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Periovulatory glycoprotein secretion in the macaque fallopian tube
Periovulatory glycoprotein fallopian tube Robert P. S. Jansen, V. K. Bajpai, Ph.D. Sun Antonio, Anatomic electron includes secreted
M.B., B.S., B.Sc.(Med.),
secretion in the macaque F.R.A.C.P.,
F.R.A.C.O.G.,
and
Texas
demonstration of a specific midcycle fallopian tube secretion has been sought by transmission microscopy in the Macaca mulaffa fascicularis with a method of in vivo perfusion-fixation that the polycation alcian blue (plus glutaraldehyde) to precipitate and stabilize anionic extracellular glycoproteins. Of nine monkeys entered into the study, six ovulated regularly. With the help of
daily plasma total estrogen radioimmunoassays and serial laparoscopies, these six monkeys were killed and the pelvic organs were perfused at specific times in the menstrual cycle: the midfollicular phase (one), during the midcycle estrogen peak immediately before ovulation (one), 24 hours after ovulation (one), 2 to 4 days after ovulation (two), and premenstrually (one). The other three monkeys were oophorectomized and were treated with estradiol benzoate, 10 pgikglday for 7 days (two), or with estradiol benzoate, 10 fig/kg/day for 7 days, and progesterone in oil, 1 mg/kg/day from day 3 to day 7. Evenly distributed, granular, precipitated material was found in the isthmic lumen both immediately before and 24 hours after ovulation (and in the two oophorectomized, estrogen-treated monkeys, both of which had plasma total estrogen levels that exceeded those of preovulatory monkeys); this would coincide with times of sperm ascent through the isthmus, ovulation, and fertilization and with the time of temporary impedance of the ovum’s normal transport to the uterus, known to occur at the ampullary-isthmic junction. No substantial precipitable secretion was found in the ampulla at any stage of the cycle, in the isthmus before the midcycle estrogen rise, or 48 hours or more after either ovulation or initiation of progesterone treatment. These observations indicate that, in the primate fallopian tube, the unique ability of the isthmus to transport spermatozoa and ova in opposite directions sequentially is related temporally (and perhaps causally) to the presence and absence, respectively, of specific isthmic luminal secretions. The biochemical basis for this isthmic property may lie in the expected highly expanded state of acid mucus glycoproteins in the secretions.
(AM.
J. OBSTET.
GYNECOL.
147598,
1983.)
The fallopian tube isthmus lies between the site of fertilization, the ampulla, and the site of implantation, the uterus. The isthmus must, therefore, be capable of effecting transport of spermatozoa and ova in opposite directions. This dual transport capability is sequential. After distal sperm ascent and fertilization of the newly ovulated egg in the ampulla, the egg has its proximal transport delayed in the ampulla at or near the ampullary-isthmic junction for at least 48 hours in rhesus monkeys’ and in humans” before the isthmus is passed and the ovum reaches the uterus. The physiologic basis for this ovum transport delay
From the Depnrtmmt of Obstetrics and Gynecology, Unizwsity of Texas Health Science Centtl at San Antonio. Supported by University of Texas Institutional Grant 626.167121 I5 (to R. P. S. J.) and, in part, by National Institutes of Health Grants P30 HD10202 (Centerfor Research in Reproductive Biology: Bioassay Core, Morphology Core, Radioimmunoassay Core) and HDO9039 (Physiology and Pharmacology of Ovum Transport). V. K. B. was on a World Health Organization Postdoctoral Fellowship. Presented at the Thirtieth Annual Meeting of the Society for Gynecologic Investigation, Washington, D. C., March 17-20, 1983. Reprint requests: Dr. Robert P. S. Jansm, The Fertility Laboratory, King George I’ Memorial Hospital, Camper-down, Sydney 2050, Australia. 598
at the ampullary-isthmic junction is not understood. A similar delay occurs in rabbits, and extensive studies of smooth muscle and ciliary activity have failed wholly to account either for the bidirectionality of the sperm-and ovum-transporting capabilities of the isthmus or for the delay of the ovum at the ampullary-isthmic junction.“-” Earlier studies indicated that the approximate time during which sperm transport takes place distally, and during which proximally directed ovum transport is interrupted, coincides with the presence of specific, precipitable secretion in the isthmic lumen. These findings were based on surface electron microscopic appearances in estrous rabbit? and midcycle humans.’ but there was concern that tubal constriction at this time might have artifactually preserved luminal secretions that at other stages of the cycle were washed out. Subsequently, more precise transmission electron microscopic studies of tissues fixed by vascular perfusion with glutaraldehyde and the polycation alcian blue (to precipitate and stabilize secreted glycoproteins) confirmed the presence of material identifiable histochemically as acid mucus glycoprotein in the isthmic lumen of estrous rabbits.8 Because these procedures for in situ fixation of secreted glycoproteins are highly toxic, these studies could not be extended to human
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Fig. 1. Early follicular phase isthmic epithelium showing ciliated and nonciliated activity is present, but ribosomes are abundant in the cytoplasm. tissu es. The rhesus monkey species Macaca f ascii xdaris (crab-eating macaque) has been instc tad. Mi aterial and methods Nine sexually M onkeys. (crab-eating macaques) keys
mulattu studied
mature cynomolgus monwere bought from Primate
599
cells: no secretory
Imports (now Charles River Primate Imports, PC31-t Washington, New York). The crab-eating macaque, or M. mulattafusciculur~, is closely related to the larger a nd more familiar rhesus monkey, M. muluttu mukzttu; th leir interbreeding both in captivity and where their natu ral ranges are contiguous has shown these monkeys to be two races of the same species,Y and it has been con5 il‘d -
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and Bajpai
Fig. 2. Low-power view of early follicular phase (Aj and late folhcular phase (B) tubal isthmus in cross section, showing a substantial increase in stainable acid mucus glycoproteins. [High-iron diamine (pH l.O), alcian blue (pH 2.5), and nuclear fast red.] ered that for reproductive studies data obtained for one should be applicable to the other.” After purchase the animals were quarantined for a further 6 weeks before introduction to the primate colony at the University of Texas Health Science Center at San Antonio, where they were individually caged under controlled conditions of 75” F, 50% relative humidity, and 14 hours of fluorescent light per day. Subsequent reproductive cyclicity was documented daily with the monkeys under ketamine-induced tranquility (ketamine hydrochloride, 3 to 4 mg intramuscularly) (1) by plasma total estrogen radioimmunoassays (RIAs) after femoral vein puncture and (2) by daily vaginal swabbing for menstrual blood. Six of the nine monkeys ovulated regularly and were entered into the study after observation in a control cycle. Plasma total estrogen and plasma progesterone were assayed daily in duplicate during the study cycle according to methods described previously”; estrogen results were available on the same day. Serial laparoscopies performed daily (and sometimes twice daily) with the animals under ketamine-induced narcosis were started when rising plasma total estrogen levels indicated preovulatory follicular development. The characteristic appearances of immediately preovulatory and postovulatory follicles have been described,” preovulatory follicles being characterized by formation of translucent areas in the follicular blister and postovulatory follicles bearing a hemorrhagic stigma to mark the site of ovulation. Frequent laparoscopies in rhesus monkeys have been shown not to interfere with the physical and endocrine events associated with ovulation.“. I2 Monkeys were put to death before and after the time of ovulation as follows: in the midfollicularpha,se (n = 1; early follicular development, no plasma total estrogen rise had yet been evident); immediately before ovulation
November 1, 1983 Am. J. Obstet. Cynecol.
