Reprod. Fertil. Dev., 1992, 4 , 249-57
Oviducal Fluid Protein Patterns in the Rhesus Monkey (Macaca mulatta) during the Menstrual Cycle
A. Paliwal, B. ~ a l a v i ~ aand * V. P. Kamboj Division of Endocrinology, Central Drug Research Institute, Lucknow - 226 001, India. A To whom reprint requests should be addressed.
Abstract Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviducal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviducal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviducal fluid changes during the menstrual cycle.
Introduction Oviducal fluid provides the environment in which the initial events of reproduction occur; transport of gametes, maturation of the ovum, sperm capacitation and the subsequent acrosome reaction, cumulus cell dispersion, degeneration of supernumerary sperm and penetration of the zona pellucida and vitellus by sperm etc. Recognition of the importance of the luminal environment has led numerous investigators to study the biochemical nature of the oviducal milieu and its regulation by hormones. Several studies have shown that both transudatory and secretory events contribute to the formation of oviducal fluid (Leese 1988). In all the mammalian species studied, the oviducal epithelium secretes at least one reproductive stage-specific oestrogen-dependent macromolecule (Oliphant and Ross 1982; Sutton et al. 1984; Kapur and Johnson 1985; Fazleabas and Verhage 1986; Leveille et al. 1987; Verhage et al. 1988). However, the tuba1 proteins of the rhesus monkey, an animal that has a close phylogenic relationship and a physiological similarity to the human, have received little attention. This study was undertaken in order to determine the protein profile of Fallopian tube flushings during different stages of the menstrual cycle in the rhesus monkey. Materials and Methods Animals Adult female rhesus monkeys (body weight 4-6 kg) used in this study were housed in the Institute's primate colony and maintained in a 12 L : 12 D photoperiod, at an ambient temperature of 25 2OC.
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They were fed on commercial pellet diet (Lipton India, Bangalore), seasonal fruits, soaked grams (Cicer arietinum) and water ad libitum. The animals were monitored for regular cyclicity for two months. Groups of two animals were killed by an intravenous injection of a saturated solution of MgClz on Days 8, 14, 19 and 25 of the menstrual cycle. Day 1 of the menstrual cycle was the day that menstrual bleeding was first observed.
Fallopian Tube Flushings Both oviducts were excised from each animal at the time of slaughter. They were trimmed of excess non-oviducal tissue under sterile conditions in the laboratory and blotted on sterile gauze to minimize contamination with blood. Each oviduct was flushed with 0.5 mL sterile saline (0.9% w/v) by inserting a 22-gauge needle attached to a 1 mL syringe into the ampullary lumen and collecting the lavage from the isthmic end. Flushings obtained from both the tubes of each animal were pooled, centrifuged at 1700 g for 15 min at 4OC and then stored at -20°C until needed. Serum Before slaughter 2 mL blood was drawn from each animal to obtain serum for comparative studies. Tissue Cytosol Tuba1 tissue was weighed, rinsed 3 or 4 times in ice-cold 50 mM Tris-HC1 buffer (pH 7.4) containing 1 mM mercaptoethanol, 1 mM ethylenediamine tetraacetic acid (EDTA) and 10% glycerol, and then minced. The minced tissue was homogenized in similar buffer in an ultraturrax homogenizer (Kinematica P T 10-35, Switzerland). The homogenate was centrifuged at 105 000 g for 60 min at 4°C in an ultracentrifuge (International centrifuge IEC/B-60, USA). The supernatant (cytosol) was aspirated and stored at -20°C for 2-3 days. Determination of Protein Concentration The protein concentrations of Fallopian tube fluids and cytosol samples were determined by the method described by Bradford (1976), using a protein assay kit-11 (Bio-Rad Laboratories, Richmond, CA). Electrophoretic Procedure Serum, Fallopian tube flushings and cytosolic samples were analysed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% gels according to the method described by Laemmli (1970). Molecular weight markers (Bio-Rad Laboratories) were run on each gel as standards. A total of 80 pg protein was loaded into each lane of the gel. Electrophoresis was carried out at 30 mA constant current mode. Immediately after the completion of an electrophoresis run, gels were stained with periodic acid Schiff's (PAS) reagent (Fairbanks et al. 1971) and then with coomassie brilliant blue. PAS and coomassie blue stained protein patterns were scanned at 560 and 550 nm respectively by means of a thin layer chromatography (TLC) scanner (model CS-930, Shimadzu, Japan).
