for 60 days in MEM Earle's, a conventional medium used for assisted conception procedures, supplemented with fetal bovine serum. C O N F I R M A T I O N OF CELL TYPE A major problem encountered in epithelial cell culture is contamination by fibroblast overgrowth. Different methods have been devised to select epithelial cells preferentially and prevent fibroblast proliferation (Djahanbakhch et al., 1994). Phase contrast microscopy is widely used to identify cells as epithelial-like or fibroblast-like (Figure 1). A more accurate assessment of cell type can be made by demonstrating the staining characteristics of intermediate filament proteins: cytokeratin (epithelial cells), vimentin (mesodermal cells), and desmine (muscle cells). It is important to include positive and negative controls. Our control experiments included one in which the primary antibody was excluded. Anti-cytokeratin (Figure 2) and anti-vimentin antibodies were used in parallel experiments on cells from the same culture well. After staining with the primary antibody, antibody-containing cells can be observed using different methods e.g., PAP or Biotin-Avitin. Electron microscopy can identify specific characteristics of epithelial cells e.g., microvilli (sometimes covered by glycocalyx), intercellular junction complexes and polarized cytoplasmic structures (Figures 3 and 4).
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Polarized cells typically show a columnar pattern and prominent epithelial patches while non-polarized cells were seen to be flat.
Figure 1. Light microscopy of Fallopian tube epithelial cells in the primary culture on day 7 (Magnification X200).
Figure 2. Immm~ofluorencence staining of cultured Fallopian tube epithelial cells with anti-cytokeratin antibody (Magnification X400).
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~
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Figure 3. Transmission electron microscopy of polarized Fallopian tube epithelial cells. Microvilli can be seen on the apical surface, n: nucleus; nl: nucleolus; arrow: endoplasmic reticulum; arrowhead: mitochondria (Magnification X7700).
Figure 4. Scanning electron microscopy of polarized Fallopian tube epithelial cells (Magnification X900).
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Functional Aspects of Tubal Epithelium The role of tubal proteins in the events of early r e p r o d u c t i o n is tmknown, though there is evidence of interaction between gametes, the p r e - e m b r y o and tubal fluid which contains the p r o d u c t s of secretion of the epithelial cells. In sheep, Gandolfi et al. (1989) d e m o n s t r a t e d that the oviduct p r o d u c e d two p o l y p e p t i d e s , referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), these were detected within the first 4-5 days of estrus. Labeling studies showed that these proteins were b o u n d to the zona pellucida of oocytes collected from the oviduct, and were absent from oocytes which had no contact with the oviduct. The increased capacitation of bovine s p e r m a t o z o a has been d e m o n s t r a t e d in oviduct fluid (Parrish et al., 1989). H u m a n s p e r m a t o z o a with p o o r motility have been shown to have an fi~creased ability to penetrate a zona-free h a m s t e r oocyte in the presence of a m e d i u m b a s e d on the composition of h u m a n tubal fluid c o m p a r e d with H a m F10 m e d i u m alone (Cai a n d Marik, 1990). The value of using culture m e d i a with the characteristics of h u m a n tubal fluid is less clear, with some reporting i m p r o v e d p r e g n a n c y rates (Quinn et al., 1985) while others found no i m p r o v e m e n t (Cummins et al., 1986).
250 r
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Figure 5. Values represent m e a n • S.E. of PP14 secreted by the Fallopian tube epithelial cells in p r i m a r y cultures (PC) a n d subcultures (SC), ( * p < 0.05, M a n n - W h i t n e y U Test).
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300 [] Proliferative
250
I Secretory
0
r"
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~ 150 o
~
100
~
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Figure 6. Values represent mean +_ S.E. of PP14 secreted by the cultured Fallopian tube epithelial cells from w o m e n in the proliferative (follicular) phase (n = 4) and the secretory (luteal) phase (n = 4). The result did not show a significant difference both in the primary cultures (PC) and subcultures (SC).
We have shown that Fallopian tube epithelial cells cultured in a conventional IVF m e d i u m are effective in achieving sperm capacitation (Kervancioglu et al., 1994b) and reversing the blockage of blastocyst formation (Djahanbakhch et al., 1994). Although this effect has been reported (Menezo et al., 1990; Pearlstone et al., 1993) using other established cell lines (e.g., Vero ceils), cells from the genital tract, particularly Fallopian tube epithelial cells have been shown to be superior in promoting sperm function (Guerin et al., 1991; Kervanciogtu et al., 1994b) and favoring embryo development (Myers et al., 1994). Currently, the exact mechanism responsible for the beneficial effects of the coculture system is unknown. In animal studies, it has been shown that contact between the spermatozoa and oviduct epithelial cells is beneficial for sperm survival both in vivo (Smith and Yanagimachi, 1990) and m vitro (Pollard et al., 1991). The other possibility is that cultured cells may reduce oxygen tension in the culture m e d i u m or neutralise toxic reactive oxygen species by serving as sacrificial scavengers (Thibodeaux and Godke, 1992; Kervancioglu et al., 1994b). It is also possible that secretory proteins m a y be responsible for this beneficial effect. In cell cultures, the nature of the secretory behavior of these cells is not known. Thus, it is important to determine how these cells behave in vitro and whether their secretory behavior changes in subsequent subculture in both fresh and frozen-thawed cells.
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We have studied the secretory function of Fallopian tube epithelial cells in vitro and chose to measure placental protein 14 (PP14) as a marker for this purpose, as PP14 is one of the major products of the secretory endometrium and is also secreted by the Fallopian tube epithelial cells (Maguiness et al., 1992; Saridogan et al., 1997). There was a significant amount of PP14 secretion into the culture media in primary cultures. PP14 secretion decreased considerably after the subculture 1 and was not significantly different from the control values after subculture 2 (Figure 5). Epithelial cells from w o m e n in the proliferative phase and the luteal phase secreted similar amounts of PP14 into the culture media (Figure 6). There was no significant difference between PP14 secretion by the non-polarized and polarized ceils in both the primary cultures and subcultures (Figure 7). PP14 secretion between fresh and frozen-thawed Fallopian tube epithelial cells in the subcultures showed a significant difference, but PP14 secretion by frozen-thawed cells in the subcultures was not significantly different from the control values (Figure 8). Although one would expect that polarized and non-polarized cells behave differently and secrete different amounts of proteins, polarized and non-polarized cells showed similar amounts of PP14 secretion in this study. Likewise epithelial cells obtained from the Fallopian tube at different stages of the ovarian cycle showed similar amounts of PP14 secretion, confirming our previous observation that PP14 synthesis and secretion by the Fallopian tube mucosa occurs throughout the ovarian cycle without any significant change.
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Figure 7. Values represent mean _4- S.E. of PP14 secreted bv the polarized and nonpolarized Fallopian tube epithelial cells in vitro (PC: primary culture; SC: subculture).
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Figure 8. Values represent mean +_ S.E. of PP14 secreted by fresh and frozen-thawed Fallopian tube epithelial cells in subcultures ( * p < 0.05, Mann-Whitney U Test).
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Figure 9. Transmission electron microscopy of non-polarized frozen-thawed Fallopian tube epithelial cells, n: nucleus; arrow: endoplasmic reticulum; arrowhead: mitochondria (Magnification X7200). (With permission from Human Reproduction.)