(n = 1; 1.5 cm follicle present, preovulatory increase in plasma total estrogen level had begun); about 24 hours after ovulation (n = 1; follicle appeared immediately preovulatory at laparoscopy 48 hours previously; plasma total estrogen level had fallen after midcycle peak); 2 to 4 days after ovulation (n = 2); and in the late luteal phase (n = 1; just over 1 week after ovulation was seen laparoscopically, with endometrium histologically in the early menstrual phase). Three monkeys failed to ovulate during the time they were observed, and bilateral oophorectomies were performed at least 1 month before hormonal therapy and sacrifice. One monkey was treated with estradiol benzoate, 10 pg/kg/ day for 7 days (plasma total estrogen levels were greater than those of the midcycle peak of ovulating monkeys); the other two monkeys received, in addition to estrogen, 4 days’ progesterone in oil, 1 mg/kg/day from day 3 to day 7. With the animals under ketamine-induced narcosis, a midline ventral laparotomy was performed. The active preovulatory or postovulatory ovary was noted. Both ovarian arteries, but not the ovarian veins, were tied to encourage collateral circulation from the uterine arteries to the fallopian tubes. The aortic bifurcation was dissected, and the median sacral artery and vein were ligated. The pelvic sidewalls were then dissected and the internal iliac arteries were tied beyond the origin of the uterine artery on each side. The femoral artery and vein were tied on each side after groin dissection. Dissection of the aorta was advanced cranially, and both the aorta and inferior vena cava were exposed; loose ligatures were placed around these vessels at a level between the superior and inferior mesenteric arteries. The inferior vena cava was cannulated to allow free drainage of blood from the pelvic organs. Immediately after the aortic ligature was tied, the aort was cannulated with a Teflon cannula connected to a controlled-pressure perfusion apparatus.‘” Perfusion was begun with glutaraldehyde (1.5% in 0.1 M cacodylate buffer at pH 7.4, with an osmolality of 350 mOsm/L, determined with an Osmette A freezingpoint-depression automatic osmometer, Precision Instruments, Inc.) at a constant pressure of 125 mm Hg. As the glutaraldehyde infusion was begun, the monkeys were killed with an overdose of pentobarbital administered through a mesenteric vein. The ovarian veins were slit open to allow drainage. Ultrastructural studies. The uterus and tubes, together with (in most cases) the bladder and lower colon, were thus fixed in situ with glutaraldehyde for 5 minutes. The perfusing solution was then changed to one containing, in addition to the glutaraldehyde and cacodylate buffer as above, 1% alcian blue (freshly prepared and filtered, with an osmolality of 465 mOsm/L). The previously blanched and hardened tissues turned
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5
Fig. 3. A, Preovulatory
tubal isthmus showing glycogen and acid mucus glycoproteins. (PAS and hematoxylin.) B, Diastase digestion removes glycogen but glycoproteins remain. (PAS and hematoxylin.) C, Acid mucus glycoproteins revealed. (High-iron diamine (pH 1 .O), alcian blue (pH 2.5), and nuclear fast red.)
blue as the perfusate was changed from glutaraldehyde to the alcian blue-glutaraldehyde mixture, which was continued for 8 to 10 minutes. This is essentially the method described by Behnke and Zelander14 for preserving tissue mucopolysaccharides and is identical to the methodology used in our earlier study in estrous rabbitsx The fixed fallopian tubes were removed and prepared for transmission electron microscopy as described previously,” with 2 hours’ further fixation in the alcian blue-glutaraldehyde perfusate before postfixation with osmium tetroxide (2% in O.lM cacodylate buffer) for 60 minutes. At least two segments from the ampulla and two segments from the isthmus of one tube were examined; the contralateral tube was processed for light microscopy. Thin sections were examined, after staining with 1% uranyl acetate and 1% lead citrate, with a Siemens 101 electron microscope at 80 kV. Histochemical studies. From each monkey studied ultrastructurally, the contralateral glutaraldehyde-fixed fallopian tube was dehydrated, embedded in paraffin, and sectioned for light microscopic examination. Several methods were used to identify cells producing neutral, sialyated (moderately acid), and sulfated (highly acid) mucus glycoproteins: (1) periodic acid-Schiff (PAS) and Mayer’s hematoxylin, (2) high-iron diamine at pH 1.0 and alcian blue at pH 2.5, and (3) these staining reactions after diastase (Fluka, Geneva; 0.1% for 90 minutes at 37” C) to dissolve glycogen, pronase (Calbiochem; 0.1% for 2 hours at 37” C) to remove mucus glycoproteins, or neuraminidase (Sigma; 0.25 U/ml, pH 5.4, for 24 hours at 37” C) to partly remove sialic acid. Light microscopic methods were controlled by staining a section of mouse rectosigmoid colon on each occasion. The mouse rectosigmoid has surface
mucus and crypt goblet cells that stain for sulfated (highly acid) mucus glycoproteins, as well as a distinct population of deep crypt cells that stain only for sialyated (moderately acid) mucus glycoproteins.‘j Details of the methods and expected results are provided elsewhere.x Results Estrogen influence. Tubal morphology from the early follicular macaque was relatively undifferentiated (Fig. 1). Ciliogenesis was evident in the ampulla, whereas cilia were established in the isthmus. Nonciliated cells displayed free ribosomes and variable rough endoplasmic reticulum, indicating early synthesizing activity, but Golgi activity appeared minimal and serous (dark, homogeneous) granules were sparse and restricted to the ampulla. Histochemical studies indicated a corresponding lack of stainable acidic glycoproteins (Fig. 2,A). No precipitable material was present in the lumen. The preovulatory and the estradiol-treated macaque fallopian tubes showed evidence of substantial secretory activity. Acid mucus glycoproteins were evident in the mucosa from the isthmus (Fig. 2, B), ampulla, and fimbrial end (in decreasing order of abundance and electronegativity). Staining with PAS (Fig. 3, A) was partly but not completely diminished by pretreatment with diastase (Fig. 3,B), indicating the presence of both glycogen and glycoprotein. Black-staining reactions with high-iron diamine were found only in the isthmus (Fig. 3, C), ampullary acid mucus glycoproteins staining only with alcian blue at pH 2.5; isthmic mucus glycoprotein was partly resistant to neuraminidase, whereas neuraminidase virtually abolished ampullary staining. Ampullary ultrastructure showed that ciliated cells had developed fully (Fig. 4). Nonciliated cells had dif-
602
Jansen and Bajpai
Fig. 4. Preovulatory monkey ampulla. Prominent apparatus (G); domed cytoplasmic apices filled no precipitated secretion evident in the lumen.
fere :ntiated into secretory cells with apices that bulged into I the lumen and were filled with secretory granules ofv arying density. The Golgi apparatus was prominent and appeared (Fig. 4) to be involved in concentrating seer ,etory material. The rough endoplasmic reticulum
November Am. J. Obtet.