Results Monkeys were sacrificed on different days of the menstrual cycle. At autopsy on Day 8, visual observation of the ovaries showed growing follicles. Day 14 was marked by the presence of a mature corpus haemorrhagicum. The monkeys autopsied on Days 19 and 25 presented the gross morphological changes that are compatible with that stage of the menstrual cycle. These changes were later confirmed by histological observations. There was a consistency in the qualitative protein patterns with little variation between the two animals in any given group. The densitometric scans of PAS and coomassie blue stained protein patterns of Fallopian tube flushings obtained on Days 8, 14, 19 and 25 of the cycle are shown superimposed in Figs 1-4. The coomassie blue stained electrophoretic patterns (7.5% SDS-PAGE) of Fallopian tube flushings obtained on Days 8, 14, 19 and 25 of the menstrual cycle are shown in Fig. 5 and the protein concentrations of the flushings are shown in Table 1. The protein profiles were complex and, although most of the proteins were present throughout the cycle, a few showed a cyclic variation in intensity. Thus 85 and 95 kDa proteins (peak numbers
Protein Patterns in Monkey Oviducal Fluid
Fig. 1. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 8 Fallopian tube flushing (7.5% SDS-PAGE).
-
I 42
66
97 116
Molecular weight x 1 o
200
-~
8 and 9) which were prominent on Days 7 and 14 (Figs 1 and 2) were far less prominent (negligible) on Days 19 and 25 of the menstrual cycle (Figs 3 and 4). Other proteins which showed a relatively high intensity during the follicular stage of the cycle had molecular
Fig. 2. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 14 Fallopian tube flushing (7.5% SDS-PAGE).
1 42
66
97 116
Molecular weight x 1o
-~
200
A. Paliwal et al.
Fig. 3. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 19 Fallopian tube flushing (7.5% SDS-PAGE).
42
66
97 116
200
1
Molecular weight x 10-3
masses of approximately 46, 54, 56 and 70 kDa (peak numbers 1, 3, 4 and 6; Figs 1 and 2). However, noteworthy change was observed in the intensity of the 130 kDa protein (peak number 10; Figs 1-4). It showed a progressive increase in intensity from Day 8 to 14
Fig. 4. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 25 Fallopian tube flushing (7.5% SDS-PAGE).
Molecular weight x 10-3
Protein Patterns in Monkey Oviducal Fluid
Fig. 5. Coomassie blue stained electrophoretic patterns (7 ~ 5 % SDS-PAGE) of Fallopian tube flushings obtained on Days 8 (lanes 2 and 3), 14 (lanes 4 and 5), 19 (lanes 6 and 7) and 25 (lanes 8 and 9) of the menstrual cycle. Marker proteins are in lane 1 and serum is in lane 10. Molecular mass markers are myosin (200 kDa), 0-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa) and ovalbumin (42 kDa). Proteins of molecular mass 130, 95 and 85 kDa are marked with arrows.
Table 1. Protein concentration (mg m~-') of oviducal flushings on different days of the menstrual cycle
Monkey no.
