J. Embryol. exp. Morph. Vol. 23, 1, pp. 1-19, 1970 \ Printed in Great Britain The fate of the zona pellucida in mice By A N N E M c L A R E N 1 Agricultural Research Council Unit of Animal Genetics, Edinburgh In mice and rats, the egg is surrounded by a mucoprotein coat, the zona pellucida, which is lost shortly before implantation. The mechanism of loss has long been a matter of controversy. In rats, loss of the zona has variously been reported to be by lysis (Défrise, 1933; Dickson, 1963) or by an active hatching mechanism (Dickmann & Noyes, 1961). A similar lack of unanimity is encountered in discussions on the mechanism of loss of the mouse zona pellucida in utero, though, under conditions of in vitro culture, the hatching of blastocysts from the zona has been not only observed, but even filmed (Cole & Paul, 1965; Cole, 1967). The time of loss of the mouse zona pellucida was established rather precisely for the Q randomly bred strain of mice by Orsini & McLaren (1967). In normal pregnancies, about half the blastocysts were free of the zona shortly before midnight on the fourth day of pregnancy. The observations of Dickson (1966) suggest a similar time of loss for the Swiss Webster albino strain of mice. Loss of the zona was postponed up to 24 h in females ovariectomized on the 2nd or 3rd day of pregnancy, or in blastocysts retained in the oviduct by ligation of the utero-tubal junction (Orsini & McLaren, 1967). Under these conditions, shed zonae as well as blastocysts could be recovered by flushing the uterus or oviduct, and blastocysts were sometimes seen lying within ruptured zonae. Similar findings are characteristic of mouse embryos cultivated in vitro (Whitten, 1957) and have also been reported in the rat after ovariectomy (Alloiteau & Psychoyos, 1966). A study of lactating female mice (McLaren, 1967), in which implantation was delayed, confirmed the preliminary report of Rumery & Blandau (1966) that loss of the zona under these conditions also is postponed up to 24 h. Histological examination of serially sectioned uteri on the 5th and 6th days of pregnancy with concurrent lactation showed some blastocysts still within the zona, some free of the zona, and some partially free, as well as some shed zonae (McLaren, 1968). By the 8th day, all blastocysts were zona-free; in most of the uteri examined shed zonae were also found. Implantation could be induced either by removing the litter or by injecting small amounts of oestrogen: these treatments resulted in disappearance of zonae from the uterine lumen within about 18 h. 1 Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh 9. 1-2 2 A. M C L A R E N The present paper is concerned with three topics: (1) observations on the zona during lactational or experimental delay of implantation; (2) the fate of the zona surrounding the unfertilized egg in pseudopregnant females ; (3) the fate of the zona during normal pregnancy. The observations throw some light on the situation of the mouse with respect to the 'hatching versus lysis' controversy. MATERIALS AND METHODS All mice belonged to the randomly bred Q strain and were nulliparous, except for those in lactational delay, which were primiparous. Pregnancy and pseudopregnancy were induced by mating with males of proven fertility or with vasectomized males respectively. For lactational delay only females suckling eight or more young were used. Ovariectomy was performed under ether anaesthesia; on each side the ovary and part of the ovarian fat pad were removed, leaving the oviduct more or less intact. No ovarian remnants or regenerating fragments were ever seen at autopsy. Pontamine Sky Blue (0-2 ml of a 0-5 % solution of Gurr's 5BX) was injected intravenously 15 min before the female was killed. Uterine horns were removed from the animal, and then either trimmed free of fat and mesentery and flushed through with phosphate-buffered saline into a watch-glass, or pinned out on foil-covered card and fixed in neutral formol saline. Flushings were examined carefully at a 25-fold magnification, and any eggs or zonae found were then further examined at a 100-fold magnification. After fixation, uteri were sectioned serially at 8/t, and the sections stained with Mallory's triple stain, methyl green and toluidine blue or haematoxylin and eosin. With Mallory's triple stain, the zona pellucida stains bright blue. Except where the attachment reaction (Nilsson, 1969) was already beginning (e.g. Figs. 12, 14-16, 26-28), shrinkage during fixation resulted in an artefactual space within the uterine lumen. Correspondence of the 'corrugations' (McLaren, 1968) on opposite sides of the lumen showed that in vivo the two epithelia had been apposed. Where an object (e.g. an egg or zona) was enclosed in the lumen, it was represented by an impression or mould in one or both of the epithelial surfaces (e.g. Figs. 7, 8, 13). Only when the egg or zona was still within its own mould could one be confident that it had not appreciably shifted its position during fixation or subsequent histological preparation. 