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Cell freezing has been shown to have a detrimental effect on PP14 secretion although cell integrity a p p e a r s to be protected. F r o z e n - t h a w e d cells had similar light and electron microscopic features (Figure 9). Despite the detrimental effect on frozen-thawed cells of PP 14 secretion, frozen-thawed cells still exert a beneficial effect on the co-culture system and fertilization rates in severe male infertility increase significantly after uMng frozen-thawed Fallopian tube epithelial cells (Kervancioglu et al., 1997). The exact m e c h a n i s m of h o w the co-culture system exerts its beneficial effect is unknown. O u r data suggest that the positive effect of the co-culture system m a y not be solely related to the secretory ability of the epithelial cells. However, our findings on PP14 secretion by Fallopian tube epitheliaI cells in vitro m a y not necessarily be extrapolated to all other secretory p r o d u c t s of other cells. SUMMARY The oviduct p r o v i d e s a specialized e n v i r o n m e n t for the survival and transport of the m a m m a l i a n gametes to the site of fertilization within well defined time limits. The oviduct e n v i r o n m e n t facilitates the process of fertilization and d e v e l o p m e n t of the preembryo. Furthermore, the spermatozoa and the p r e - e m b r y o , which differ antigenically from the mother, are not attacked by the i m m u n e system, which differentiates them from pathogens. The m e c h a n i s m s by which all these complex processes are controlled are not fully understood. The d i s a d v a n t a g e of cell culture systems for physiological study is that the original histological a p p e a r a n c e is lost along with some endocrine and paracrine activities. Cells c u l t u r e d on glass or plastic surfaces lose the polarization n o r m a l l y present in intact tissues. Premature senescence and de-differentiation m a y occur. It has been shown that extracellular matrix m a y stimulate proliferation and prevent dedifferentiation. The use of polarized cell cultures also enable the study of ceilular orientation and protein secretion. Culture systems for polarized and n o n - p o l a r i z e d Fallopian tube epithelial ceils have been d e v e l o p e d in our laboratory. The subsequent experimental studies have shown that the presence of oviduct epithelial cells has a specific and significant stimulatory effect on s p e r m capacitation, reversing effects on the blockage of blastocyst formation. The exact m e c h a n i s m of h o w the co-culture system achieves this is yet to be elucidated and in this regard, the unique oviduct proteins are possible candidates. Further studies of the epithelial cell cultures directed at the nature of the secretory behavior of these cells, are necessary in order to d e t e r m i n e how these cells behave in
vitro. REFERENCES Boice, M.L., Geistert, R.D., Blair, R.M. and Verhage, H.G. (1990) Identification and characterization of bovine oviductal glycoproteins synthesized at estrus. BioI Reprod. 43, 457-465. Bongso, A., Soon-Chye, N., Sathananthan, H., Lian, N.P., Rauff, M. and Ratnam, S. (1989) I m p r o v e d quality of h u m a n e m b r y o s when co-cultured with h u m a n a m p u l l a r y cells, llum. Reprod. 4, 706-713.
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Brenner, R.M. (196q) The biology of oviductal cilia. In: The Marmnaliat~ Oviduct: C~,nparative BioIo~,y and Methodolo\,y, (eds) E.S.E. Hafez, E.S.E. and R.J. Blandau, The University of Chicago Press, Chicago, pp. 203-229. Buhi, W.C., Van Wert, J.W., Alvarez, I.M., Dones-Smith, M.M. and Bernhisel, M.A. (1989a) Synthesis and secretion of proteins by postpartum human oviductal tissue in culture. Fertil. SteriI. 51, 75-80. Buhi, W.C., Vallet, J.L. and Bazer, F.W. (1989b) De-novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture. I. Exp. Zool. 252, 79-88. Buhi, W.C., Ashworth, C.J., Bazer, F.W. and Alvarez, I.M. (1992) In vitro synthesis of oviductal secretory proteins bv estrogen-treated ovariectomized gilts. J. Exp. Zool. 262, 426-435. Buhi, W.C., O'Brien, B., Alvarez, I.M., Erdos, G. and Dubois, D. (1993) hnmunogold localization of porcine oviductal secretory proteins within the zona pellucida, perivitelline space, and plasma membrane of oviductal and uterine oocytes and early embryos. Biol. Reprod. 48, 1274-1283. Butzow, R. (1989) The human Fallopian tube contains placental protein 5. Hum. Reprod. 4, 17-20. Byres, S.W., Hadley, M.A., Djakiew, D. and Dym, M. (1986) Growth and characterization of polarized monolayers of epididymal epithelial cells and Sertoli cells in dual environmental chambers. J. Androl. 7, 59-68. Cai, X.Q. and Marik, J.J. (1990) Comparison of the effects on penetration capacity of human spermatozoa between using Ham's F-10 medium and a medium based on the composition of human tubal fluid. Androlo(4y 7, 539-542. Carson, D.D., Tang, J.P., Julian, J. and Glasser, S.R. (1988) Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells. J. Cell Biol. 107, 2425-2435. Clark, G.F., Oehninger, S., Patankar, M.S., Koistinen, R., Dell, A., Morris, H.R., Koistinen, H. and Seppala, M. (1996) A role for glycoconjugates in human development: the human feto-embryonic defense system hypothesis, ttum. Reprod. 11,467-473. Critoph, F.N. and Dennis, K.J. (1977) The cellular composition of the human oviduct epithelium. Br. J. Obset. Gynaecol. 84, 219-221. Crow, J., Amso, N.N., Lewin, J. and Shaw, R.M. (1994) Morphology and ultrastructure of Fallopian tube epithelium at different stages of the menstrual cycle and menopause. Hum. Reprod. 9, 2224-2233. Cummins, J.M., Breen, T.M., Fuller, F.M., Harrison, K.L., Wilson, L.M., Hennessey, J.S., Shaw, J.M. and Shaw, G. (1986) Comparison of two media in a human in vitro fertilization program: Lack of significant differences in pregnancy rate. J. In Vitru Fertil. Emb. Trans. 3, 326-328.
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Dell, A., Morris, H.R., Easton, R., Panico, M., Oehninger, S., Koistinen, R., Seppala, M. and Clark, G.F. (1995) Structural analysis of the oligosaccharides associated with glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J. Biol. Chem. 270, 24116-24126. Djahanbakhch, O., Kervancioglu, E., Maguiness, S.D. and Martin, J.E. (I994) Fallopian tube epithelial celt culture. In: The Fallopian Tube: Clinical and Surgical Aspects (eds) J.G. GrudzLnskas, M.G. Chapman, T. Chard and O. Djahanbakhch,. Springer-Verlag Company, London, pp. 37-51. Fazleabas, A.T. and Verhage, H.C. (1986) The detection of oviduct-specific proteins in the baboon (Papio anubis). Biol. Reprod. 35,455-462. Ferenczy, A., Richart, R.M., Agate, F.J., Purkerson, M.L. and Dempsey, E.W. (1972) Scanning electron microscopy of the human Fallopian tube. Science 175, 783-784. Fredericks, C.M. (1986) Morphological and functional aspects of the oviduct epithelium. In: The Fallopian Tube: Basic Studies and Clinical Contrzbuttons, (ed.) A.M. Siegler, Futura, New York, pp. 67-80. Freshney, R.I. (1987) Culture of animal cells. A manual of basic technique. Alan R. Liss, Inc., New York, pp. 107-127. Gandolfi, F., Brevini, T.A., Richardson, L., Brown, C.R. and Moor, R.M. (1989) Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 106, 303-312. Gospodarowicz, D., Delgado, D. and Vlodavsky, I. (1980) Permissive effect of the ECM on cell proliferation in vitro. Proc. Natl. Acad. Sci. USA, 77, 4094-4098. Guerm, J.F., Ouhibi, N., Regnier-Vigouroux, G. and Menezo, Y. (1991) Movement characteristics and hyperactivation of human sperm on different epithelial cell monolayers. Int. J. Androl. 14, 412-422. Hadley, M.A., Byres, S.W., Suarez-Quian, C.A., Kleinman, H.K. and Dym, M. (1985) Extracellular matrix regulated Sertoli cell differentiation, testicular cord formation and germ cell development in vitro. J. Cell Biol. 101, 1511-1522. Henriksen, T., Tanbo, T., Abyholm, T., Oppedal, B.R., Clussen, O.P. and Hovig, T. (1990) Epithelial cells from human Fallopian tube in culture. Hum. Reprod. 5, 25-31. Hyde, B.A. and Black, D.L. (1986) Synthesis and secretion of sulphated glycoproteins by rabbit oviduct explants in vitro. J. Reprod. Fertil. 78, 83-91. Jansen, R.P. (1984) Endocrine response in the Fallopian tube. Endocr. Rev. 5, 525-551. Jansen, R.P. (1980) Cyclic changes in the human Fallopian tube isthmus and their functional importance. Am. J. Obstet. Gynecol. 136, 292-308.