Day 8
Day 14
Day 19
Day 25
and a decrease in intensity thereafter so as to be undetectable on Days 19-25. This protein migrated as a diffuse band on the 7 . 5 % gels and stained more intensely with PAS stain than did the other protein bands. The relative percentage data, computed by scanning through the relative peak areas, revealed that this protein formed about 23% of the protein present in tuba1 flushings obtained on Day 14. A comparative analysis of the electrophoretic (protein) pattern of Fallopian tube flushings with that of serum (obtained from the same animal at the same time as the flushings, just before the animal was killed), revealed the presence of certain proteins in the oviducal fluid that had mobilities the same as those of serum proteins (Fig. 6). Careful examination of the superimposed patterns revealed that relatively high concentrations of 48-54, 74-95 and 156 kDa proteins (peak numbers 2-3, 7-9 and 11 in serum), were also present in oviducal fluid. However, oviducal fluid also contained at least 6-7 proteins with a molecular mass of 46, 56, 60-64, 70 and 130 kDa (peak numbers 1, 4, 5, 6 and 10). These proteins appear to be unique to oviducal fluid in that they were not detected in serum.
A. Paliwal et al.
Fig. 6 . A , superimposed densitometric scan of the coomassie blue stained electrophoretic patterns (7.5 % SDSPAGE) of (........) serum and (-) Fallopian tube flushings obtained from the same monkey at Day 8 of the menstrual cycle.
Molecular weight x 10-3
1
2
3
4
5
Fig. 7. Coomassie blue stained electrophoretic patterns of tissue cytosols of Fallopian tubes obtained on Days 8 (lanes 2 and 3), 14 (lanes 4 and 5), 19 (lanes 6 and 7) and 25 (lanes 8 and 9) of the menstrual cycle.
Unlike the Fallopian tube flushings, tissue cytosols obtained from the four phases of the menstrual cycle had similar electrophoretic (protein) patterns with the exception of the 130 kDa glycoprotein. Although less distinct, this glycoprotein was only present in the soluble cytosolic fractions of oviducts obtained on Days 8, 14 and 19 of the menstrual cycle (Fig. 7).
Protein Patterns in Monkey Oviducal Fluid
Discussion The results of the present study clearly demonstrate that monkey oviducal flushings contain both serum and nonserum proteins. The electrophoretic pattern of proteins changes during the menstrual cycle and certain phase specific proteins were identified. Most noteworthy among these proteins was the 130 kDa protein that appeared to be an oviductspecific secretory product; it was present in both the oviducal flushings and the tissue cytosol but was absent from the serum of the same animals. As these studies are preliminary, tissue infusions to remove contaminating blood were not carried out. This was probably present in tissue cytosols but the method of collection of tubal flushings minimized contamination with blood. The finding of a 'mid cyclic' 130 kDa protein is in agreement with the reported findings in other primate species such as the baboon (Fazleabas and Verhage 1986), and human (Verhage et al. 1988). In an attempt to rule out the possibility of serum proteins contaminating the tubal flushings, Fazleabas and Verhage (1986) used explant culture techniques to demonstrate that a 130 kDa macromolecule was present only in the tissue culture medium of oviducts obtained during the mid to late follicular stages in the baboon and during midcycle in the human. In an in vitro study on the oviducts of ovariectomized, oestradiol and/or progesterone treated baboons, the oestradiol dependent synthesis of a 100-130 kDa glycoprotein was demonstrated; in addition, various oviducal tubal regions exhibited differences in their ability to synthesize the glycoprotein (Verhage and Fazleabas 1988). Like the oviducal proteins of the human and baboon, the 130 kDa protein present in the monkey oviducal flushings is also a glycoprotein (as indicated by the diffuse electrophoretic pattern and the significant PAS staining of the band). Considering the similarity of the three primate species, it is perhaps not surprising that the subunit size of the secretory protein is similar. However, immunological homology has yet to be established. By means of immunocytochemical methods, Verhage et al. (1990) located a 130 kDa glycoprotein within the secretory granules of the oviducal epithelium of the baboon. Secretory cells of human (Verhage et al. 1988) and monkey (Pathak et al. 1978) tubal epithelium also contain secretory granules in the apical region but, only during mid cycle; it is possible that these secretory granules may contain a 130 kDa glycoprotein which is released into the lumen of the oviduct. Major reproductive phase-specific proteins have now been identified in the mouse (Kapur and Johnson 1985), hamster (Leveille et al. 1987), rabbit (Oliphant and Ross 1982), sheep (Sutton et al. 1984), baboon (Fazleabas and Verhage 1986), pig (Buhi et al. 1989), cattle (Joshi 1988), human (Verhage et al. 1988) and rhesus monkey. Although