1,1983 Gyre~ol.
activity in the endoplasmic reticulum (er) and Golgi with secretory granules of varying electron density;
was also prominent, had dilated cisternae, and was filled with homogeneous pale-staining material. L ittle precipitated secretion was present in the ampul lary lumen. In the isthmus, the rough endoplasmic reticulum was variably occupied with secretion, but
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glycoprotein
secretion
603
Fig. 5. Preovulatory isthmus from the same monkey as shown in Fig. 4. Granular and membranous products of apocrine secretion pack the lumen.
g1-anular a1xxrine
and membranous products of (apparently) secretion packed the lumen (Fig. 5). Similar t-e:sults were found in ovariectomized, estradiol-treated m lacaques, indicating the estradiol dependence of tubal secretion. Secretory activity was morphologically greatest in the
immediately postovulatory macaque. Although 1 folli cular cells were being swept down the ampulla (Fig. 6), the isthmic lumen was packed tight with prel cipita ted secretion (Fig. 7), which in many transmission elect] ron microscopic fields studied appeared to have (:ome directly from rough endoplasmic reticulum Iracuo les,
604
Jansen
and Bajpai
November Am. J. Obstet.
Fig. 6. Immediately after ovulation, cumulus cells from the ruptured ampullary lumen. Nonciliated cells have lost their apical secretion. free ribosomes being evident among the secretion (Fig. 8). Much glycogen was detectable both on transmission electron microscopy and on histochemistry; although PAS reactivity was strong, diastase caused a marked loss of staining and reactivity of cells; staining with high-iron diamine and alcian blue was less than before ovulation. Progesterone influence. At 2 days after ovulation secretory vacuoles were still visible in some nonciliated cells, but dedifferentiation was in evidence; little luminal secretion was present in the isthmus other than in some mucosal crypts and there was none in the ampulla. Early luteal phase (2 to 4 days) and 4-day progesterone-treated (estradiol-primed) macaques showed similar tubal morphology. Secretory granules and vacuoles had been compacted and other cytoplasmic organelles were in disarray; the cytoplasm itself appeared concentrated: the isthmic lumen was without precipitable contents (Fig. 9). Histochemistry confirmed the rapid loss of stainable mucus glycoproteins. In the late luteal phase (Fig. 10) dedifferentiation was marked and lysozome activity was prominent; regurgitated menstrual products were seen in some sections. Comment Epithelial glycoproteins are likely to be important in many aspects of reproduction. An earlier study has
1, 1983 Gynecol.
follicle have been fixed in the
confirmed that the rabbit fallopian tube produces mucus glycoproteins.’ Histochemistry in this species showed that the production of highly acid mucus glycoprotein (apparently of the type that coats the ovum in the rabbit) is confined to the isthmus and, to a lesser extent, the mucosal crypts of the ampullary-isthmic junction; the ampulla is not involved. Using a method of perfusion-fixation that includes the polycation alcian blue in conjunction with glutaraldehyde to precipitate and stabilize glycoproteins, we demonstrated that this mucus occupied the isthmic lumen but not the ampullary lumen in estrous rabbits. The present study was designed to confirm or refute the existence of tubal luminal secretions in primates, after scanning electron microscopic evidence for such secretions had been obtained in midcycle women.7 Evenly distributed, granular, precipitated material was found in the macaque isthmic lumen both immediately before and 24 hours after ovulation and in two oophorectomized, estrogen-treated monkeys. These conditions would coincide with times of sperm ascent through the isthmus, ovulation, and fertilization and with the time of temporary impedance at the ampullary-isthmic junction of the ovum’s normal transport to the uterus. On the other hand, no substantial precipitable secretion was found in the ampulla at any stage of the cycle, in the isthmus before the midcycle estrogen rise, or 48 hours or more after either ovulation or initiation of progesterone treatment.
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147 5
605
Fig. 7. Isthmic material distinct
epithelium from the same tube as shown in Fig. 6. The lumen is packed with secretory that seems to be in direct continuity with dilated endoplasmic reticulum vacuoles and is from the small electron-dense serous granules in the cell cytoplasm.
l-Ilnlike the rabbit, in which no acid mucus glycoprotein s are detectable histochemically in the ampulla or fiml briae, stainable acid mucus glycoproteins are present in the distal (lateral) parts of the primate tube at mid Icycle; they do differ from isthmic mucus glycopro-
teins in their staining reactions, being slightly le:ss acidic and more susceptible to neuraminidase. The primate tube at midcycle also differs from the rabbit tube at estrus in that it contains glycogen, which accou nts for a major part of the tube’s carbohydrate content zand PAS
606
Jansen
November 1, 1983 Am. J. Obstet. Gynecol.
and Bajpai
Fig. 8. High-power isthmic epithelium immediately after endoplasmic reticulum vacuoles and appear in the dense in the cytoplasm.
ovulation. Rihosomes have left the swollen luminal secretion. Free glycogen is evident
Fig. 9. Early luteal phase isthmic
material
cytoplasmic
secretory
activity
epithelium. Precipitated is already inconspicuous.
lity. Glycogen and the glucose it gives rise to may portant for nutrition of the early embryo.16 Whi ereas the ultrastructural appearance of rabbit isthmi c secretion is not particularly remarkable, resembli mg (except for granule heterogeneity) typical
reactiT
be im]
is now
absent from the lumen and
mucus secretion8, I7 macaque isthmic secretions ap to be unique. As well as the more usual Golgi-medi iated production of darkly staining, homogeneous, se:rous granules, the rough endoplasmic reticulum seen 1s to accumulate electron-lucent secretion very quickly with
Volume Number
147 5
Periovulatoty
Fig. 10. Late luteal phase substantial, and secondary
isthmic epithelium. Secretory lysozomes are prominent.
the midcycle estradiol peak; release into the lumen takes place without this secretion necessarily having been processed by the Golgi apparatus; and free ribosomes accompany the secreted material into the lumen. This unusual mechanism of mucus secretion has previously been described only in the slime gland of members of the invertebrate phylum Onychophora.‘* It is possible that this apparently wasteful type of secretion, one in which there is a considerable loss of cytoplasmic components additional to the secreted material, is a necessary price to pay for what seems to be a substantial but fleeting midcycle phenomenon. Previous transmission electron microscopic studies of macaque fallopian tubes have concentrated on ciliated cellsig, ” and systematic observations of human tubal isthmic secretory cells through the periovulatory period, when what may be a very short-lived phenomenon is likely to take place, have not been published. Histochemical observations* show that acid mucus glycoproteins are even less abundant in human tubes than in macaque and baboon tubes, which could indicate that human tubes may not share this striking midcycle property with lower primates, but, on the other hand, human isthmic luminal secretions seen in an earlier scanning electron microscopic study’ constituted the stimulus for the present experiments, and some early transmission electron microscopic studies have included short descriptions of an abundant, low-density *Jansen,
R. P. S.: Unpublished
data.