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Julkunen, M., Wahlstrom, T. and Seppala, M. (1986) Human Fallopian tube contains placental protein 14. Am. J. Obstet Gynecol. 154, 1076-1079. Jung-Testas, I., Garcia, T. and Baulieu, E.E. (1986) Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures. J. Steroid Biochem. 24, 273-279. Kamarainen, M., Riittinen, L., Seppala, M., Palotie, A. and Andersson, L.C. (1994) Progesterone-associated endometrial protein - a constitutive marker of human erythroid precursors. Blood 84, 467-473. Kamarainen, M., Leivo, I., Koistinen, R. Julkunen, M., Karvonen, U., Rutanen, E.M. and Seppala, M. (1996) Normal human ovary and ovarian tumors express glycodelin, a glycoprotein with immunosuppressive and contraceptive properties. Am. J. PathoL 148, 1435-1443. Kervancioglu, M.E., Djahanbakhch, O. and Aitken, R.J. (1994a) Epithelial cell co-culture and the reduction of sperm capacitation. Fertil. Steril. 61, 1103-1108. Kervancioglu, M.E., Saridogan, E., Martin, J.E., Maguiness, S.D. and Djahanbakhch, O. (1994b) A simple technique for the long-term non-polarized and polarized culture of human Fallopian tube epithelial cells. Biol. Cell 82, 103-107. Kervancioglu, M.E., Saridogan, E., Atasu, T., Camlibel, T., Demircan, A., Sarikamis, B. and Djahanbakhch, O. (1997) Human Fallopian tube epithelial ceU co-culture increases fertilization rates in male factor infer~lity but not in ~ubaI or unexplained infe~lih,. Hum. Rwrod. 12, 1253-1258. Kutteh, W.H., Hatch, K.D., Blackwell, R.E. and Mestecky, J. (1988) Secretory immune system of the female reproductive tract. I. Immunoglobulin and secretory component-containing cells. Obstetric Gynecolo~{y 71, 56-60. l~aird, S.M., Li, T.C. and Bolton, A.E. (1993) The production of the placental protein 14 and interleukin 6 by human endometrial cells in culture. Hum. Reprod, 8, 793-798. Ludwig, H. and Metzer, H. (1976) The human reproductive tract. A scaiming electron microscopic atlas. Springer-Verlag, Heidelberg, pp. 79-105. Maguiness, S.D., Djahanbakhch, O. and Grudzinskas, J.G. (1992) Oviducts proteins. Contemp, Rev. Obstet. Gynecol. 4, 42-50. Maguiness, S.D., Shrimanker, K., Djahanbakhch, O., Deeks, J.J., Teisner, B. and Grudzinskas, J.G. (1993a) In vitro synthesis of total protein and placental protein 14 by the Fallopian tube mucosa: variation m relation to anatomical site, the ovarian cycle and the menopause. Hum. Reprod. 8, 678-683. Maguiness, S.D., Stmmanker, K., Djahanbakhch, O., Teisner, B. and Grudzinskas, J.G. (1993b) Evidence for the in vitro de-novo synthesis of immunoglobulin and a previously underscribed 17 kDa protein (TEP-2) by the mucosa of the Fallopian tube. Hum. Reprod. 8, 1199-1202.
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Mahmood, T., Saridogan, E., Smutna, S., Habib, A.M. and Djahanbakhch, O (1!)98)The effect of ovarian steroids on epithelial ciliary beat trequency in the hun~an Fallopian tube. t from. Rcprod, 13, 2001-2004 Malayer, J.R., Hansen, P.J. and Buhi, W.C. (1988) Secretion ot proteins bv cultured bovine oviducts collected from estrus through early diestrus. I. Exp. Zool. 248, M5-353. Menezo, Y.J., Guerin, J.F. and Czyba, J.C. (1990) Improvement of human early embryo development in vitro by co-culture on monolavers of Vero cells. BtoI. Rep~od. 42, 301-306. Murray, M.K. (1992) Biosynthesis and immunocvtochemical localization of an estrogendependent glycoprotein and associated morphological alterations in the sheep ampulla oviduct. Biol. Reprod. 47, 889-902. Murray, M.K. (1993) An estrogen-dependent glycoprotein is synthesized and released from the oviduct in a temporal- and region-specific manner during earl~ pregnancy in the ewe. Biol. Reprod. 48, 446-453. Myers, M.W., Broussard, J.R., Menezo, Y., Prough, S.G., Blackwell, J., Godke, R.A. and Thibodeaux, J.K. (1994) Established cell lines and their conditioned media support bovine embryo development during in vitro culture. Hum. Reprod. 9, 1927-1931. Oehnhlger, S., Coddington, C.C., Hodgen, G.D. and Seppala, M. (1995) Factors affecting fertilization: Endometriai protein 14 (PP14) reduces the capacity of human spermatozoa to bind to the human zona pellucida. FertiI. SteriI. 63, 377-383. Ouhibi, N., Menezo, Y., Benet, G. and Nicollet, B. (1989) Culture of epithelial cells derived from the oviduct of different species. Hum. Reprod, 4, 229-235. Olajide, F. and Chard, T. (1992) Biological and clinical significance of the endometrial protein PP14 in reproductive endocrinology. Obstet. Gynecol. 5urv. 47, 252-257. Parrish, J.J., Susko-Parrish, J.I,., Handrow, RR., Sims, M.M. and First, N.L. (1989) Capacitation of bovine spermatozoa by oviduct fluid. Biol. Reprod. 40, 1020-1025. Patek, E., Nilsson, L. and Johannisson, E. (1972a) Scacmmg electron microscopic study of the human Fallopian tube. Report I. The proliferative and secretory stages. Fertil. Steril. 23, 459-465. Patek, E., Nitsson, L. and Johannisson, E. (1972b) Scaca~ing electron microscopic study of the human Fallopian tube. Report 1I. Fetal life, reproductive life, and menopause. FertiL Steril. 23, 719-733. Pauerstein, C.J. and Eddy, C.A. (1979) Morphology of the Fallopian tube. In: Tho Bio/oy,y of the Fluids of the Female Genital Tract, (eds.) B.K. Belier, F.K. and G.F.B. Schumacher, Elsevier, North Holland, pp. 299-317.