evidence is accumulating that specific oviducal macromolecules associate themselves with ova and embryos during their transit through the oviduct (Boice et al. 1990), it is not yet known (to us) if this sequestration process is specific. Regardless of the mechanism whereby it is achieved, the intimate association of oviducal antigens with mammalian eggs may have biological significance. The interaction of gametes and subsequent fertilization events may be influenced by specific-oviducal secretory macromolecules. In the hamster, the ability of sperm to bind to ova is reduced and in vitro fertilization is inhibited if the ovulated ova are treated with a monoclonal antibody prepared against the characterized hamster oviducal glycoprotein (Sakai et al. 1988). However, it is known that follicular and ovulated oocytes can be fertilized in vitro without exposure to the oviduct. This suggests that oviducal antigens may play an obligatory role in facilitating the fertilization process in vitro. It is tempting to speculate that oviducal glycoproteins play a role in embryonic development and survival. Several studies have demonstrated that embryonic development in vitro is facilitated by coincubation with isolated oviducts (Krisher et al. 1989), oviducal cells in suspension (Eyestone and First 1989) or monolayer culture (Rexroad and Powell 1988) as well as with medium previously incubated with oviducal cells (Krisher et al. 1989). Also, in vivo development is enhanced if sheep embryos are cocultured with oviducal cells prior
A. Paliwal et al.
to transfer, as compared to embryos cultured with other cell types or with media alone (Eyestone and First 1989). Further studies are needed in order to determine the functional significance of the oviducal secretory proteins. The 130 kDa protein found in the monkey oviduct may be used as a marker for detecting oestrogenic activity in primates. Acknowledgments
The authors wish to thank Professor B. N. Dhawan for his interest in these studies. Financial assistance given by the Ministry of Health and Family Welfare, Government of India, is gratefully acknowledged.
References Boice, M. L., McCarthy, T. J., Mavrogianis, P. A., Fazleabas, A. T., and Verhage, H. G . (1990). Localization of oviductal glycoproteins with the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons. (Papio arubis). Biol. Reprod. 43, 340-6. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-54. Buhi, W. C., Vallet, J . L., and Bazer, F. W. (1989). De novo synthesis and release of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture. J. Exp. 2001.252, 79-88. Eyestone, W. H . , and First, N. L. (1989). Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil. 85, 715-20. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971). Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-16. Fazleabas, A. T., and Verhage, H. G . (1986). The detection of oviduct-specific proteins in the baboon (Papio anubis). Biol. Reprod. 35, 455-62. Joshi, M . S. (1988). Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow. J. Reprod. Fertil. 83, 249-61. Kapur, R. P., and Johnson, L. V. (1985). An oviductal fluid glycoprotein associated with ovulated mouse ova and early embryos. Dev. Biol. 112, 89-93. Krisher, R. L., Petters, R. M., Johnson, B. H., Bavistar, B. D., and Archibong, A. E. (1989).Development of porcine embryos from one-cell stage to blastocyst in mouse oviducts maintained in organ culture. J. Exp. Zool. 249, 235-9. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature (Lond.) 227, 680-5. Leese, H. J . (1988). The formation and function of oviductal fluid. J. Reprod. Fertil. 82, 843-56. Leveille, M. C., Roberts, K. D., Chavalier, S., Chapdelaine, A., and Bleau, G. (1987). Uptake of an oviductal antigen by the hamster zona pellucida. Biol. Reprod. 36, 227-38. Oliphant, G., and Ross, P. R. (1982). Demonstration of production and isolation of three sulfated glycoproteins from the rabbit oviduct. Biol. Reprod. 26, 537-44. Pathak, R. K., Bajpai, V. K., Shipstone, A. C., Chandra, H., and Karkun, J. N. (1978). Studies on primate fallopian tube. Part I. Observations on the tuba1 mucosa of immature rhesus monkey (Macaca mulatta). Indian J. Exp. Biol. 16, 1244-62. Rexroad, C. E. Jr, and Powell, A. M. (1988). Co-culture of ovine ova with oviductal cells in medium 199. J. Anim. Sci. 66, 947-53. Sakai, Y., Araki, Y., Yamashita, T., Kurata, S., Oikawa, T., Hiroi, M., and Sendo, F. (1988). Inhibition of in vitro fertilization by a monoclonal antibody reacting with the zona pellucida of the oviductal egg but not with that of the ovarian egg of the golden hamster. J. Reprod. Zmmunol. 14, 177-89. Sutton, R., Nancarrow, C. D., Wallace, A. L. C., and Rigby, N. W. (1984). Identification of an oestrus-associated glycoprotein in oviductal fluid of the sheep. J. Reprod. Fertil. 72, 415-22. Verhage, H. G . , and Fazleabas, A. T. (1988). The in vitro synthesis of estrogen dependent proteins by the baboon (Papio anubis) oviduct. Endocrinology 123, 552-8.