activity
is absent,
glycoprotein
loss of differentiation
secretion
607
is
isthmic secretion present in human fallopian tubes in some circumstances. “* ** Further observations of the human tubal isthmus around the time of ovulation would be fruitful. The functional importance of isthmic luminal mucus glycoproteins in primate tubal physiology is not yet clear. First, the secretion is strategically placed to offer opportunities for interaction with spermatozoa penetrating the isthmus. It is of teleologic interest that primates achieve this precipitable luminal secretion, which is so similar in appearance to that of estrous rabbits, by such a different mechanism. In the rabbit, isthmic secretions have been shown in vitro to inhibit sperm motility reversibly, implying an important role for tubal secretions in controlling sperm penetration of the isthmus.23 Second, for as long as mucus glycoproteins accumulate and persist in the isthmic lumen, their presumably highly expanded and negatively charged state in vivo could be expected to impede entry into the isthmus of either the acid proteoglycan-rich cumulus mass or the acid glycoproteinaceous zona pellucida. The present study indicates that isthmic luminal secretion has disappeared by the time of ovum transport through the isthmus. Notwithstanding quantitative and qualitative changes in myosalpingeal contractility24p 25 that have been invoked to explain the interrupted quality of normal ovum transport, muscular activity imparts a high degree of randomness on the immediate position of the
608
Jansen
ovum,
and Bajpai
in spite
November 1, 1983 Am. J. Obstet. Gynecol.
of which
there
ability in overail transport therefore, plausible that absence
of isthmic
luminal
to the bidirectionality capability
of the
experienced junction.
by
little uterusz6 presence
mucus
may
contribute
of the sperm
and
ovum
isthmus the
is surprisingly
rates to the the sequential
ovum
and
to the at
the
variIt is, and
13.
both transport
physiologic
14.
delay
ampullary-isthmic
REFERENCES 1. Eddy, C. A., Garcia, R. G., Kraemer, D. C., and Pauerstein, C. J.: Detailed time course of ovum transport in the rhesus monkey (Macaca mu&a), Biol. Reprod. 13:363, 1976. 2. Croxatto, H. B., Ortiz, M. E., Diaz, S., Hess, R., Balmaceda, J., and Croxatto, H.-D.: Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following luteinizing hormone peak, AM.J. OBSTET. GYNECOL. 134:629, 1978. 3. Hodgson, B. J., and Eddy, C. A.: The autonomic nervous system and its relationship to tubal ovum transport-a reappraisal, Gynecol. Invest. 6:162, 1975. 4. Croxatto, H. B., and Ortiz, M.-A. S.: Egg transport in the fallopian tube, Gynecol. Invest. 6:215, 1975. 5. Blandau, R. J., and Verdugo, P.: An overview of gamete transport-comparative aspects, in Harper, M. J. K., Pauerstein, C. J.. Adams, C. E., Coutinho, E. M.. Croxatto, H. B., and Paton, D. M., editors: Ovum Transport and Fertility Regulation, Copenhagen, 1976, Scriptor, pp. 138-146. 6. Jansen. R. P. S.: Fallopian tube isthmic mucus and ovum transport, Science 201:349, 1978. 7. Jansen, R. P. S.: Cyclic changes in the human fallopian tube isthmus and their functional importance, AM. J. OBSTET.GYNECOL. 136:292,1980. 8. Jansen. R. P. S., and Bajpai, V. K.: Oviductal acid mucus glycoproteins in the estrous rabbit: an ultrastructural and histochemical study, Biol. Reprod. 26:155, 1982. J.: Rhesus and crab-eating macaques: intergra9. Fooden, dation in Thailand, Science 143:363, 1964. 10. Dukelow. W. R.: Similarities and dissimilarities of the reproductive physiology of Macaca mu&a and M. fascicularis. Lab. Primate Newsiett. 14:1, 1975. 11. Pauerstein, C. J., Eddy, C. A., Croxatto, H. D., Hess, R., Siler-Khodr, T. M., and Croxatto, H. B.: Temporal relationships of estrogen, progesterone, and luteinizing hormone levels to ovulation in women and infrahuman primates, AM.J. OBSTET. GYNECOL. 130:876, 1978. 12. Dukelow. W. R.: The morphology of follicular develop-
15.
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17. 18.
19.
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21.
22.
23.
24.
25.
26.
ment and ovulation in non-human primates, J. Reprod. Fertil. (Supp1.)29:23, 1975. Palay, S. L., McGee-Russell, S. M., Gordon, S., Jr., and Grillo, M. A.: Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide, J. Cell Biol. 12:385, 1967. Behnke, O., and Zelander, T.: Preservation of intercellular substances by the cationic dye alcian blue in preparative procedures for electron microscopy, J. Ultrastruct. Res. 31:424, 1970. Spicer, S. S.: Diamine methods for differentiating mucosubstances histochemically, J. Histochem. Cytochem. 13: 211, 1965. Jansen, R. P. S.: Fallopian tube secretions: implications for tubal microsurgery, Aust. N. Z. J. Obstet. Gynaecol. 41:140, 1981. Jones, R., and Reid, L.: Secretory cells and their glycoproteins in health and disease, Br. Med. BuIl. 349, 1978. Lavallard, R., and Campiglia, S.: Donnees cytochimiques et ultrastructurales sur les tubes secreteurs des glandes de la glu chez Pti~atzls acacioi Marcus et Marcus (Onychophore), 2. Zellforsch. 118:12, 1971. Anderson, R. G. W.: The three-dimensional structure of the basal body from the rhesus monkey oviduct, J. Cell Biol. 54:246, 1972. Odor, D. L., Gaddum-Rosse, P., Rumery, R. E., and Blandau, R. J.: Cyclic variations in the oviductal ciliated cells during the menstrual cycle and after estrogen treatment in the pig-tailed monkey, Macaca nemestrina, Anat. Rec. 198:35, 1980. Hashimoto, M., Shimoyama, T., Kosaka, M., Komori, A., Hirasawa, T., Yokoyama, Y., and Akashi, K.: Electron microscope studies on the epithelial cells of the human fallopian tube (Report I), J. Jpn. Obstet. Gynecol. Sot. 9:200, 1962. Fredricsson, B., and Bjorkman, N.: Morphologic alterations in the human oviduct epithelium induced by contraceptive steroids, Fertil. Steril. 2419, 1973. Johnson, L. L., Katz, D. F., and Overstreet, J. W.: The movement characteristics of rabbit spermatozoa before and after activation, Gam. Res. 4275, 1981. Verdugo, P., Blandau, R. J.. Tam, P. Y., and Halbert, S. A.: Stochastic elements in the development of deterministic models of egg transport, in Harper, M. J. K., Pauerstein, C. J., Adams, C. E., Coutinho, E. M., Croxatto, H. B., and Paton, D. M., editors: Ovum Transportation and Fertility Regulation, Copenhagen, 1976, _ Scriptor, pp. 126-137.’ Portnow, I., Hodnson, B. I., and Talo, A.: Simulation of oviductal-ovum Gansporc Can. J. Physiol. Pharmacol. 55:972, 1977. Chatkoff, M. L.: A biophysicist’s view of ovum transport, Gynecol. Invest. 6:105, 1975.