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Pearlstone, A.C., Wiker, S.R., Chart, S.Y.W., Wang, C. and Tucker, M.J. (1993) The effects of Vero (Green monkey kidney) cell co-culture on the motility patterns of cryopreserved human spermatozoa. Fertil. Steril. 59, 1105-1111. Pollard, J.W., Plante, C., King, W.A., Hansen, P.J., Betteridge, K.J. and Suarez, S.S. (1991) Fertilizing capacity of bovine sperm may be maintained by binding to oviductal epithelial cells. Biol. Reprod. 44, 192-197. Quinn, P., Kerin, J.F. and Warnes, G.M.(1985) Improved pregnancy rate in human in vitro fertilisation with the use of a medium based on the composition of human tubal fluid. Fertil. Steril. 44, 493-498. Saridogan, E., Djahanbakhch, O., Kervancioglu, M.E., Kahyaoglu, F., Shrimanker, K. and Grudzinskas, J.G. (1997) Placental protein 14 production by human Fallopian tube epithelial cells in vitro, ttum. Reprod. 12, 1500-1507. Seppala, M., Julkunen, M., Riittinen, L. and Koistinen, R. (1992) Endometrial proteins: A reappraisal. Hum. Reprod. 7 (Suppl. 1), 31-38. Sjoberg, J., Wahlstrom, T., Grudzinskas, J.G. and Sinosich, M.J. (1986) Demonstration of pregnancy-associated plasma protein-A (PAPP-A)-Iike material in the Fallopian tube. Fertil. Steril. 45, 517-521. Smith, T.T. and Yanagimachi, R. (1990) The viability of Hamster spermatozoa stored in the isthmus of the oviduct: the importance of sperm-epithelium contact for sperm survival. Biol. Reprod. 42, 450-457. Takeuchi, K., Murayama, T., Yamamoto, S., Oki, T. and Nagata, Y. (1991) Isolation and monolayer culture of human Fallopian tube epithelial cells. In Vitro Cell. Dev. Biol, 26, 720-724. Thibodeaux, J.K. and Godke, R.A. (1992) In vitro enhancement of early-stage embryos with co-culture. Arch. Pathol. Lab. Med. 116, 364-372. Tiitinen, A., Wahlstrom, T., Julkunen, M. and Seppala, M. (1986) The content and immunohistochemical localization of placental protein 10 (PP10) in the Fallopian tube. Br. J. Obstet. Gynaecol. 93, 924-927. Verhage, H.G. and Fazleabas, A.T, (1988) The m vitro synthesis of estrogen-dependent proteins by the baboon (Papio anubis) oviduct. Endocrinology 123, 552-558. Verhage, H.G., Fazleabas, A.T. and Donnelly, K. (1988) The in vitro synthesis and release of proteins by the human oviduct. Endocrinology 122, 1639-1645. Verhage, H.G., Bareither, M.L., Jaffe, R.C. and Akbar, M. (1979) Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women. Am. J. Anat. 156, 505-521.
S E C R E T O R Y F U N C T I O N OF T H E F A L L O P I A N T U B E E P I T H E L I A L CELLS I N V I T R O - A Review Ovrang Djahanbakhch, Ertan Saridogan, M. Ertan Kervancioglu, Tariq M a h m o o d , Lin Li and Jurgis G. Grudzinskas Department of Obstetrics and Gynaecology St. Bartholomew's and The Royal London School of Medicine and Dentistry London, United Kingdom
INTRODUCTION The oviduct has m a n y roles in the process of natural conception: the major two being retrieval of the oocyte released by the ruptured ovarian follicle and facilitation of final maturation of the gametes. Both the sperm and oocyte are transported in the Fallopian tube to the site of fertilization and meet within a well-defined time limit. Fertilization takes place at the isthmic-ampullary junction, and then the pre-embryo is transported to the uterine cavity at the optimal time for nidation. Significantly, the sperm and pre-embryo, which differ antigenically from the mother, are not attacked by the immune system. The factors controlling gamete and pre-embryo movement, maturation and interactions with the maternal immune system are not fully understood. The physiological study of the Fallopian tube has been hampered by its relative inaccessibility. Our knowledge comes from several sources: animal studies, studies on h u m a n Fallopian tube specimens removed surgically, studies of the tubal fluid obtained by tubal cannulation, in vitro short term explants or epithelial cell cultures from the Fallopian tubes, and in vivo endoscopic, electrophysiological and telemetric studies on Fallopian tubes. All these methods have inherent advantages and disadvantages and when data from these studies are analyzed, limitations of the methodology used have to be considered e.g., conclusions drawn from animal studies cannot necessarily be extrapolated directly to h u m a n reproduction. The aim of this paper is to review studies on the cellular and metabolic aspects of Fallopian tube epithelial cells.
Histological Organization of Fallopian Tube Mucosa The mucosa of the Fallopian tube is convoluted into folds longitudinally around the lumen. The size and complexity of these folds increases progressively along the lumen towards the fimbriated end where there are many secondary and tertiary folds superimposed on the primary fold. Each fold consists of a single epithelial cell layer on the basement membrane. This is surrounded by a connective tissue layer, the lamina propria, which separates the epithelial layer from the muscle layer, the tunica muscularis. In the epithelial layer four types of cells have been identified, the most c o m m o n being are ciliated and secretory cells. Less c o m m o n l y seen are the smaller peg
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(intercalary) and basal (indifferent) cells. In the lamina propria there are plasma cells, the most c o m m o n of which are present throughout the length of the tube, and secrete lgA (Kutteh et al., 1988). Plasma cells of the IgG or IgM class are also present, but are less frequently seen. The ciliated cells are cuboid in shape and contain a fine granular cytoplasm. These cells are further characterized by their vesiculated endoplasmic reticulum, long mitochondria and cytoplasmic droplets. The nucleus is usually centrally located and is round or oval in shape. A perinuclear halo of clear cytoplasm is often seen. The cilia are attached to a refractile row of basal granules beneath the cell membrane. They are approximately 7 F-m long and consist of two central filaments surrounded by nine double lateral filaments. Ciliated cells are most commonly seen on the apical aspect of the mucosal folds. Along the length of the mucosal folds some workers have reported a uniform distribution, while others suggest a progressive decrease in their occurrence from the fimbriated end to the isthmus (Ferenczy et al., 1972; Patek et al., 1972a,b; Critoph and Dennis, 1977). Secretory cells are distributed throughout the length of the Fallopian tube, being most c o m m o n in the ampulla where they constitute approximately 50% of epithelial cells. Secretory cells have a more granular cytoplasm (secretory granules), a well-developed endoplasmic reticulum and Golgi bodies. The mitochondria are smaller than in ciiiated cells and the nucleus is dark and oval with its longitudinal axis parallel to that of the cell. As with the secretory epithelial cells of the endometrium, the position of the nucleus varies throughout the ovarian cycle. Intercalary cells appear as small cells with little cytoplasm and a long dark nucleus, compressed between adjacent cells as they are seen most often in the luteal phase of the ovarian cycle (Fredericks, 1986). The basal cells arc small, rounded and also have sparse cytoplasm surrounding a dark nucleus and are located adjacent to the basement membrane. The Effect of Ovarian Steroids on Tubal M o r p h o l o g y The general morphology of the Fallopian tube mucosa varies during the ovarian cycle, though the variations are much less pronounced than those observed in the endometrium. The changes are qualitatively similar in different parts of the tube, but there are quantitative differences. As mitosis is rare in the Fallopian tube epithelmm, there is little or no cyclic change in cell number (Jansen, 1984). The epithelial thickness is uniformly low during menstruation and increases during the follicular phase reaching its m a x i m u m height (30 ~tm) during the late follicular phase (Verhage et al., 1979). At this time both ciliated and non-ciliated cells are of equal height. In the luteal phase, the epithelium loses its height gradually to a m i n i m u m (10-15 lam) during menstruation (Fredericks, 1986). There are minor differences seen during the cyclic changes of the ciliated and non-ciliated cell in height. Patek et al. (1972b) reported ciliated cells of the h u m a n tubal mucosa to be reduced in height during the menstrual phase. The height is increased in the proliferative phase when the ciliated cells are covered by well-shaped, regularly distributed microvilli. The m a x i m u m height is reached in the periovulatory period, at this time both cell types are of equal height, with non-ciliated cell apices forming domes between the tufts of cilia. After ovulation the cilia become more prominent as the secretory cells shorten. Then the ciliated cells become broader and lower, while the nonciliated cells are left with pedunculated apices during the luteal phase (Jansen, 1984).