3 Fate of the zona pellucida RESULTS 1. Delay of implantation (a) Lactational delay. The zona is eventually lost from the blastocyst, but persists in the uterine lumen (McLaren, 1967), only to disappear rapidly once implantation has been induced (McLaren, 1968). At this time the lumen is Table 1. Zonae detected during histological examination of uteri from pregnant females in lactational delay ]Position of zona A Days p.c. 4i 5i 7i 1\ (12-15 h after removal of litter) No. of females No. of blastocysts No. of zonae 3 1 3 2 30 12 20 23 23 10 11 11 At a Surrounding Adjacent distance blasto-- to blasto- from cyst cyst blastocyst 7 0 0 0 10 8 11 9 6 2 0 2 Table 2. Relative positions of zonae and blastocysts during lactational delay Days p.c. 4*-5± 7+ No. of zonae Mean distance from zona to nearest blastocyst (JJ) Variance No. of zonae Mean distance from zona to nearest blastocyst iß) Variance Zonae and blastocysts still in original positions in uterus No information on original positions of zonae and blastocysts 3 40-0 ±23-7 13 205-6 ±47-4 1 687-0 11 100-2 ±15-8 2735-8 29154-3 9 165-4 ±55-7 27955-3 virtually closed and, since the blastocysts remain in position, it is hardly likely that the zonae are expelled from the uterus. Their disappearance is therefore presumably due to lysis. A total of 55 zonae were examined at different stages of lactational delay, as shown in Table 1. The appearance of the zonae, once they had been shed, was similar at all stages. Fig. 1 shows a zona which was still present 15 h after 4 A. M C L A R E N 'removal of the litter. It shows no signs of thinning or dissolution (cf. figs. 2-4 of McLaren, 1967), suggesting that the lytic process, once started, acts relatively fast. No zonae were present 19 h or more after removal of the litter. The relative positions of zonae and blastocysts are also indicated in Table 1. Table 2 summarizes data on the distance of each zona from the nearest blastocyst, estimated either by counting sections or, where appropriate, by measuring across the uterine lumen, using an eyepiece micrometer. In those instances where both zona and blastocyst could be seen to have retained their original positions, as indicated by the impressions left in the wall of the lumen (see Materials and Table 3. Zonae detected during histological examination of uteri from six pregnant females ovariectomized 2\ days p.c. and killed 5 days later Blastocysts With zona attached Naked 4 0 1 5 4 0 3 5 6 Total 0 0 6 5 1 16 Female 1 2 3 Free zonae 2 1 'Dispersed' ova 1 0 7 0 2 4 1 0 0 8 3 0 1 11 Methods), the distances are significantly shorter (P < 001 at 4^-5^ days p.c.) and also less variable (P < 0-001 at 1\ days p.c.) than where no impressions could be seen. This suggests that the latter category includes instances in which zona or blastocyst or both have changed their positions during fixation or subsequent histological preparation. Omitting this category, the mean distance from zona to blastocyst is greater at 1\ days p.c. than at 4-^-5^ days p.c., significantly so when the lower proportion of zonae actually adjacent to blastocysts is taken into account. Empty zonae of essentially similar structure to those described in the present paper have also been observed by O. Nilsson (personal communication) in the uteri of lactating Q-strain mice, sent from Edinburgh to Uppsala. The uteri were fixed by aortic perfusion of glutaraldehyde, post-fixed in osmic acid and embedded in Epon. This technique gives relatively good preservation of structure, with minimal disturbance of cellular relations. The zonae did not appear to be adjacent to blastocysts, but it was not possible to estimate the distance between zona and blastocyst using this technique. (b) Experimental delay. In females ovariectomized at 2\ and killed at 1\ days p.c., some of the zonae were still adjacent to or partially surrounding blastocysts (Table 3), but most of the blastocysts were naked, with the shed zonae situated at a distance. The lumen is not so tightly closed after ovariectomy as Fate of the zona pellucida 5 during lactation, so it was seldom possible to be sure of the original position of the zonae. The proportion of naked