Protein Patterns in Monkey Oviducal Fluid
Verhage, H. G., Fazleabas, A. T., and Donnelly, K. (1988). The in vitro synthesis and release of proteins by the human oviduct. Endocrinology 127, 1639-45. Verhage, H. G., Mavrogianis, P. A., Boice, M. L., Li, W., and Fazleabas, A. T. (1990). Oviductal epithelium of the baboon, hormonal control and the immunogold localization of oviduct-specific glycoproteins. Am. J. Anat. 187, 81-90.
Manuscript received 10 July 1991; revised 21 October 1991; accepted 16 April 1992
Oviducal Fluid Protein Patterns in the Rhesus Monkey (Macaca mulatta) during the Menstrual Cycle
A. Paliwal, B. ~ a l a v i ~ aand * V. P. Kamboj Division of Endocrinology, Central Drug Research Institute, Lucknow - 226 001, India. A To whom reprint requests should be addressed.
Abstract Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviducal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviducal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviducal fluid changes during the menstrual cycle.
Introduction Oviducal fluid provides the environment in which the initial events of reproduction occur; transport of gametes, maturation of the ovum, sperm capacitation and the subsequent acrosome reaction, cumulus cell dispersion, degeneration of supernumerary sperm and penetration of the zona pellucida and vitellus by sperm etc. Recognition of the importance of the luminal environment has led numerous investigators to study the biochemical nature of the oviducal milieu and its regulation by hormones. Several studies have shown that both transudatory and secretory events contribute to the formation of oviducal fluid (Leese 1988). In all the mammalian species studied, the oviducal epithelium secretes at least one reproductive stage-specific oestrogen-dependent macromolecule (Oliphant and Ross 1982; Sutton et al. 1984; Kapur and Johnson 1985; Fazleabas and Verhage 1986; Leveille et al. 1987; Verhage et al. 1988). However, the tuba1 proteins of the rhesus monkey, an animal that has a close phylogenic relationship and a physiological similarity to the human, have received little attention. This study was undertaken in order to determine the protein profile of Fallopian tube flushings during different stages of the menstrual cycle in the rhesus monkey. Materials and Methods Animals Adult female rhesus monkeys (body weight 4-6 kg) used in this study were housed in the Institute's primate colony and maintained in a 12 L : 12 D photoperiod, at an ambient temperature of 25 2OC.
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They were fed on commercial pellet diet (Lipton India, Bangalore), seasonal fruits, soaked grams (Cicer arietinum) and water ad libitum. The animals were monitored for regular cyclicity for two months. Groups of two animals were killed by an intravenous injection of a saturated solution of MgClz on Days 8, 14, 19 and 25 of the menstrual cycle. Day 1 of the menstrual cycle was the day that menstrual bleeding was first observed.