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There is disagreement about the occurrence of specific ultrastructural changes in ciliated cells during the menstrual cycle. However, there may be an increase in the number and size of cytoplasmic granules and mitochondria, as the cycle progresses (Fredericks, 1986). In the human oviduct, there is never complete shedding and regeneration of cilia unlike the infundibulum of the rhesus monkey oviduct (Brecmer, 1969). Verhage et al., (1979) reported cyclic changes in the cilia of ampullary and fimbrial cells of the human Fallopian tube. They described deciliation of 10-12% of cells during the luteal phase, with regeneration in the early follicular phase of the next cycle. During the menstrual phase the cilia appeared "droopy". In the follicular phase the cilia became erect and vigorous. The rate of beat of the cilia was seen to be greatest at, or just after, ovulation, suggesting that ciliary activity is related to the circulating level of ovarian steroids (Critoph and Dennis, 1977). Using a method that detects variation in light intensity we studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have observed that incubation with progesterone (10 gM) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This progesterone induced reduction in ciliary beat frequency was prevented by the progesterone receptor antagonist mifepristone. Estradiol alone had no effect on ciliary beat frequency but did prevent the reduction in ciliary beat frequency seen with progesterone when the tissue was incubated with the two steroids together (Mahmood et at., 1998). In humans, secretory cells of the tubal epithelium are generally low in height during the menstrual phase. However, some appear in clusters, are round or sphere shaped, and bulge into the tubal lumen (Patek et al., 1972b), these features being retained from the late luteal phase of the previous menstrual cycle During the follicular phase, secretory cells increase in height and are covered with microvilli. There are cytoplasmic apical projections or domes at the luminal surface, these finger-like projections are prominent in the follicular phase and in the imcnediate preovulatory phase this surface activity reaches a peak, with very irregular protrusions, some of which are higher than the adjacent cilia (Crow et al., 1994). The apical projections become wasted and even completely separated from the underlying cells. This form of apocrine or decapitation secretion results in release of secretory granules with large cell fragments, sometimes including the nucleus and a number of whole cells. Secretory material in the lumen covers the cell surface and obscures the cilia. This secretory activity is particularly pronounced in the isthmus (Jansen, 1980). In the periovulatory period the microvilli become swollen and adhere to each other forming tuft-like structures. After ovulation a decrease in secretion becomes apparent, and cilia start regaining their prominence By days 18 and 19, the luminal secretion disappears, the secretory cell dominance is lost and the cilia are prominent and erect (Jansen, 1980)~ These changes are associated with ultrastructural alterations which reflect changing secretory activity (Ludwig and Metzer, 1976; Pauerstein and Eddy, 1979). At the onset of the proliferative phase the Golgi apparatus is compact, the endoplasmic reticulum limited and the mitochondria diminished. These structures become more prominent during the follicular phase and about cycle day 10 granules of ribonucleoprotein (Palade granules) become numerous. By the end of the follicular phase, secretory granules appear beneath the cell membrane adjacent to the lumen. In the luteal phase the endoplasmic reticulum dilates and secretory droplets and granules appear, while the Golgi apparatus expands the mitochondria decrease in number. Some secretory cells rupture and release their contents during the midluteal phase. Liposomes
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appear in increased numbers in the luteal phase. Secretory activity during the menstrual cycle is more prominent in the isthmus than in the ampulla (Ludwig and Metzer, 1976; Pauerstein and Eddy, 1979).
Fallopian Tube Epithelial Secretory Proteins The oviduct synthesizes and secretes both specific proteins and proteins of general Mullerian origin, some of which vary according to anatomical site, stage of the estrus or ovarian cycle, and during pregnancy. There are as yet few systematic, controlled studies of specific protein synthesis and secretion in normal human Fallopian tube and there are no simple assays to measure specific tubal epithelial proteins to evaluate these substances as potential markers of Fallopian tube function. For clinicians these phenomena will be important both for the possible development of new methods of contraception and in the assessment of sub-fertile women who do not have anatomical tubal factors as a cause of their infertility. Fallopian tube mucosal explants or epithelial cells are used to study the in vitro synthesis and secretion of proteins. In these systems a radiolabeled amino acid or glucosamine is added to the culture medium which is incorporated into newly synthesized proteins. Using this technique, estrogen-dependent high molecular weight oviduct secretory glycoproteins have been described in baboons (Fazleabas and Verhage, 1986; Verhage and Fazleabas, 1988), humans (Buhi et al., 1989a; Verhage et al., 1988), sheep (Murray, 1992, 1993), pigs (Buhi et al., 1989b, 1992, 1993) and cows (Boice et al., 1990). This glycoprotein has later been characterized and called 'oviductin'. In addition, a 66 kDa oviduct specific protein was demonstrated in rabbits (Hyde and Black, 1986), 16 and 88 kDa proteins in baboons (Fazleabas and Verhage, 1986), 30, 55, 85-97 and 97 kDa proteins in cows (Malayer et al., 1988), 20 and 60 kDa proteins in pigs (Buhi et al., 1989b). In humans we have reported in vitro de novo synthesis of immunoglobulin and previously undescribed 17 and 25 kDa proteins (Maguiness et al., 1993b). These 25 and 17 kDa proteins (designated as tubal epithelial protein-I, TEP-1 and TEP-2, respectively) were not secreted by the endometrium. Furthermore, TEP-2 was not secreted by the Fallopian tube from postmenopausal women, but only by premenopausal women, suggesting dependence on ovarian function. Using immunohistochemical localization and radioimmunoassay, Pregnancy Associated Placental Protein-A, PAPP-A (Sjoberg et al., 1986), and Placental Protein 5, PP 5 (Butzow, 1989) have been demonstrated in the human Fallopian tube. This methodology, refined by the use of gel filtration, has also identified the presence of Placental Protein 10, PP 10 (Tiitinen et al., 1986) and Placental Protein 14, PP 14 (Julkunen et al., 1986). Placental protein 14 is one of the major secretory proteins of the endometrium and is also known as progesterone-dependent endometrial protein (Seppala et al., 1992). Recent publications refer to this protein as glycodelin A due to its glyeoprotein structure (Dell et al., 1995) and it is present in hemopoietic cells and the ovarian tissue (Kamarainen et al., 1994; 1996). It has been reported that PP 14 has immunosuppressive effects (Olajide and Chard, 1992) and also inhibits sperm binding to zona pellucida in humans (Oehninger et al., 1995). Recently, it has been hypothesized that glycodelin A is involved in the human feto-embryonic defense system by providing an immunosuppressive environment which protects the embryo from immediate attack by
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e n d o m e t r i a l natural killer cells (Clark et al., 1996). In vitro de novo synthesis and the release of PP 14 by the Fallopian tube mucosal explants has been observed d u r i n g the ovarian cycle (Verhage et al., 1988; Maguiness et al., 1993a). Verhage et al. (1988) reported that in vitro PP14 secretion was detected only in the late luteal phase in the Fallopian tube and similarly, it was shown that PP 14 secretion b y the e n d o m e t r i a l cells in vitro increased almost 10 times in the late luteal phase (Laird et al., 1993). However, we have d e m o n s t r a t e d that PP 14 synthesis and secretion by the Fallopian tube mucosa occurred t h r o u g h o u t the ovarian cycle i n d e p e n d e n t of any cyclical changes (Maguiness et al., 1993a).