blastocysts represented by shed zonae (8/16) was similar to that at the corresponding stage of pregnancy with concurrent lactation (22/43, see Table 1). It is not possible to say whether the missing zonae disappeared in vivo or were lost during the course of histological preparation. The 'dispersed' ova, which are commonly found after ovariectomy (see Orsini & McLaren, 1967), were all surrounded by intact zonae, confirming that no zona lysin is present in the uterus of recently ovariectomized females. Table 4. Persistence of blastocysts and zonae after transfer to the uteri offemales ovariectomized 6 weeks earlier Interval between transfer and autopsy (days) 1 2 3 7 Total Control No. of females Total no. blastocysts transferred 2 2 4 2 10* 10 30 36 60 32 158 0 Flushed horn Sectioned horn A f r Blastocysts Zonae Unfertilized 0 2 1 0 3 0 0 0 1 0 1 0 0 0 0 0 0 3 Blastocysts 0 3 n i 15 0 Zonae Unfertilized 0 1 9 1 11 0 0 2 0 3 5 10 * A total of .12 females received blastocysts; two have been omitted1, as co nsiderable haemorrhage into the uterine lumen occurred. Oestrogen and progesterone influence may persist for several days after ovariectomy. To study the effect on the zona of a hormonally 'neutral' environment, cyclic females were ovariectomized, and 6 weeks later blastocysts (from donors 3^ days p.c.) were transferred to their uteri. After varying intervals, the females were killed, one horn of the uterus was flushed with saline and the other serially sectioned and examined histologically (Table 4). A control group of ovariectomized females received no blastocysts. Very few blastocysts or zonae were detected by the flushing procedure. On histological examination, 15 naked blastocysts and 11 free zonae were found, out of 79 eggs transferred (Figs. 5, 6). The uteri were very much reduced in size 6 weeks after ovariectomy, and considerable difficulty was experienced in making the transfers; some of the large loss of eggs is therefore likely to be due either to failure to introduce the eggs into the uterus, or to subsequent expulsion of eggs through the needle hole. Most of the eggs were found towards the cervical end of the horn, so some may also have been lost through the cervix. It is clear, in any case, that those blastocysts which remained in the uterus shed their zonae, and that most of these shed zonae persisted for several days at least. Unfertilized eggs in intact zonae were observed in 2/10 of the control females and 6 A. M C L A R E N also in 2/10 of those receiving blastocysts (Figs. 2-4). Possibly the unfertilized eggs only persist in the uterus if the ovaries are removed at one particular stage of the oestrous cycle. The zonae surrounding these eggs must have survived for at least 6-7 weeks. There is thus no reason to believe that lysis of the zona takes place in the hormonally 'neutral' mouse uterus. 2. The unfertilized egg Unfertilized eggs survive longer in pseudopregnant than in unmated cyclic females, and have been observed in uterine washings on the 4th and 5th day of pseudopregnancy (McLaren & Orsini, 1968). To examine the fate of the zona pellucida, a series of pseudopregnant females, some intact and others ovariectomized at 2\ days/?.c, were killed at intervals during the 5th and 6th day of pseudopregnancy; one horn of each was flushed with saline, while the other was serially sectioned and examined histologically (Table 5). In some of the females 10 % serum was injected into one horn at 3£ days p.c., as for egg transfer. In the intact animal this procedure induces partial decidualization. In the ovariectomized females, examination of the serially sectioned uteri revealed six to eight unfertilized eggs per horn. These consisted of a degenerating vitellus, surrounded by a thick, rigid zona (Figs. 7, 8). After injection of fluid into the horn, the number of surviving unfertilized eggs decreased but their appearance did not alter (Figs. 9, 10). In the intact females, only just EXPLANATION OF FIGURES Figs. 1-31 are all transverse sections of mouse uteri fixed in formol saline and stained with Mallory's trichrome, which stains the zona pellucida blue. Space within the uterine lumen is due to shrinkage during fixation. Colour differences between figures are chiefly an artefact of the colour photography. FIGURES 1-8 Fig. 1. Zona pellucida at antimesometrial end of uterine lumen. Female in lactational delay, 1\ days p.c., litter killed 15 h earlier. x460. Figs. 2-4. Unfertilized eggs in uteri of females ovariectomized 7 weeks earlier. Fig. 2. Two eggs stuck together, x 450. Fig. 3. Three eggs stuck together, in the extreme cervical