M.B., B.S., B.Sc.(Med.),
secretion in the macaque F.R.A.C.P.,
F.R.A.C.O.G.,
and
Texas
demonstration of a specific midcycle fallopian tube secretion has been sought by transmission microscopy in the Macaca mulaffa fascicularis with a method of in vivo perfusion-fixation that the polycation alcian blue (plus glutaraldehyde) to precipitate and stabilize anionic extracellular glycoproteins. Of nine monkeys entered into the study, six ovulated regularly. With the help of
daily plasma total estrogen radioimmunoassays and serial laparoscopies, these six monkeys were killed and the pelvic organs were perfused at specific times in the menstrual cycle: the midfollicular phase (one), during the midcycle estrogen peak immediately before ovulation (one), 24 hours after ovulation (one), 2 to 4 days after ovulation (two), and premenstrually (one). The other three monkeys were oophorectomized and were treated with estradiol benzoate, 10 pgikglday for 7 days (two), or with estradiol benzoate, 10 fig/kg/day for 7 days, and progesterone in oil, 1 mg/kg/day from day 3 to day 7. Evenly distributed, granular, precipitated material was found in the isthmic lumen both immediately before and 24 hours after ovulation (and in the two oophorectomized, estrogen-treated monkeys, both of which had plasma total estrogen levels that exceeded those of preovulatory monkeys); this would coincide with times of sperm ascent through the isthmus, ovulation, and fertilization and with the time of temporary impedance of the ovum’s normal transport to the uterus, known to occur at the ampullary-isthmic junction. No substantial precipitable secretion was found in the ampulla at any stage of the cycle, in the isthmus before the midcycle estrogen rise, or 48 hours or more after either ovulation or initiation of progesterone treatment. These observations indicate that, in the primate fallopian tube, the unique ability of the isthmus to transport spermatozoa and ova in opposite directions sequentially is related temporally (and perhaps causally) to the presence and absence, respectively, of specific isthmic luminal secretions. The biochemical basis for this isthmic property may lie in the expected highly expanded state of acid mucus glycoproteins in the secretions.
(AM.
J. OBSTET.
GYNECOL.
147598,
1983.)
The fallopian tube isthmus lies between the site of fertilization, the ampulla, and the site of implantation, the uterus. The isthmus must, therefore, be capable of effecting transport of spermatozoa and ova in opposite directions. This dual transport capability is sequential. After distal sperm ascent and fertilization of the newly ovulated egg in the ampulla, the egg has its proximal transport delayed in the ampulla at or near the ampullary-isthmic junction for at least 48 hours in rhesus monkeys’ and in humans” before the isthmus is passed and the ovum reaches the uterus. The physiologic basis for this ovum transport delay
From the Depnrtmmt of Obstetrics and Gynecology, Unizwsity of Texas Health Science Centtl at San Antonio. Supported by University of Texas Institutional Grant 626.167121 I5 (to R. P. S. J.) and, in part, by National Institutes of Health Grants P30 HD10202 (Centerfor Research in Reproductive Biology: Bioassay Core, Morphology Core, Radioimmunoassay Core) and HDO9039 (Physiology and Pharmacology of Ovum Transport). V. K. B. was on a World Health Organization Postdoctoral Fellowship. Presented at the Thirtieth Annual Meeting of the Society for Gynecologic Investigation, Washington, D. C., March 17-20, 1983. Reprint requests: Dr. Robert P. S. Jansm, The Fertility Laboratory, King George I’ Memorial Hospital, Camper-down, Sydney 2050, Australia. 598
at the ampullary-isthmic junction is not understood. A similar delay occurs in rabbits, and extensive studies of smooth muscle and ciliary activity have failed wholly to account either for the bidirectionality of the sperm-and ovum-transporting capabilities of the isthmus or for the delay of the ovum at the ampullary-isthmic junction.“-” Earlier studies indicated that the approximate time during which sperm transport takes place distally, and during which proximally directed ovum transport is interrupted, coincides with the presence of specific, precipitable secretion in the isthmic lumen. These findings were based on surface electron microscopic appearances in estrous rabbit? and midcycle humans.’ but there was concern that tubal constriction at this time might have artifactually preserved luminal secretions that at other stages of the cycle were washed out. Subsequently, more precise transmission electron microscopic studies of tissues fixed by vascular perfusion with glutaraldehyde and the polycation alcian blue (to precipitate and stabilize secreted glycoproteins) confirmed the presence of material identifiable histochemically as acid mucus glycoprotein in the isthmic lumen of estrous rabbits.8 Because these procedures for in situ fixation of secreted glycoproteins are highly toxic, these studies could not be extended to human
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Fig. 1. Early follicular phase isthmic epithelium showing ciliated and nonciliated activity is present, but ribosomes are abundant in the cytoplasm. tissu es. The rhesus monkey species Macaca f ascii xdaris (crab-eating macaque) has been instc tad. Mi aterial and methods Nine sexually M onkeys. (crab-eating macaques) keys
mulattu studied
mature cynomolgus monwere bought from Primate
599
cells: no secretory
Imports (now Charles River Primate Imports, PC31-t Washington, New York). The crab-eating macaque, or M. mulattafusciculur~, is closely related to the larger a nd more familiar rhesus monkey, M. muluttu mukzttu; th leir interbreeding both in captivity and where their natu ral ranges are contiguous has shown these monkeys to be two races of the same species,Y and it has been con5 il‘d -
600
Jansen
and Bajpai
Fig. 2. Low-power view of early follicular phase (Aj and late folhcular phase (B) tubal isthmus in cross section, showing a substantial increase in stainable acid mucus glycoproteins. [High-iron diamine (pH l.O), alcian blue (pH 2.5), and nuclear fast red.] ered that for reproductive studies data obtained for one should be applicable to the other.” After purchase the animals were quarantined for a further 6 weeks before introduction to the primate colony at the University of Texas Health Science Center at San Antonio, where they were individually caged under controlled conditions of 75” F, 50% relative humidity, and 14 hours of fluorescent light per day. Subsequent reproductive cyclicity was documented daily with the monkeys under ketamine-induced tranquility (ketamine hydrochloride, 3 to 4 mg intramuscularly) (1) by plasma total estrogen radioimmunoassays (RIAs) after femoral vein puncture and (2) by daily vaginal swabbing for menstrual blood. Six of the nine monkeys ovulated regularly and were entered into the study after observation in a control cycle. Plasma total estrogen and plasma progesterone were assayed daily in duplicate during the study cycle according to methods described previously”; estrogen results were available on the same day. Serial laparoscopies performed daily (and sometimes twice daily) with the animals under ketamine-induced narcosis were started when rising plasma total estrogen levels indicated preovulatory follicular development. The characteristic appearances of immediately preovulatory and postovulatory follicles have been described,” preovulatory follicles being characterized by formation of translucent areas in the follicular blister and postovulatory follicles bearing a hemorrhagic stigma to mark the site of ovulation. Frequent laparoscopies in rhesus monkeys have been shown not to interfere with the physical and endocrine events associated with ovulation.“. I2 Monkeys were put to death before and after the time of ovulation as follows: in the midfollicularpha,se (n = 1; early follicular development, no plasma total estrogen rise had yet been evident); immediately before ovulation
November 1, 1983 Am. J. Obstet. Cynecol.