Oviduct Epithelial Cell Culture Cell culture p r o v i d e s a useful means of s t u d y i n g various aspects of the function of epithelial cells of the Fallopian tube such as metabolism, protein synthesis, and response to d r u g s and infection. The effect of different physicochemical conditions and physiological agents such as h o r m o n e s and other regulatory substances on the Fallopian tube can be studied in cells obtained from different anatomical sites at each stage of the ovarian cycle, in cell culture the characterization and h o m o g e n e i t y of the sample can be tightly controlled. Single cell type can be i n d i v i d u a l l y assessed or the interaction of two different cell types can be o b s e r v e d (Byres et al., 1986). The use of p o l a r i z e d cell cultures allows the direction of substrate uptake and protein secretion to be studied (Carson et al., 1988). The translocation of h o r m o n e receptor complexes, fluctuations in cellular metabolic pools and cell-to-cell interactions can be e x a m i n e d (Freshney, 1987). The d i s a d v a n t a g e s of cell culture systems are that the original histology is lost, together with any paracrine influences. Cells cultured on an u n c o a t e d plastic surface lose the polarization n o r m a l l y present in intact tissues. P r e m a t u r e senescence and dedifferentiation m a y occur in the cells cultured on either glass or plastic surfaces ( G o s p o d a r o w i c z et al., 1980). It has been shown that extracellular matrices (ECM) stimulate proliferation a n d prevent de-differentiation ( H a d l e y et al., 1985). We currently use a commercially available ECM on a permeable filter for culture of epithelial cells (Kervancioglu et al., 1994b). I S O L A T I O N A N D CULTURE OF F A L L O P I A N TUBE EPITHELIAL CELLS The Fallopian tube epithelial cells can be isolated by several methods. The m e t h o d we have used to isolate epithelial cells from the Fallopian tube involves dissecting off the tubal mucosa and cutting it into small pieces a p p r o x i m a t e l y 1 m m ~ with fine scissors. The tissue pieces are w a s h e d to remove any traces of blood then, placed in culture m e d i u m s u p p l e m e n t e d with fetal bovine s e r u m and incubated at 37~ in an a t m o s p h e r e of 5% C O , / 9 5 % air and 95% h u m i d i t y ( p r i m a r y culture 1). Between d a y 7 and 10 of the p r i m a r y culture the tissue explants are r e m o v e d and the p r i m a r y culture p r o c e d u r e r e p e a t e d ( p r i m a r y culture 2). The cells which have become adherent to the p r i m a r y culture dishes are w a s h e d with calcium and m a g n e s i u m free H a n k s ' balanced salt solution, then trypsinized and subcultured into fresh culture m e d i u m . Subsequent cultures are p a s s a g e d at confluence (4-5 days) by transferring half of the cell population, using the same trypsinization technique, into two identical culture flasks containing subculture m e d i u m (Kervancioglu et al., 1994b).
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Mechanical isolation (Bongso et al., 1989) involves dislodging epithelial cells by using a needle and syringe to flush the lumen of the Fallopian tube with Hank's Balanced Salt Solution (HBSS). If the cell numbers obtained using this method were inadequate the authors resorted to gently scraping the epithelial laver. Enzymatic disaggregation of the epithelial layer can be achieved using trypsin (Ouhibi et al., 1989), collagenase (Takeuchi et al., 1991), or a combination of the two (Jung-Testas et al., 1986). Care needs to be taken with these enzymes as trypsin has been shown to have a deleterious effect oil cells if the reaction is performed at 37~ (Freshney, 1987). At lower temperatures (4~ a higher yield of viable cells can be obtained after 18 hours. If serum free m e d i u m is used the effect of trypsin has to be neutralized with an inhibitor, e.g., soyabean trypsin inhibitor. Polarized cell culture can be achieved by seeding isolated cells onto porous filters in inserts such as Millicell PC coated with an extra-cellular matrix (ECM) such as matrigel or collagen. The coated inserts are then placed into multiwell dishes and culture medium added to the upper and lower compartments (Kervancioglu et al., 1994b). MEDIA REQUIREMENTS A N D M A I N T E N A N C E
Various media have been used for the culture of h u m a n Fallopian tube epithelial cells: Chang, B2, MEM, M199, RPMI 1640. With the exception of Cbang's m e d i u m (Bongso et al., 1989) all other media require the addition of 7.5% to 20% serum. When cells become confluent they are detached from their substrate with trypsin/EDTA and transferred to fresh media in new culture dishes to low further cell proliferation in subculture. Without serum supplementation, primary culture has been carried on for up to 56 days in the presence of albumin, insulin and transferrin (Henriksen et al., 1990). However, the cell n u m b e r did not increase after day 10 and subculture was not successful. Supplements used by others include lactalbumin, hydrolyzed epidermal growth factor and serotonin (Takeuchi et al., 1991). We have successfully cultured and subcultured tubal epithelial cells for 60 days in MEM Earle's, a conventional medium used for assisted conception procedures, supplemented with fetal bovine serum. C O N F I R M A T I O N OF CELL TYPE A major problem encountered in epithelial cell culture is contamination by fibroblast overgrowth. Different methods have been devised to select epithelial cells preferentially and prevent fibroblast proliferation (Djahanbakhch et al., 1994). Phase contrast microscopy is widely used to identify cells as epithelial-like or fibroblast-like (Figure 1). A more accurate assessment of cell type can be made by demonstrating the staining characteristics of intermediate filament proteins: cytokeratin (epithelial cells), vimentin (mesodermal cells), and desmine (muscle cells). It is important to include positive and negative controls. Our control experiments included one in which the primary antibody was excluded. Anti-cytokeratin (Figure 2) and anti-vimentin antibodies were used in parallel experiments on cells from the same culture well. After staining with the primary antibody, antibody-containing cells can be observed using different methods e.g., PAP or Biotin-Avitin. Electron microscopy can identify specific characteristics of epithelial cells e.g., microvilli (sometimes covered by glycocalyx), intercellular junction complexes and polarized cytoplasmic structures (Figures 3 and 4).
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Polarized cells typically show a columnar pattern and prominent epithelial patches while non-polarized cells were seen to be flat.
Figure 1. Light microscopy of Fallopian tube epithelial cells in the primary culture on day 7 (Magnification X200).
Figure 2. Immm~ofluorencence staining of cultured Fallopian tube epithelial cells with anti-cytokeratin antibody (Magnification X400).
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Djahanbakhch et al.
~
i~i~ :, ii!~i~ii~i~'~84184,~IS%~ 84184184
Figure 3. Transmission electron microscopy of polarized Fallopian tube epithelial cells. Microvilli can be seen on the apical surface, n: nucleus; nl: nucleolus; arrow: endoplasmic reticulum; arrowhead: mitochondria (Magnification X7700).
Figure 4. Scanning electron microscopy of polarized Fallopian tube epithelial cells (Magnification X900).
Fallopian Secretion
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Functional Aspects of Tubal Epithelium The role of tubal proteins in the events of early r e p r o d u c t i o n is tmknown, though there is evidence of interaction between gametes, the p r e - e m b r y o and tubal fluid which contains the p r o d u c t s of secretion of the epithelial cells. In sheep, Gandolfi et al. (1989) d e m o n s t r a t e d that the oviduct p r o d u c e d two p o l y p e p t i d e s , referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), these were detected within the first 4-5 days of estrus. Labeling studies showed that these proteins were b o u n d to the zona pellucida of oocytes collected from the oviduct, and were absent from oocytes which had no contact with the oviduct. The increased capacitation of bovine s p e r m a t o z o a has been d e m o n s t r a t e d in oviduct fluid (Parrish et al., 1989). H u m a n s p e r m a t o z o a with p o o r motility have been shown to have an fi~creased ability to penetrate a zona-free h a m s t e r oocyte in the presence of a m e d i u m b a s e d on the composition of h u m a n tubal fluid c o m p a r e d with H a m F10 m e d i u m alone (Cai a n d Marik, 1990). The value of using culture m e d i a with the characteristics of h u m a n tubal fluid is less clear, with some reporting i m p r o v e d p r e g n a n c y rates (Quinn et al., 1985) while others found no i m p r o v e m e n t (Cummins et al., 1986).
250 r
200 0 c-
Z
.
!
l-
c~ 150 c-E 0
b
100
h--
5O
I 12-
/
0
PC1
PC2
SC1
SC2
SC3
SC4
SC5
SC6
Figure 5. Values represent m e a n • S.E. of PP14 secreted by the Fallopian tube epithelial cells in p r i m a r y cultures (PC) a n d subcultures (SC), ( * p < 0.05, M a n n - W h i t n e y U Test).
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Djahanbakhch et al.
300 [] Proliferative
250
I Secretory
0
r"
200 r
~ 150 o
~
100
~
5O
PC1
PC2
SC1
SC2
SC3
SC4
SC5
SC6
Figure 6. Values represent mean +_ S.E. of PP14 secreted by the cultured Fallopian tube epithelial cells from w o m e n in the proliferative (follicular) phase (n = 4) and the secretory (luteal) phase (n = 4). The result did not show a significant difference both in the primary cultures (PC) and subcultures (SC).