end of the uterine horn, where the two horns are adjacent to one another, x 114. Fig. 4. Single egg. x 450. Figs. 5, 6. Egg transfers to females ovariectomized 6 weeks earlier. Fig. 5. Shed zona in uterus of ovariectomized female which had received zona-clad blastocysts 3 days before, x 450. Fig. 6. Naked blastocyst and attached zona in uterus of ovariectomized female which had received zona-clad blastocysts 7 days before. Trophoblast cells appear vacuolated and unhealthy but zona shows no signs of lysis, x 440. Figs. 7, 8. Unfertilized eggs, in thick intact zonae, in uteri of females mated to vasectomized males, ovariectomized 2\ days p.c., and killed 4 days 16 h p.c. Note that the uterine lumen must have been closed beforefixation,as the wall of the uterus appears to be moulded around the eggs, x 450. Fate of the zona pellucida 1 8 A. M C L A R E N Fate of the zona pellucida 9 over two eggs per horn could be found at 4£ days p.c., and these looked more fragile, with thin zonae (Figs. 11-14). After injection of fluid, the number again decreased, and those eggs that were seen consisted of barely identifiable remnants, crushed by the tightly apposed luminal walls (Figs. 15-18). At 5|- days p.c., no more eggs could be detected in the intact females. In all groups, flushing with saline was considerably less effective in detecting eggs than was serial sectioning. Table 5. Survival of unfertilized eggs in intact and ovariectomized pseudopregnant females (from examination of serially sectioned uteri) Ovariectomies were done 2\ days p.c. ; 10 % calf serum in phosphate-buffered saline was injected 3 | days p.c., as for egg transfer. Time of No. No. autopsy of of (days p.c.) $? horns No injection Ovariectomized Intact 10 % serum injected Ovariectomized Intact A\ 5i 4i 5± 4i 4i 8 2 9 3 3 6 8 2 9 3 3 6 Mean no. unfertilized eggs per horn 6-5 7-5 2-2 0-0 4-7 1-2 State of eggs Thick zona Thick zona Thin zona — Thick zona Remnants only FIGURES 9-18 Figs. 9,10. Unfertilized eggs, showing disintegrating vitellus but thick intact zona, in uterus of female mated to a vasectomized male, ovariectomized 2\ days p.c., fluid injected into one uterine horn 3i days p.c., killed 4 days 15 h p.c. x 500. Figs. 11-14. Unfertilized eggs in uteri of females mated to vasectomized males and killed 4 days 16 h p.c. Fig. 11. Three eggs stuck together. Vitellus in varying stages of disintegration. Zona thin (cf. Figs. 7-10). x470. Fig. 12. Egg in thin zona, held at antimesometrial end of uterine lumen. Note that the edges of the lumen are closely apposed, x 470. Fig. 13. Egg in very thin zona (cf. Figs. 11, 12). Note 'impression' in wall of uterus where egg was held before fixation, x 490. Fig. 14. Remains of egg, squeezed between closely apposed edges of uterine lumen. x460 Figs. 15-18. Unfertilized eggs in uteri of females mated to vasectomized males, fluid injected into one uterine horn 3 | days p.c., killed 4 days 15 h p.c. (cf. Figs. 7-10). Fig. 15. Remains of egg, squeezed between closely apposed edges of uterine lumen. x480. Fig. 16. Remains of egg, at antimesometrial end of uterine lumen. The edges of the uterine lumen are closely apposed, x 480. Fig. 17. Very degenerate remains of egg, free in lumen, x 470. Fig. 18. Very degenerate remains of egg, between closely apposed edges of uterine lumen. x490. 10 A. M C L A R E N Fate of the zona pellucida 11 These observations suggest that lysis of the zona of the unfertilized egg takes place towards the end of the 5th day of pseudopregnancy, unless oestrogen sensitization of the uterus is prevented by removal of the ovaries. The zonae of unfertilized eggs persist longer than those around blastocysts : this was observed when implantation was induced in lactating females by removal of the litter, and also during the course of implantation in normal pregnancy (see next section). 3. Normal pregnancy. To investigate the situation in normal, non-delayed pregnancy, females were killed at around the time of 50 % zona loss, and one or both horns of the uterus flushed with saline. The blastocysts recovered were examined under a dissecting microscope ( x 100), and the presence or absence of the zona was recorded. In cases of doubt the 'salt test' was applied (McLaren, 1967). FIGURES 19-31 Uteri of pregnant females at the end of the 4th day of gestation, just as implantation is beginning. Figs. 19-20. Shed zonae in the uterine lumen, 3 days 20 h and 3 days 23 h p.c. x 440. Figs. 21-22. Blastocyst in ' implantation chamber' at antimesometrial end of uterine lumen, 3 days 20 h p.c. In Fig. 21 this blastocyst appears to be emerging from its