Fallopian Tube Flushings Both oviducts were excised from each animal at the time of slaughter. They were trimmed of excess non-oviducal tissue under sterile conditions in the laboratory and blotted on sterile gauze to minimize contamination with blood. Each oviduct was flushed with 0.5 mL sterile saline (0.9% w/v) by inserting a 22-gauge needle attached to a 1 mL syringe into the ampullary lumen and collecting the lavage from the isthmic end. Flushings obtained from both the tubes of each animal were pooled, centrifuged at 1700 g for 15 min at 4OC and then stored at -20°C until needed. Serum Before slaughter 2 mL blood was drawn from each animal to obtain serum for comparative studies. Tissue Cytosol Tuba1 tissue was weighed, rinsed 3 or 4 times in ice-cold 50 mM Tris-HC1 buffer (pH 7.4) containing 1 mM mercaptoethanol, 1 mM ethylenediamine tetraacetic acid (EDTA) and 10% glycerol, and then minced. The minced tissue was homogenized in similar buffer in an ultraturrax homogenizer (Kinematica P T 10-35, Switzerland). The homogenate was centrifuged at 105 000 g for 60 min at 4°C in an ultracentrifuge (International centrifuge IEC/B-60, USA). The supernatant (cytosol) was aspirated and stored at -20°C for 2-3 days. Determination of Protein Concentration The protein concentrations of Fallopian tube fluids and cytosol samples were determined by the method described by Bradford (1976), using a protein assay kit-11 (Bio-Rad Laboratories, Richmond, CA). Electrophoretic Procedure Serum, Fallopian tube flushings and cytosolic samples were analysed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% gels according to the method described by Laemmli (1970). Molecular weight markers (Bio-Rad Laboratories) were run on each gel as standards. A total of 80 pg protein was loaded into each lane of the gel. Electrophoresis was carried out at 30 mA constant current mode. Immediately after the completion of an electrophoresis run, gels were stained with periodic acid Schiff's (PAS) reagent (Fairbanks et al. 1971) and then with coomassie brilliant blue. PAS and coomassie blue stained protein patterns were scanned at 560 and 550 nm respectively by means of a thin layer chromatography (TLC) scanner (model CS-930, Shimadzu, Japan).
Results Monkeys were sacrificed on different days of the menstrual cycle. At autopsy on Day 8, visual observation of the ovaries showed growing follicles. Day 14 was marked by the presence of a mature corpus haemorrhagicum. The monkeys autopsied on Days 19 and 25 presented the gross morphological changes that are compatible with that stage of the menstrual cycle. These changes were later confirmed by histological observations. There was a consistency in the qualitative protein patterns with little variation between the two animals in any given group. The densitometric scans of PAS and coomassie blue stained protein patterns of Fallopian tube flushings obtained on Days 8, 14, 19 and 25 of the cycle are shown superimposed in Figs 1-4. The coomassie blue stained electrophoretic patterns (7.5% SDS-PAGE) of Fallopian tube flushings obtained on Days 8, 14, 19 and 25 of the menstrual cycle are shown in Fig. 5 and the protein concentrations of the flushings are shown in Table 1. The protein profiles were complex and, although most of the proteins were present throughout the cycle, a few showed a cyclic variation in intensity. Thus 85 and 95 kDa proteins (peak numbers
Protein Patterns in Monkey Oviducal Fluid
Fig. 1. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 8 Fallopian tube flushing (7.5% SDS-PAGE).
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I 42
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97 116
Molecular weight x 1 o
200
-~
8 and 9) which were prominent on Days 7 and 14 (Figs 1 and 2) were far less prominent (negligible) on Days 19 and 25 of the menstrual cycle (Figs 3 and 4). Other proteins which showed a relatively high intensity during the follicular stage of the cycle had molecular
Fig. 2. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 14 Fallopian tube flushing (7.5% SDS-PAGE).
1 42
66
97 116
Molecular weight x 1o
-~
200
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Fig. 3. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 19 Fallopian tube flushing (7.5% SDS-PAGE).