(n = 1; 1.5 cm follicle present, preovulatory increase in plasma total estrogen level had begun); about 24 hours after ovulation (n = 1; follicle appeared immediately preovulatory at laparoscopy 48 hours previously; plasma total estrogen level had fallen after midcycle peak); 2 to 4 days after ovulation (n = 2); and in the late luteal phase (n = 1; just over 1 week after ovulation was seen laparoscopically, with endometrium histologically in the early menstrual phase). Three monkeys failed to ovulate during the time they were observed, and bilateral oophorectomies were performed at least 1 month before hormonal therapy and sacrifice. One monkey was treated with estradiol benzoate, 10 pg/kg/ day for 7 days (plasma total estrogen levels were greater than those of the midcycle peak of ovulating monkeys); the other two monkeys received, in addition to estrogen, 4 days’ progesterone in oil, 1 mg/kg/day from day 3 to day 7. With the animals under ketamine-induced narcosis, a midline ventral laparotomy was performed. The active preovulatory or postovulatory ovary was noted. Both ovarian arteries, but not the ovarian veins, were tied to encourage collateral circulation from the uterine arteries to the fallopian tubes. The aortic bifurcation was dissected, and the median sacral artery and vein were ligated. The pelvic sidewalls were then dissected and the internal iliac arteries were tied beyond the origin of the uterine artery on each side. The femoral artery and vein were tied on each side after groin dissection. Dissection of the aorta was advanced cranially, and both the aorta and inferior vena cava were exposed; loose ligatures were placed around these vessels at a level between the superior and inferior mesenteric arteries. The inferior vena cava was cannulated to allow free drainage of blood from the pelvic organs. Immediately after the aortic ligature was tied, the aort was cannulated with a Teflon cannula connected to a controlled-pressure perfusion apparatus.‘” Perfusion was begun with glutaraldehyde (1.5% in 0.1 M cacodylate buffer at pH 7.4, with an osmolality of 350 mOsm/L, determined with an Osmette A freezingpoint-depression automatic osmometer, Precision Instruments, Inc.) at a constant pressure of 125 mm Hg. As the glutaraldehyde infusion was begun, the monkeys were killed with an overdose of pentobarbital administered through a mesenteric vein. The ovarian veins were slit open to allow drainage. Ultrastructural studies. The uterus and tubes, together with (in most cases) the bladder and lower colon, were thus fixed in situ with glutaraldehyde for 5 minutes. The perfusing solution was then changed to one containing, in addition to the glutaraldehyde and cacodylate buffer as above, 1% alcian blue (freshly prepared and filtered, with an osmolality of 465 mOsm/L). The previously blanched and hardened tissues turned
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Periovulatory glycoprotein secretion
147
601
5
Fig. 3. A, Preovulatory
tubal isthmus showing glycogen and acid mucus glycoproteins. (PAS and hematoxylin.) B, Diastase digestion removes glycogen but glycoproteins remain. (PAS and hematoxylin.) C, Acid mucus glycoproteins revealed. (High-iron diamine (pH 1 .O), alcian blue (pH 2.5), and nuclear fast red.)
blue as the perfusate was changed from glutaraldehyde to the alcian blue-glutaraldehyde mixture, which was continued for 8 to 10 minutes. This is essentially the method described by Behnke and Zelander14 for preserving tissue mucopolysaccharides and is identical to the methodology used in our earlier study in estrous rabbitsx The fixed fallopian tubes were removed and prepared for transmission electron microscopy as described previously,” with 2 hours’ further fixation in the alcian blue-glutaraldehyde perfusate before postfixation with osmium tetroxide (2% in O.lM cacodylate buffer) for 60 minutes. At least two segments from the ampulla and two segments from the isthmus of one tube were examined; the contralateral tube was processed for light microscopy. Thin sections were examined, after staining with 1% uranyl acetate and 1% lead citrate, with a Siemens 101 electron microscope at 80 kV. Histochemical studies. From each monkey studied ultrastructurally, the contralateral glutaraldehyde-fixed fallopian tube was dehydrated, embedded in paraffin, and sectioned for light microscopic examination. Several methods were used to identify cells producing neutral, sialyated (moderately acid), and sulfated (highly acid) mucus glycoproteins: (1) periodic acid-Schiff (PAS) and Mayer’s hematoxylin, (2) high-iron diamine at pH 1.0 and alcian blue at pH 2.5, and (3) these staining reactions after diastase (Fluka, Geneva; 0.1% for 90 minutes at 37” C) to dissolve glycogen, pronase (Calbiochem; 0.1% for 2 hours at 37” C) to remove mucus glycoproteins, or neuraminidase (Sigma; 0.25 U/ml, pH 5.4, for 24 hours at 37” C) to partly remove sialic acid. Light microscopic methods were controlled by staining a section of mouse rectosigmoid colon on each occasion. The mouse rectosigmoid has surface
mucus and crypt goblet cells that stain for sulfated (highly acid) mucus glycoproteins, as well as a distinct population of deep crypt cells that stain only for sialyated (moderately acid) mucus glycoproteins.‘j Details of the methods and expected results are provided elsewhere.x Results Estrogen influence. Tubal morphology from the early follicular macaque was relatively undifferentiated (Fig. 1). Ciliogenesis was evident in the ampulla, whereas cilia were established in the isthmus. Nonciliated cells displayed free ribosomes and variable rough endoplasmic reticulum, indicating early synthesizing activity, but Golgi activity appeared minimal and serous (dark, homogeneous) granules were sparse and restricted to the ampulla. Histochemical studies indicated a corresponding lack of stainable acidic glycoproteins (Fig. 2,A). No precipitable material was present in the lumen. The preovulatory and the estradiol-treated macaque fallopian tubes showed evidence of substantial secretory activity. Acid mucus glycoproteins were evident in the mucosa from the isthmus (Fig. 2, B), ampulla, and fimbrial end (in decreasing order of abundance and electronegativity). Staining with PAS (Fig. 3, A) was partly but not completely diminished by pretreatment with diastase (Fig. 3,B), indicating the presence of both glycogen and glycoprotein. Black-staining reactions with high-iron diamine were found only in the isthmus (Fig. 3, C), ampullary acid mucus glycoproteins staining only with alcian blue at pH 2.5; isthmic mucus glycoprotein was partly resistant to neuraminidase, whereas neuraminidase virtually abolished ampullary staining. Ampullary ultrastructure showed that ciliated cells had developed fully (Fig. 4). Nonciliated cells had dif-
602
Jansen and Bajpai
Fig. 4. Preovulatory monkey ampulla. Prominent apparatus (G); domed cytoplasmic apices filled no precipitated secretion evident in the lumen.
fere :ntiated into secretory cells with apices that bulged into I the lumen and were filled with secretory granules ofv arying density. The Golgi apparatus was prominent and appeared (Fig. 4) to be involved in concentrating seer ,etory material. The rough endoplasmic reticulum
November Am. J. Obtet.