zona, but the adjacent section (Fig. 22) shows that the 'zona' is actually a degenerating unfertilized egg, and that the blastocyst is still at least in part surrounded by its own close-fitting zona (arrowed), x 470. Fig. 23. Blastocyst in 'implantation chamber' at antimesometrial end of uterine lumen, 3 days 21 h p.c. The thin zona (arrowed) is no longer adjacent to the blastocyst except at the abembryonic pole, but this is probably a fixation artefact : during fixation the lumen opened up, rupturing the zona and pulling the two sides of it away from each other and from the blastocyst, x 450. Figs. 24-25. Blastocyst at antimesometrial end of uterine lumen, 3 days 20 h p.c. The inner cell mass, cut through in Fig. 24, is devoid of zona; in contrast the abembryonic region (Fig. 25) is surrounded by a closely fitting, apparently intact, zona. x460. Figs. 26-27. Blastocyst at antimesometrial end of uterine lumen, 3 days 23 h p.c. In this case fixation has not opened up the lumen, so one can see how tightly the blastocyst is held. In Fig. 26 no zona material can be seen between trophoblast and uterine epithelium; in Fig. 27 blue zonamaterial can be distinctly seen in at least two places (arrowed), x 460. Fig. 28. Blastocyst in antimesometrial 'implantation chamber', 3 days 23 h p.c. Zona material can be seen around part of the periphery of the blastocyst (arrowed), but is absent elsewhere, x 460. Fig. 29. Blastocyst with fragments of zona material (arrowed) still adhering to it, 3 days 22 h p.c. x 450. Fig. 30. Blastocyst 3 days 22 h p.c., with closely fitting zona present around the abembryonic trophoblast (arrowed), but absent over the inner cell mass, x 450. Fig. 31. Blastocyst 3 days 22 h p.c., surrounded by zona which appears swollen and vacuolated, as during digestion with pronase. 12 A. McLAREN Of 41 females killed between 8.30 p.m. (G.M.T.) on the 4th day of pregnancy and 1.00 a.m. on the 5th, 14 showed Pontamine Blue areas in both horns of the uterus, 9 in one horn only, and 18 in neither horn. Flushing (Table 6) revealed that the eggs (blastocysts and morulae) from females without Pontamine Blue areas were virtually all still within the zona, though one blastocyst was oval Table 6. Zona loss during normal pregnancy (eggs recovered by flushing) Females were killed, 15 min after Pontamine Blue injection, between 8.30 p.m. (G.M.T.) on the 4th day of pregnancy and 1.00 a.m. on the 5th day. Eggs were recovered, by flushing, from one or or both horns of the uterus. No. of females No Pontamine Blue areas in either horn No Pontamine Blue areas in flushed horn Pontamine Blue areas in both horns No. of PontaNo. of mine No. of uterine Blue eggs rehorns areas covered In zona Zona ruptured Out of zona Empty zonae 18 23 0 116 114 1 1 0 9 9 0 44 35 1 8 1 14 14 40 32 22 0 10 0 Table 7. Zona loss during normal pregnancy (from serial sections) The uterine horns entered in this table are contralateral to those listed in the lower two lines of Table 6. No. of uterine horns No. of Pontamine Blue areas No. of eggs found No Pontamine Blue areas in flushed horns 9 26 33 Pontamine Blue areas in both horns 13 52 76 Zona ruptured Out of zona Empty zonae 5 4 24 1 23 14 39 6 In zona rather than spherical in shape, and the zona of another was ruptured. When Pontamine Blue areas were seen in one horn only, 80 % of the eggs recovered were in zonae; one of these again was oval, and another had a ruptured zona. One was seen to hatch from its zona during the flushing procedure, leaving an empty zona. When Pontamine Blue areas were seen in both horns, 69 % of the eggs recovered were in zonae ; two of these, in different females, were oval and two others had sticky, easily deformable zonae. Fate of the zona pellucida 13 In all but one of the 23 females in which at least one horn showed Pontamine Blue areas, the non-flushed horn was fixed and serially sectioned. The findings are summarized in Table 7. The percentage of eggs still in zonae, even where both horns showed Pontamine Blue areas (30 %), was significantly lower than in the flushings, confirming the conclusion of Orsini & McLaren (1967) that blastocysts become less easy to flush from the uterus once the zona is lost. The 18 eggs in the 'zona ruptured' category consist of 11 in which zona material was present over some of the blastocyst surface but absent elsewhere (Figs. 2431), and seven in which the blastocyst was partly out of a ruptured but otherwise intact zona (Fig. 23). In some instances, what appeared to be a partly shed zona turned out on closer inspection to be an adjacent unfertilized egg (Figs. 21-22). Compared