42
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200
1
Molecular weight x 10-3
masses of approximately 46, 54, 56 and 70 kDa (peak numbers 1, 3, 4 and 6; Figs 1 and 2). However, noteworthy change was observed in the intensity of the 130 kDa protein (peak number 10; Figs 1-4). It showed a progressive increase in intensity from Day 8 to 14
Fig. 4. Superimposed densitometric scan of coomassie blue and PAS stained electrophoretic pattern of a Day 25 Fallopian tube flushing (7.5% SDS-PAGE).
Molecular weight x 10-3
Protein Patterns in Monkey Oviducal Fluid
Fig. 5. Coomassie blue stained electrophoretic patterns (7 ~ 5 % SDS-PAGE) of Fallopian tube flushings obtained on Days 8 (lanes 2 and 3), 14 (lanes 4 and 5), 19 (lanes 6 and 7) and 25 (lanes 8 and 9) of the menstrual cycle. Marker proteins are in lane 1 and serum is in lane 10. Molecular mass markers are myosin (200 kDa), 0-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa) and ovalbumin (42 kDa). Proteins of molecular mass 130, 95 and 85 kDa are marked with arrows.
Table 1. Protein concentration (mg m~-') of oviducal flushings on different days of the menstrual cycle
Monkey no.
Day 8
Day 14
Day 19
Day 25
and a decrease in intensity thereafter so as to be undetectable on Days 19-25. This protein migrated as a diffuse band on the 7 . 5 % gels and stained more intensely with PAS stain than did the other protein bands. The relative percentage data, computed by scanning through the relative peak areas, revealed that this protein formed about 23% of the protein present in tuba1 flushings obtained on Day 14. A comparative analysis of the electrophoretic (protein) pattern of Fallopian tube flushings with that of serum (obtained from the same animal at the same time as the flushings, just before the animal was killed), revealed the presence of certain proteins in the oviducal fluid that had mobilities the same as those of serum proteins (Fig. 6). Careful examination of the superimposed patterns revealed that relatively high concentrations of 48-54, 74-95 and 156 kDa proteins (peak numbers 2-3, 7-9 and 11 in serum), were also present in oviducal fluid. However, oviducal fluid also contained at least 6-7 proteins with a molecular mass of 46, 56, 60-64, 70 and 130 kDa (peak numbers 1, 4, 5, 6 and 10). These proteins appear to be unique to oviducal fluid in that they were not detected in serum.
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Fig. 6 . A , superimposed densitometric scan of the coomassie blue stained electrophoretic patterns (7.5 % SDSPAGE) of (........) serum and (-) Fallopian tube flushings obtained from the same monkey at Day 8 of the menstrual cycle.
Molecular weight x 10-3
1
2
3
4
5
Fig. 7. Coomassie blue stained electrophoretic patterns of tissue cytosols of Fallopian tubes obtained on Days 8 (lanes 2 and 3), 14 (lanes 4 and 5), 19 (lanes 6 and 7) and 25 (lanes 8 and 9) of the menstrual cycle.
Unlike the Fallopian tube flushings, tissue cytosols obtained from the four phases of the menstrual cycle had similar electrophoretic (protein) patterns with the exception of the 130 kDa glycoprotein. Although less distinct, this glycoprotein was only present in the soluble cytosolic fractions of oviducts obtained on Days 8, 14 and 19 of the menstrual cycle (Fig. 7).