1,1983 Gyre~ol.
activity in the endoplasmic reticulum (er) and Golgi with secretory granules of varying electron density;
was also prominent, had dilated cisternae, and was filled with homogeneous pale-staining material. L ittle precipitated secretion was present in the ampul lary lumen. In the isthmus, the rough endoplasmic reticulum was variably occupied with secretion, but
Volume Number
147 5
Periovulatory
glycoprotein
secretion
603
Fig. 5. Preovulatory isthmus from the same monkey as shown in Fig. 4. Granular and membranous products of apocrine secretion pack the lumen.
g1-anular a1xxrine
and membranous products of (apparently) secretion packed the lumen (Fig. 5). Similar t-e:sults were found in ovariectomized, estradiol-treated m lacaques, indicating the estradiol dependence of tubal secretion. Secretory activity was morphologically greatest in the
immediately postovulatory macaque. Although 1 folli cular cells were being swept down the ampulla (Fig. 6), the isthmic lumen was packed tight with prel cipita ted secretion (Fig. 7), which in many transmission elect] ron microscopic fields studied appeared to have (:ome directly from rough endoplasmic reticulum Iracuo les,
604
Jansen
and Bajpai
November Am. J. Obstet.
Fig. 6. Immediately after ovulation, cumulus cells from the ruptured ampullary lumen. Nonciliated cells have lost their apical secretion. free ribosomes being evident among the secretion (Fig. 8). Much glycogen was detectable both on transmission electron microscopy and on histochemistry; although PAS reactivity was strong, diastase caused a marked loss of staining and reactivity of cells; staining with high-iron diamine and alcian blue was less than before ovulation. Progesterone influence. At 2 days after ovulation secretory vacuoles were still visible in some nonciliated cells, but dedifferentiation was in evidence; little luminal secretion was present in the isthmus other than in some mucosal crypts and there was none in the ampulla. Early luteal phase (2 to 4 days) and 4-day progesterone-treated (estradiol-primed) macaques showed similar tubal morphology. Secretory granules and vacuoles had been compacted and other cytoplasmic organelles were in disarray; the cytoplasm itself appeared concentrated: the isthmic lumen was without precipitable contents (Fig. 9). Histochemistry confirmed the rapid loss of stainable mucus glycoproteins. In the late luteal phase (Fig. 10) dedifferentiation was marked and lysozome activity was prominent; regurgitated menstrual products were seen in some sections. Comment Epithelial glycoproteins are likely to be important in many aspects of reproduction. An earlier study has
1, 1983 Gynecol.
follicle have been fixed in the
confirmed that the rabbit fallopian tube produces mucus glycoproteins.’ Histochemistry in this species showed that the production of highly acid mucus glycoprotein (apparently of the type that coats the ovum in the rabbit) is confined to the isthmus and, to a lesser extent, the mucosal crypts of the ampullary-isthmic junction; the ampulla is not involved. Using a method of perfusion-fixation that includes the polycation alcian blue in conjunction with glutaraldehyde to precipitate and stabilize glycoproteins, we demonstrated that this mucus occupied the isthmic lumen but not the ampullary lumen in estrous rabbits. The present study was designed to confirm or refute the existence of tubal luminal secretions in primates, after scanning electron microscopic evidence for such secretions had been obtained in midcycle women.7 Evenly distributed, granular, precipitated material was found in the macaque isthmic lumen both immediately before and 24 hours after ovulation and in two oophorectomized, estrogen-treated monkeys. These conditions would coincide with times of sperm ascent through the isthmus, ovulation, and fertilization and with the time of temporary impedance at the ampullary-isthmic junction of the ovum’s normal transport to the uterus. On the other hand, no substantial precipitable secretion was found in the ampulla at any stage of the cycle, in the isthmus before the midcycle estrogen rise, or 48 hours or more after either ovulation or initiation of progesterone treatment.
Volume Number
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147 5
605
Fig. 7. Isthmic material distinct
epithelium from the same tube as shown in Fig. 6. The lumen is packed with secretory that seems to be in direct continuity with dilated endoplasmic reticulum vacuoles and is from the small electron-dense serous granules in the cell cytoplasm.
l-Ilnlike the rabbit, in which no acid mucus glycoprotein s are detectable histochemically in the ampulla or fiml briae, stainable acid mucus glycoproteins are present in the distal (lateral) parts of the primate tube at mid Icycle; they do differ from isthmic mucus glycopro-
teins in their staining reactions, being slightly le:ss acidic and more susceptible to neuraminidase. The primate tube at midcycle also differs from the rabbit tube at estrus in that it contains glycogen, which accou nts for a major part of the tube’s carbohydrate content zand PAS
606
Jansen
November 1, 1983 Am. J. Obstet. Gynecol.
and Bajpai
Fig. 8. High-power isthmic epithelium immediately after endoplasmic reticulum vacuoles and appear in the dense in the cytoplasm.
ovulation. Rihosomes have left the swollen luminal secretion. Free glycogen is evident
Fig. 9. Early luteal phase isthmic
material
cytoplasmic
secretory
activity
epithelium. Precipitated is already inconspicuous.
lity. Glycogen and the glucose it gives rise to may portant for nutrition of the early embryo.16 Whi ereas the ultrastructural appearance of rabbit isthmi c secretion is not particularly remarkable, resembli mg (except for granule heterogeneity) typical
reactiT
be im]
is now
absent from the lumen and
mucus secretion8, I7 macaque isthmic secretions ap to be unique. As well as the more usual Golgi-medi iated production of darkly staining, homogeneous, se:rous granules, the rough endoplasmic reticulum seen 1s to accumulate electron-lucent secretion very quickly with
Volume Number
147 5
Periovulatoty
Fig. 10. Late luteal phase substantial, and secondary
isthmic epithelium. Secretory lysozomes are prominent.
the midcycle estradiol peak; release into the lumen takes place without this secretion necessarily having been processed by the Golgi apparatus; and free ribosomes accompany the secreted material into the lumen. This unusual mechanism of mucus secretion has previously been described only in the slime gland of members of the invertebrate phylum Onychophora.‘* It is possible that this apparently wasteful type of secretion, one in which there is a considerable loss of cytoplasmic components additional to the secreted material, is a necessary price to pay for what seems to be a substantial but fleeting midcycle phenomenon. Previous transmission electron microscopic studies of macaque fallopian tubes have concentrated on ciliated cellsig, ” and systematic observations of human tubal isthmic secretory cells through the periovulatory period, when what may be a very short-lived phenomenon is likely to take place, have not been published. Histochemical observations* show that acid mucus glycoproteins are even less abundant in human tubes than in macaque and baboon tubes, which could indicate that human tubes may not share this striking midcycle property with lower primates, but, on the other hand, human isthmic luminal secretions seen in an earlier scanning electron microscopic study’ constituted the stimulus for the present experiments, and some early transmission electron microscopic studies have included short descriptions of an abundant, low-density *Jansen,
R. P. S.: Unpublished
data.