with 63 zona-free blastocysts, only seven empty zonae were seen (Figs. 19, 20), two of them adjacent to zona-free blastocysts. The relation of blastocysts with and without a zona to the Pontamine Blue areas is discussed in McLaren (1969 a), in which the histological findings described above were summarized. DISCUSSION The data described above suggest that there exist two mechanisms for the loss of the zona pellucida in mice: an active 'hatching' mechanism, whereby the blastocyst emerges leaving the zona ruptured but otherwise intact, and a lytic mechanism. Depending on the circumstances of egg and uterus, loss of the zona may involve either or both of these processes. During lactational delay, or after ovariectomy (whether recent or several weeks earlier), the two components are separated in time. Only the hatching mechanism operates initially, with the result that naked blastocysts and evacuated zonae co-exist in the uterine lumen. The activity of the blastocyst which causes rupture of the zona and subsequent emergence probably involves the pulsating movements recorded under in vitro conditions by Borghese & Cassini (1963) and Cole & Paul (1965). When a stimulus to induce implantation is given, within a few hours the zonae have disappeared, presumably by lysis. The fact that blastocysts in lactating females retain their zonae for up to 24 h longer than in normal pregnancy suggests that zona loss by hatching alone is a more protracted process than when lysis plays a part. Unfertilized eggs in pseudopregnant females illustrate the complementary situation. No hatching is possible, since the unfertilized egg possesses no powers of contraction or activity of its own, so cannot emerge from the zona. The vitellus degenerates within the zona, and the zona is finally eliminated by lysis. The zona persists longer than in normal pregnancy, perhaps because some zona lysin normally emanates from the blastocyst, or perhaps because it does not have to undergo the stretching that accompanies blastocyst expansion. (As the diameter of the egg increases from 70 fi to 100 [i, the thickness of the zona must be halved, without the intervention of any lytic process.) 14 A. M C L A R E N The postulated scheme is summarized in Table 8. In normal pregnancy, blastocyst activity and the lytic factor are both present, and collaborate to produce loss of the zona late on the 4th or early on the 5th day of pregnancy. In the absence of either factor, zona loss is delayed. It is therefore not entirely correct to assert that 'uterine factors play a minimal role' in the escape of the mouse blastocyst from its zona (Cole, 1967). Indeed, it is difficult to be certain how often, if ever, the blastocyst in normal pregnancy actually emerges from the zona before the process of lysis is complete. The argument that hatching is precluded in normal pregnancy by the close contact between blastocyst and epithelium (Potts & Wilson, 1967) is not conclusive, because at the time of zona loss the uterine lumen is usually still at the 'pre-attachment' stage which also characterizes the uterus during delay (Potts Table 8. Factors affecting loss of the zona pellucida Situation Ovariectomized or lactating females Pseudopregnant females (unfertilized eggs) Normal pregnancy Activity of egg Zona lysin Time of zona loss Present Absent Late on 5th day Absent Present Late on 5th day Present Present Beginning of 5th day & Psychoyos, 1967); yet in delay hatching certainly occurs. Evacuated zonae are occasionally flushed from the uterus during the period of zona loss in normal pregnancy (Orsini & McLaren, 1967), but it may be that the violent dislodgement associated with flushing ruptures the already weakened zona and expels the contained blastocyst. Similarly, the histological evidence, that empty and ruptured zonae and 'hatching' blastocysts are seen in the uterus at the time of zona loss but not earlier, could be a result of the shrinkage due to fixation ; the partially lysed zona may stick to the uterine epithelium so that it ruptures when the epithelial surfaces are forced apart by the action of the fixative (Fig. 23). The appearance of occasional distorted and sticky zonae as the time of zona loss approaches shows that sometimes at least the lytic process is well advanced before the blastocyst has emerged, and it seems likely that this process may facilitate or even forestall emergence. As the two factors are so closely synchronized, a small difference in relative timing of trophoblast activity and release of lysin could determine whether the zona is dissolved while it still surrounds the