Protein Patterns in Monkey Oviducal Fluid
Discussion The results of the present study clearly demonstrate that monkey oviducal flushings contain both serum and nonserum proteins. The electrophoretic pattern of proteins changes during the menstrual cycle and certain phase specific proteins were identified. Most noteworthy among these proteins was the 130 kDa protein that appeared to be an oviductspecific secretory product; it was present in both the oviducal flushings and the tissue cytosol but was absent from the serum of the same animals. As these studies are preliminary, tissue infusions to remove contaminating blood were not carried out. This was probably present in tissue cytosols but the method of collection of tubal flushings minimized contamination with blood. The finding of a 'mid cyclic' 130 kDa protein is in agreement with the reported findings in other primate species such as the baboon (Fazleabas and Verhage 1986), and human (Verhage et al. 1988). In an attempt to rule out the possibility of serum proteins contaminating the tubal flushings, Fazleabas and Verhage (1986) used explant culture techniques to demonstrate that a 130 kDa macromolecule was present only in the tissue culture medium of oviducts obtained during the mid to late follicular stages in the baboon and during midcycle in the human. In an in vitro study on the oviducts of ovariectomized, oestradiol and/or progesterone treated baboons, the oestradiol dependent synthesis of a 100-130 kDa glycoprotein was demonstrated; in addition, various oviducal tubal regions exhibited differences in their ability to synthesize the glycoprotein (Verhage and Fazleabas 1988). Like the oviducal proteins of the human and baboon, the 130 kDa protein present in the monkey oviducal flushings is also a glycoprotein (as indicated by the diffuse electrophoretic pattern and the significant PAS staining of the band). Considering the similarity of the three primate species, it is perhaps not surprising that the subunit size of the secretory protein is similar. However, immunological homology has yet to be established. By means of immunocytochemical methods, Verhage et al. (1990) located a 130 kDa glycoprotein within the secretory granules of the oviducal epithelium of the baboon. Secretory cells of human (Verhage et al. 1988) and monkey (Pathak et al. 1978) tubal epithelium also contain secretory granules in the apical region but, only during mid cycle; it is possible that these secretory granules may contain a 130 kDa glycoprotein which is released into the lumen of the oviduct. Major reproductive phase-specific proteins have now been identified in the mouse (Kapur and Johnson 1985), hamster (Leveille et al. 1987), rabbit (Oliphant and Ross 1982), sheep (Sutton et al. 1984), baboon (Fazleabas and Verhage 1986), pig (Buhi et al. 1989), cattle (Joshi 1988), human (Verhage et al. 1988) and rhesus monkey. Although evidence is accumulating that specific oviducal macromolecules associate themselves with ova and embryos during their transit through the oviduct (Boice et al. 1990), it is not yet known (to us) if this sequestration process is specific. Regardless of the mechanism whereby it is achieved, the intimate association of oviducal antigens with mammalian eggs may have biological significance. The interaction of gametes and subsequent fertilization events may be influenced by specific-oviducal secretory macromolecules. In the hamster, the ability of sperm to bind to ova is reduced and in vitro fertilization is inhibited if the ovulated ova are treated with a monoclonal antibody prepared against the characterized hamster oviducal glycoprotein (Sakai et al. 1988). However, it is known that follicular and ovulated oocytes can be fertilized in vitro without exposure to the oviduct. This suggests that oviducal antigens may play an obligatory role in facilitating the fertilization process in vitro. It is tempting to speculate that oviducal glycoproteins play a role in embryonic development and survival. Several studies have demonstrated that embryonic development in vitro is facilitated by coincubation with isolated oviducts (Krisher et al. 1989), oviducal cells in suspension (Eyestone and First 1989) or monolayer culture (Rexroad and Powell 1988) as well as with medium previously incubated with oviducal cells (Krisher et al. 1989). Also, in vivo development is enhanced if sheep embryos are cocultured with oviducal cells prior
A. Paliwal et al.
to transfer, as compared to embryos cultured with other cell types or with media alone (Eyestone and First 1989). Further studies are needed in order to determine the functional significance of the oviducal secretory proteins. The 130 kDa protein found in the monkey oviduct may be used as a marker for detecting oestrogenic activity in primates. Acknowledgments
The authors wish to thank Professor B. N. Dhawan for his interest in these studies. Financial assistance given by the Ministry of Health and Family Welfare, Government of India, is gratefully acknowledged.
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Protein Patterns in Monkey Oviducal Fluid
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Manuscript received 10 July 1991; revised 21 October 1991; accepted 16 April 1992