activity
is absent,
glycoprotein
loss of differentiation
secretion
607
is
isthmic secretion present in human fallopian tubes in some circumstances. “* ** Further observations of the human tubal isthmus around the time of ovulation would be fruitful. The functional importance of isthmic luminal mucus glycoproteins in primate tubal physiology is not yet clear. First, the secretion is strategically placed to offer opportunities for interaction with spermatozoa penetrating the isthmus. It is of teleologic interest that primates achieve this precipitable luminal secretion, which is so similar in appearance to that of estrous rabbits, by such a different mechanism. In the rabbit, isthmic secretions have been shown in vitro to inhibit sperm motility reversibly, implying an important role for tubal secretions in controlling sperm penetration of the isthmus.23 Second, for as long as mucus glycoproteins accumulate and persist in the isthmic lumen, their presumably highly expanded and negatively charged state in vivo could be expected to impede entry into the isthmus of either the acid proteoglycan-rich cumulus mass or the acid glycoproteinaceous zona pellucida. The present study indicates that isthmic luminal secretion has disappeared by the time of ovum transport through the isthmus. Notwithstanding quantitative and qualitative changes in myosalpingeal contractility24p 25 that have been invoked to explain the interrupted quality of normal ovum transport, muscular activity imparts a high degree of randomness on the immediate position of the
608
Jansen
ovum,
and Bajpai
in spite
November 1, 1983 Am. J. Obstet. Gynecol.
of which
there
ability in overail transport therefore, plausible that absence
of isthmic
luminal
to the bidirectionality capability
of the
experienced junction.
by
little uterusz6 presence
mucus
may
contribute
of the sperm
and
ovum
isthmus the
is surprisingly
rates to the the sequential
ovum
and
to the at
the
variIt is, and
13.
both transport
physiologic
14.
delay
ampullary-isthmic
REFERENCES 1. Eddy, C. A., Garcia, R. G., Kraemer, D. C., and Pauerstein, C. J.: Detailed time course of ovum transport in the rhesus monkey (Macaca mu&a), Biol. Reprod. 13:363, 1976. 2. Croxatto, H. B., Ortiz, M. E., Diaz, S., Hess, R., Balmaceda, J., and Croxatto, H.-D.: Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following luteinizing hormone peak, AM.J. OBSTET. GYNECOL. 134:629, 1978. 3. Hodgson, B. J., and Eddy, C. A.: The autonomic nervous system and its relationship to tubal ovum transport-a reappraisal, Gynecol. Invest. 6:162, 1975. 4. Croxatto, H. B., and Ortiz, M.-A. S.: Egg transport in the fallopian tube, Gynecol. Invest. 6:215, 1975. 5. Blandau, R. J., and Verdugo, P.: An overview of gamete transport-comparative aspects, in Harper, M. J. K., Pauerstein, C. J.. Adams, C. E., Coutinho, E. M.. Croxatto, H. B., and Paton, D. M., editors: Ovum Transport and Fertility Regulation, Copenhagen, 1976, Scriptor, pp. 138-146. 6. Jansen. R. P. S.: Fallopian tube isthmic mucus and ovum transport, Science 201:349, 1978. 7. Jansen, R. P. S.: Cyclic changes in the human fallopian tube isthmus and their functional importance, AM. J. OBSTET.GYNECOL. 136:292,1980. 8. Jansen. R. P. S., and Bajpai, V. K.: Oviductal acid mucus glycoproteins in the estrous rabbit: an ultrastructural and histochemical study, Biol. Reprod. 26:155, 1982. J.: Rhesus and crab-eating macaques: intergra9. Fooden, dation in Thailand, Science 143:363, 1964. 10. Dukelow. W. R.: Similarities and dissimilarities of the reproductive physiology of Macaca mu&a and M. fascicularis. Lab. Primate Newsiett. 14:1, 1975. 11. Pauerstein, C. J., Eddy, C. A., Croxatto, H. D., Hess, R., Siler-Khodr, T. M., and Croxatto, H. B.: Temporal relationships of estrogen, progesterone, and luteinizing hormone levels to ovulation in women and infrahuman primates, AM.J. OBSTET. GYNECOL. 130:876, 1978. 12. Dukelow. W. R.: The morphology of follicular develop-
15.
16.
17. 18.
19.
20.
21.
22.
23.
24.
25.
26.
ment and ovulation in non-human primates, J. Reprod. Fertil. (Supp1.)29:23, 1975. Palay, S. L., McGee-Russell, S. M., Gordon, S., Jr., and Grillo, M. A.: Fixation of neural tissues for electron microscopy by perfusion with solutions of osmium tetroxide, J. Cell Biol. 12:385, 1967. Behnke, O., and Zelander, T.: Preservation of intercellular substances by the cationic dye alcian blue in preparative procedures for electron microscopy, J. Ultrastruct. Res. 31:424, 1970. Spicer, S. S.: Diamine methods for differentiating mucosubstances histochemically, J. Histochem. Cytochem. 13: 211, 1965. Jansen, R. P. S.: Fallopian tube secretions: implications for tubal microsurgery, Aust. N. Z. J. Obstet. Gynaecol. 41:140, 1981. Jones, R., and Reid, L.: Secretory cells and their glycoproteins in health and disease, Br. Med. BuIl. 349, 1978. Lavallard, R., and Campiglia, S.: Donnees cytochimiques et ultrastructurales sur les tubes secreteurs des glandes de la glu chez Pti~atzls acacioi Marcus et Marcus (Onychophore), 2. Zellforsch. 118:12, 1971. Anderson, R. G. W.: The three-dimensional structure of the basal body from the rhesus monkey oviduct, J. Cell Biol. 54:246, 1972. Odor, D. L., Gaddum-Rosse, P., Rumery, R. E., and Blandau, R. J.: Cyclic variations in the oviductal ciliated cells during the menstrual cycle and after estrogen treatment in the pig-tailed monkey, Macaca nemestrina, Anat. Rec. 198:35, 1980. Hashimoto, M., Shimoyama, T., Kosaka, M., Komori, A., Hirasawa, T., Yokoyama, Y., and Akashi, K.: Electron microscope studies on the epithelial cells of the human fallopian tube (Report I), J. Jpn. Obstet. Gynecol. Sot. 9:200, 1962. Fredricsson, B., and Bjorkman, N.: Morphologic alterations in the human oviduct epithelium induced by contraceptive steroids, Fertil. Steril. 2419, 1973. Johnson, L. L., Katz, D. F., and Overstreet, J. W.: The movement characteristics of rabbit spermatozoa before and after activation, Gam. Res. 4275, 1981. Verdugo, P., Blandau, R. J.. Tam, P. Y., and Halbert, S. A.: Stochastic elements in the development of deterministic models of egg transport, in Harper, M. J. K., Pauerstein, C. J., Adams, C. E., Coutinho, E. M., Croxatto, H. B., and Paton, D. M., editors: Ovum Transportation and Fertility Regulation, Copenhagen, 1976, _ Scriptor, pp. 126-137.’ Portnow, I., Hodnson, B. I., and Talo, A.: Simulation of oviductal-ovum Gansporc Can. J. Physiol. Pharmacol. 55:972, 1977. Chatkoff, M. L.: A biophysicist’s view of ovum transport, Gynecol. Invest. 6:105, 1975.