blastocyst, or shortly after it has been shed. For most of the blastocysts 'caught' histologically in the act of losing the zona (Figs. 24-31), dissolution of the zona in situ appears to be in progress. Vacuolation and disintegration of the zona in situ has also been reported by Potts & Wilson (1967). Fate of the zona pellucida 15 Two pieces of evidence suggest that, in the mouse, the zona lysin may be secreted by the uterus rather than by the blastocyst. During delay of implantation the shed zona is by no means always adjacent to the blastocyst (Table 2); yet, once implantation has been induced, the zonae are briskly lysed. Diffusion of a lytic factor from the blastocyst, for 100 pi or more along the uterine lumen, seems unlikely. A uterine lysin also accounts more conveniently for the lysis of the zona of unfertilized eggs in pseudopregnant females, where no blastocysts exist. Vitelline disintegration in itself does not appear to produce lysis of the zona, since unfertilized eggs trapped in the oviduct can undergo almost complete disintegration while the zona remains intact. An opposite conclusion is reached by Dickmann (1969) on the basis of his experiments on rats, namely that in this species the zona lysin is produced by the trophoblast and not by the uterus. He found that rat morulae transferred to a 5-day pseudopregnant uterus mostly retained the zona when recovered several hours later, although blastocysts would have already lost the zona at the corresponding stage of pregnancy. Again, rat blastocysts taken from the uterus on the 5th day of pregnancy behaved ' autonomously ', and lost their zonae even after transfer to a female at an earlier stage of pseudopregnancy (Dickmann & Noyes, 1961) or to a progesterone-maintained ovariectomized recipient (Dickmann & de Feo, 1967). The later in the day the blastocysts were transferred, the shorter the interval before loss of the zona. It is not clear whether the autonomous behaviour of the blastocysts was restricted to 'hatching' activity, which constitutes the autonomous component of zona loss in the mouse also, or whether it included dissolution of the zonae. Other possible interpretations of these experiments have been discussed by McLaren (19696). The possibility of a real species difference between rat and mouse is raised by the observation of Folstad, Bennett & Dorfman (1969) that rat eggs cultured to blastocyst stage retain the zona under conditions in which mouse blastocysts usually hatch. However, this could merely indicate that the culture conditions were suboptimal for the rat eggs, since in vitro hatching activity tends to vary from experiment to experiment, even with mouse eggs (Brinster, 1965), and may reflect the pH of the medium (Bowman & McLaren, 1969). Whatever its source, the zona lysin of the mouse appears not to be released except under the influence of oestrogen. In the hormonally 'neutral' uterus of a female ovariectomized several weeks before, as well as in the progesteronedominated environment of a lactating female, the shed zonae persist indefinitely, and lysis only occurs when a state of oestrogen sensitization sufficient to allow implantation is achieved. In the hamster, where oestrogen seems not to be necessary for implantation, Orsini (1963) reports that loss of the zona (presumably by lysis) is progesterone-dependent. In the rat, Dickmann (1969) postulates that the release of zona lysin is actively inhibited by progesterone and that, in a hormonally 'neutral' environment, lysis occurs. Certainly in rats ovariectomized early in pregnancy and maintained on 16 A. M C L A R E N progesterone, at least some of the shed zonae persist as they do in the mouse during lactation (Alloiteau & Psychoyos, 1966; Psychoyos, 1966; Dickmann & de Feo, 1967), in contrast to normal pregnancy, where the zonae are rapidly dissolved (Dickmann, 1967). However, the failure to recover zonae by flushing, after transfer of blastocysts to females ovariectomized several weeks before (Dickmann, 1968), could be misleading, since in the mouse the technique of uterine flushing reveals only a small proportion of the zonae actually persisting in the long-term ovariectomized female (Table 4). The nature of the zona lysin is not yet known for any species. The pH is probably not involved, at least in the mouse, since in vitro studies have shown that a pH low enough to dissolve the zona is lethal to the blastocyst (Bowman & McLaren, 1969). One must therefore postulate the existence of a lytic enzyme. The decidual reaction in the rat is associated with the breakdown of collagen in the uterine stroma (Fainstat, 