We have shown that Fallopian tube epithelial cells cultured in a conventional IVF m e d i u m are effective in achieving sperm capacitation (Kervancioglu et al., 1994b) and reversing the blockage of blastocyst formation (Djahanbakhch et al., 1994). Although this effect has been reported (Menezo et al., 1990; Pearlstone et al., 1993) using other established cell lines (e.g., Vero ceils), cells from the genital tract, particularly Fallopian tube epithelial cells have been shown to be superior in promoting sperm function (Guerin et al., 1991; Kervanciogtu et al., 1994b) and favoring embryo development (Myers et al., 1994). Currently, the exact mechanism responsible for the beneficial effects of the coculture system is unknown. In animal studies, it has been shown that contact between the spermatozoa and oviduct epithelial cells is beneficial for sperm survival both in vivo (Smith and Yanagimachi, 1990) and m vitro (Pollard et al., 1991). The other possibility is that cultured cells may reduce oxygen tension in the culture m e d i u m or neutralise toxic reactive oxygen species by serving as sacrificial scavengers (Thibodeaux and Godke, 1992; Kervancioglu et al., 1994b). It is also possible that secretory proteins m a y be responsible for this beneficial effect. In cell cultures, the nature of the secretory behavior of these cells is not known. Thus, it is important to determine how these cells behave in vitro and whether their secretory behavior changes in subsequent subculture in both fresh and frozen-thawed cells.
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We have studied the secretory function of Fallopian tube epithelial cells in vitro and chose to measure placental protein 14 (PP14) as a marker for this purpose, as PP14 is one of the major products of the secretory endometrium and is also secreted by the Fallopian tube epithelial cells (Maguiness et al., 1992; Saridogan et al., 1997). There was a significant amount of PP14 secretion into the culture media in primary cultures. PP14 secretion decreased considerably after the subculture 1 and was not significantly different from the control values after subculture 2 (Figure 5). Epithelial cells from w o m e n in the proliferative phase and the luteal phase secreted similar amounts of PP14 into the culture media (Figure 6). There was no significant difference between PP14 secretion by the non-polarized and polarized ceils in both the primary cultures and subcultures (Figure 7). PP14 secretion between fresh and frozen-thawed Fallopian tube epithelial cells in the subcultures showed a significant difference, but PP14 secretion by frozen-thawed cells in the subcultures was not significantly different from the control values (Figure 8). Although one would expect that polarized and non-polarized cells behave differently and secrete different amounts of proteins, polarized and non-polarized cells showed similar amounts of PP14 secretion in this study. Likewise epithelial cells obtained from the Fallopian tube at different stages of the ovarian cycle showed similar amounts of PP14 secretion, confirming our previous observation that PP14 synthesis and secretion by the Fallopian tube mucosa occurs throughout the ovarian cycle without any significant change.
250 T
09
_~ 20O-
[] Non-polarised
I
[] Polarised
O cO4
~r-- 150 c-o
b IO0 ~-
5o
n
-..i
PC
m
I
I
SC1
L
I
SC2
SC3
Figure 7. Values represent mean _4- S.E. of PP14 secreted bv the polarized and nonpolarized Fallopian tube epithelial cells in vitro (PC: primary culture; SC: subculture).
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D j a h a n b a k h c h et al.
bO
[
03
[] Fresh [] Frozen
o 40 c
g 3O co 20
P--'- 10
0 -
*
mR SC1
L ~ SC2
SC3
SC4
SC5
SC6
Figure 8. Values represent mean +_ S.E. of PP14 secreted by fresh and frozen-thawed Fallopian tube epithelial cells in subcultures ( * p < 0.05, Mann-Whitney U Test).
-,-
-
.s
o
..
Figure 9. Transmission electron microscopy of non-polarized frozen-thawed Fallopian tube epithelial cells, n: nucleus; arrow: endoplasmic reticulum; arrowhead: mitochondria (Magnification X7200). (With permission from Human Reproduction.)
Fallopian Secretion
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Cell freezing has been shown to have a detrimental effect on PP14 secretion although cell integrity a p p e a r s to be protected. F r o z e n - t h a w e d cells had similar light and electron microscopic features (Figure 9). Despite the detrimental effect on frozen-thawed cells of PP 14 secretion, frozen-thawed cells still exert a beneficial effect on the co-culture system and fertilization rates in severe male infertility increase significantly after uMng frozen-thawed Fallopian tube epithelial cells (Kervancioglu et al., 1997). The exact m e c h a n i s m of h o w the co-culture system exerts its beneficial effect is unknown. O u r data suggest that the positive effect of the co-culture system m a y not be solely related to the secretory ability of the epithelial cells. However, our findings on PP14 secretion by Fallopian tube epitheliaI cells in vitro m a y not necessarily be extrapolated to all other secretory p r o d u c t s of other cells. SUMMARY The oviduct p r o v i d e s a specialized e n v i r o n m e n t for the survival and transport of the m a m m a l i a n gametes to the site of fertilization within well defined time limits. The oviduct e n v i r o n m e n t facilitates the process of fertilization and d e v e l o p m e n t of the preembryo. Furthermore, the spermatozoa and the p r e - e m b r y o , which differ antigenically from the mother, are not attacked by the i m m u n e system, which differentiates them from pathogens. The m e c h a n i s m s by which all these complex processes are controlled are not fully understood. The d i s a d v a n t a g e of cell culture systems for physiological study is that the original histological a p p e a r a n c e is lost along with some endocrine and paracrine activities. Cells c u l t u r e d on glass or plastic surfaces lose the polarization n o r m a l l y present in intact tissues. Premature senescence and de-differentiation m a y occur. It has been shown that extracellular matrix m a y stimulate proliferation and prevent dedifferentiation. The use of polarized cell cultures also enable the study of ceilular orientation and protein secretion. Culture systems for polarized and n o n - p o l a r i z e d Fallopian tube epithelial ceils have been d e v e l o p e d in our laboratory. The subsequent experimental studies have shown that the presence of oviduct epithelial cells has a specific and significant stimulatory effect on s p e r m capacitation, reversing effects on the blockage of blastocyst formation. The exact m e c h a n i s m of h o w the co-culture system achieves this is yet to be elucidated and in this regard, the unique oviduct proteins are possible candidates. Further studies of the epithelial cell cultures directed at the nature of the secretory behavior of these cells, are necessary in order to d e t e r m i n e how these cells behave in
vitro. REFERENCES Boice, M.L., Geistert, R.D., Blair, R.M. and Verhage, H.G. (1990) Identification and characterization of bovine oviductal glycoproteins synthesized at estrus. BioI Reprod. 43, 457-465. Bongso, A., Soon-Chye, N., Sathananthan, H., Lian, N.P., Rauff, M. and Ratnam, S. (1989) I m p r o v e d quality of h u m a n e m b r y o s when co-cultured with h u m a n a m p u l l a r y cells, llum. Reprod. 4, 706-713.