1963), presumably by the action of a collagenase. However, the only enzymes found to dissolve the zona at concentrations tolerated by the blastocyst were trypsin and pronase; collagenase had no effect (Bowman & McLaren, unpublished observations). In any case, if the zona lysin is uterine in origin, the observations on pseudopregnant females described in the present paper suggest that its release occurs independently of the presence of blastocysts, i.e. that it is a phenomenon of uterine sensitization rather than of decidualization (McLaren, 1969 c). SUMMARY 1. In female mice undergoing lactational delay of implantation, the zona pellucida is shed from the blastocyst but persists in the uterus. Such evacuated zonae were identified histologically, and the distance between blastocyst and zona was estimated to be greater at 1\ days than at A\ days post coitum. 2. The shed zonae also persisted after ovariectomy in early pregnancy, or when blastocysts were transferred to the uteri of females ovariectomized 6 weeks earlier. Histological examination proved a more effective method of detecting these zonae than recovery by flushing. Unfertilized eggs in intact zonae were found in the uteri of a proportion of females, even 6 weeks after ovariectomy. 3. In pseudopregnant females, the unfertilized eggs were reduced to tiny remnants on the 5th day p.c., and had disappeared altogether by the 6th day. After removal of the ovaries, however, the unfertilized eggs retained their thick, prominent zonae, and were easily detectable on the 6th day p.c. 4. The process of zona loss was also examined during normal pregnancy, both in blastocysts recovered by flushing from the uterus, and in serial sections. In females where zona loss was in progress, the zonae which remained were sometimes observed to be sticky and easily deformable. Shed zonae and 'hatching' blastocysts were observed, but could have been artefacts of the technique. 5. Two factors contributing to loss of the zona in mice are proposed: Fate of the zona pellucida 17 a lytic factor, emanating probably from the oestrogen-sensitized uterus, and the expansive activity of the blastocyst itself. During lactation or after ovariectomy the first is absent, and the blastocyst eventually bursts from the zona by its own efforts; lysis acts later, at the time of implantation, to eliminate the shed zonae. The unfertilized egg does not expand; hence the zona is neither thinned nor ruptured, but is none the less slowly lysed. During normal implantation the blastocyst expands, but the thinned zona is probably most often lost by lysis before emergence can take place. RÉSUMÉ Le devenir de la zone pellucide chez la souris 1. Chez la femelle subissant un délai d'implantation dû à la lactation, la zone pellucide est détachée du blastocyste mais persiste dans l'utérus. Ces zones pellucides séparées ont été identifiées histologiquement, et la distance entre le blastocyste et la zone est plus grande à 7 jours \ qu'à 4 jours \ après le coït. 2. La zone pellucide séparée persiste après ovariectomie au début de la gestation, ou quand les blastocystes sont transférés dans l'utérus de femelles ovariectomisées 6 semaines plus tôt. L'étude histologique est une meilleure méthode de détection de ces zones pellucides que la perfusion. Des œufs non fécondés entourés de zones pellucides intactes ont été trouvés dans l'utérus de femelles 6 semaines après l'ovariectomie. 3. Chez les femelles pseudogestantes les œufs non fécondés étaient réduits 5 jours après le coït et disparus le 6e jour. Après ablation des ovaires les œufs non fécondés conservaient une zone pellucide épaisse et pouvaient être décelés au 6e jour. 4. Le processus de la perte de la zone pellucide a été étudié pendant la gestation normale chez des blastocystes obtenus par perfusion de l'utérus et après des coupes histologiques en séries. Chez les femelles où la perte de la zone pellucide était observée, certaines zones qui persistaient étaient visqueuses et facilement déformables. Des éclosions de blastocystes ont été notées mais pouvaient être dues à des artefacts. 5. Deux facteurs sont proposés pour expliquer la perte de la zone pellucide chez la souris: un facteur de lyse, provenant probablement de l'utérus sensibilisé à l'œstrogène, et l'activité d'expansion du blastocyste. Pendant la lactation ou après ovariectomie le premier facteur est absent, et éventuellement le blastocyste sort de la membrane pellucide par son propre action; la lyse intervient après, au moment de l'implantation, pour éliminer la zone pellucide séparée. 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