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Brenner, R.M. (196q) The biology of oviductal cilia. In: The Marmnaliat~ Oviduct: C~,nparative BioIo~,y and Methodolo\,y, (eds) E.S.E. Hafez, E.S.E. and R.J. Blandau, The University of Chicago Press, Chicago, pp. 203-229. Buhi, W.C., Van Wert, J.W., Alvarez, I.M., Dones-Smith, M.M. and Bernhisel, M.A. (1989a) Synthesis and secretion of proteins by postpartum human oviductal tissue in culture. Fertil. SteriI. 51, 75-80. Buhi, W.C., Vallet, J.L. and Bazer, F.W. (1989b) De-novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture. I. Exp. Zool. 252, 79-88. Buhi, W.C., Ashworth, C.J., Bazer, F.W. and Alvarez, I.M. (1992) In vitro synthesis of oviductal secretory proteins bv estrogen-treated ovariectomized gilts. J. Exp. Zool. 262, 426-435. Buhi, W.C., O'Brien, B., Alvarez, I.M., Erdos, G. and Dubois, D. (1993) hnmunogold localization of porcine oviductal secretory proteins within the zona pellucida, perivitelline space, and plasma membrane of oviductal and uterine oocytes and early embryos. Biol. Reprod. 48, 1274-1283. Butzow, R. (1989) The human Fallopian tube contains placental protein 5. Hum. Reprod. 4, 17-20. Byres, S.W., Hadley, M.A., Djakiew, D. and Dym, M. (1986) Growth and characterization of polarized monolayers of epididymal epithelial cells and Sertoli cells in dual environmental chambers. J. Androl. 7, 59-68. Cai, X.Q. and Marik, J.J. (1990) Comparison of the effects on penetration capacity of human spermatozoa between using Ham's F-10 medium and a medium based on the composition of human tubal fluid. Androlo(4y 7, 539-542. Carson, D.D., Tang, J.P., Julian, J. and Glasser, S.R. (1988) Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells. J. Cell Biol. 107, 2425-2435. Clark, G.F., Oehninger, S., Patankar, M.S., Koistinen, R., Dell, A., Morris, H.R., Koistinen, H. and Seppala, M. (1996) A role for glycoconjugates in human development: the human feto-embryonic defense system hypothesis, ttum. Reprod. 11,467-473. Critoph, F.N. and Dennis, K.J. (1977) The cellular composition of the human oviduct epithelium. Br. J. Obset. Gynaecol. 84, 219-221. Crow, J., Amso, N.N., Lewin, J. and Shaw, R.M. (1994) Morphology and ultrastructure of Fallopian tube epithelium at different stages of the menstrual cycle and menopause. Hum. Reprod. 9, 2224-2233. Cummins, J.M., Breen, T.M., Fuller, F.M., Harrison, K.L., Wilson, L.M., Hennessey, J.S., Shaw, J.M. and Shaw, G. (1986) Comparison of two media in a human in vitro fertilization program: Lack of significant differences in pregnancy rate. J. In Vitru Fertil. Emb. Trans. 3, 326-328.
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Dell, A., Morris, H.R., Easton, R., Panico, M., Oehninger, S., Koistinen, R., Seppala, M. and Clark, G.F. (1995) Structural analysis of the oligosaccharides associated with glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J. Biol. Chem. 270, 24116-24126. Djahanbakhch, O., Kervancioglu, E., Maguiness, S.D. and Martin, J.E. (I994) Fallopian tube epithelial celt culture. In: The Fallopian Tube: Clinical and Surgical Aspects (eds) J.G. GrudzLnskas, M.G. Chapman, T. Chard and O. Djahanbakhch,. Springer-Verlag Company, London, pp. 37-51. Fazleabas, A.T. and Verhage, H.C. (1986) The detection of oviduct-specific proteins in the baboon (Papio anubis). Biol. Reprod. 35,455-462. Ferenczy, A., Richart, R.M., Agate, F.J., Purkerson, M.L. and Dempsey, E.W. (1972) Scanning electron microscopy of the human Fallopian tube. Science 175, 783-784. Fredericks, C.M. (1986) Morphological and functional aspects of the oviduct epithelium. In: The Fallopian Tube: Basic Studies and Clinical Contrzbuttons, (ed.) A.M. Siegler, Futura, New York, pp. 67-80. Freshney, R.I. (1987) Culture of animal cells. A manual of basic technique. Alan R. Liss, Inc., New York, pp. 107-127. Gandolfi, F., Brevini, T.A., Richardson, L., Brown, C.R. and Moor, R.M. (1989) Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 106, 303-312. Gospodarowicz, D., Delgado, D. and Vlodavsky, I. (1980) Permissive effect of the ECM on cell proliferation in vitro. Proc. Natl. Acad. Sci. USA, 77, 4094-4098. Guerm, J.F., Ouhibi, N., Regnier-Vigouroux, G. and Menezo, Y. (1991) Movement characteristics and hyperactivation of human sperm on different epithelial cell monolayers. Int. J. Androl. 14, 412-422. Hadley, M.A., Byres, S.W., Suarez-Quian, C.A., Kleinman, H.K. and Dym, M. (1985) Extracellular matrix regulated Sertoli cell differentiation, testicular cord formation and germ cell development in vitro. J. Cell Biol. 101, 1511-1522. Henriksen, T., Tanbo, T., Abyholm, T., Oppedal, B.R., Clussen, O.P. and Hovig, T. (1990) Epithelial cells from human Fallopian tube in culture. Hum. Reprod. 5, 25-31. Hyde, B.A. and Black, D.L. (1986) Synthesis and secretion of sulphated glycoproteins by rabbit oviduct explants in vitro. J. Reprod. Fertil. 78, 83-91. Jansen, R.P. (1984) Endocrine response in the Fallopian tube. Endocr. Rev. 5, 525-551. Jansen, R.P. (1980) Cyclic changes in the human Fallopian tube isthmus and their functional importance. Am. J. Obstet. Gynecol. 136, 292-308.
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Julkunen, M., Wahlstrom, T. and Seppala, M. (1986) Human Fallopian tube contains placental protein 14. Am. J. Obstet Gynecol. 154, 1076-1079. Jung-Testas, I., Garcia, T. and Baulieu, E.E. (1986) Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures. J. Steroid Biochem. 24, 273-279. Kamarainen, M., Riittinen, L., Seppala, M., Palotie, A. and Andersson, L.C. (1994) Progesterone-associated endometrial protein - a constitutive marker of human erythroid precursors. Blood 84, 467-473. Kamarainen, M., Leivo, I., Koistinen, R. Julkunen, M., Karvonen, U., Rutanen, E.M. and Seppala, M. (1996) Normal human ovary and ovarian tumors express glycodelin, a glycoprotein with immunosuppressive and contraceptive properties. Am. J. PathoL 148, 1435-1443. Kervancioglu, M.E., Djahanbakhch, O. and Aitken, R.J. (1994a) Epithelial cell co-culture and the reduction of sperm capacitation. Fertil. Steril. 61, 1103-1108. Kervancioglu, M.E., Saridogan, E., Martin, J.E., Maguiness, S.D. and Djahanbakhch, O. (1994b) A simple technique for the long-term non-polarized and polarized culture of human Fallopian tube epithelial cells. Biol. Cell 82, 103-107. Kervancioglu, M.E., Saridogan, E., Atasu, T., Camlibel, T., Demircan, A., Sarikamis, B. and Djahanbakhch, O. (1997) Human Fallopian tube epithelial ceU co-culture increases fertilization rates in male factor infer~lity but not in ~ubaI or unexplained infe~lih,. Hum. Rwrod. 12, 1253-1258. Kutteh, W.H., Hatch, K.D., Blackwell, R.E. and Mestecky, J. (1988) Secretory immune system of the female reproductive tract. I. Immunoglobulin and secretory component-containing cells. Obstetric Gynecolo~{y 71, 56-60. l~aird, S.M., Li, T.C. and Bolton, A.E. (1993) The production of the placental protein 14 and interleukin 6 by human endometrial cells in culture. Hum. Reprod, 8, 793-798. Ludwig, H. and Metzer, H. (1976) The human reproductive tract. A scaiming electron microscopic atlas. Springer-Verlag, Heidelberg, pp. 79-105. Maguiness, S.D., Djahanbakhch, O. and Grudzinskas, J.G. (1992) Oviducts proteins. Contemp, Rev. Obstet. Gynecol. 4, 42-50. Maguiness, S.D., Shrimanker, K., Djahanbakhch, O., Deeks, J.J., Teisner, B. and Grudzinskas, J.G. (1993a) In vitro synthesis of total protein and placental protein 14 by the Fallopian tube mucosa: variation m relation to anatomical site, the ovarian cycle and the menopause. Hum. Reprod. 8, 678-683. Maguiness, S.D., Stmmanker, K., Djahanbakhch, O., Teisner, B. and Grudzinskas, J.G. (1993b) Evidence for the in vitro de-novo synthesis of immunoglobulin and a previously underscribed 17 kDa protein (TEP-2) by the mucosa of the Fallopian tube. Hum. Reprod. 8, 1199-1202.
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