The Role of the Mucin Layer in Development of the Rabbit Blastocyst' GILBERT S. GREENWALD Department of A n a t o m y , University of W a s h i n g t o n , School of Medicine, Seattle, Washington The rabbit is unique among eutherian tremes and marsupials. If this were true mammals in the thick layer of mucin that the egg might be able to develop normally coats the ovum during oviducal passage. in the complete absence of the mucin The mucin is an acid mucopolysaccharide layer. (Braden, '52) and is released from the No experiments have been performed tuba1 epithelium after ovulation (Moreaux, which directly shed light on the validity of '13; Cohnen, '27; Westman, '30; Adams, any of these hypotheses. Recently, it was '58; Fredricsson, '59). The secretion is found that the injection of estrogen imsynthesized by estrogen and apparently re- mediately post coiturn blocks the release leased under the influence of progesterone of most of the mucin from the oviducal epi(Greenwald, '58). However, Flerko ('55) thelium and consequently the eggs acquire believed that progesterone is not involved a considerably reduced mucin coating in the discharge as judged by histological (Greenwald, '57). It was thought that the criteria. diminished mucin layer might account in Several theories have been offered to part for the heavy embryonic mortality account for the extensive deposition of that follows the administration of estrogen. mucin around the rabbit egg. Gregory However, further studies indicated that ('30) believed that the mucin layer by re- acceleration of transport of ova during leasing its water content was responsible the first few days of pregnancy was the for the expansion of the rabbit blastocyst. primary factor in embryonic loss; thus the Recent findings in relation to the problem role of the reduced imucin layer has not of ovulation, suggest that depolymerization yet been evaluated (Greenwald, '59). of acid mucopolysaccharides may increase The present experiments were designed intra-follicular osmotic pressure; this may to eliminate the accelerating effects of account for the final spurt of growth of the estrogen noted above. This was accompreovulatory follicle (Zachariae, '59). This plished by transplanting three day old attractive hypothesis could be equally ap- rabbit embryos from animals treated with plicable to the marked and rapid expan- estrogen to the corriua of animals that sion of the rabbit blastocyst. were three days pseudopregnant. Such ova Another theory proposed by Assheton would have a reduced amount of mucin (1895) assigned a purely protective role and yet be subject to' a normal endocrine to the mucin layer. The expansion of the environment during implantation. rabbit egg is dependent most likely on MATERIALS AND METHODS hydrostatic pressure and the thin wall of the blastocyst may be incapable of withA total of 139 New Zealand giant white standing the build-up in pressure. The rigid female rabbits were used in the transtough mucin layer could act to prevent research was supported by grant 7063 from the rupture of the expanding blastocyst. the1 This U. S. Public Health Service.. The human chorjonic While both of the preceding theories gonadotropin (APL) used in thls study was furnished the courtesy of Ayerst Laboratories, New suggest a functional role for the mucin through York. the estradiol cyclopentylpropionate (ECP) was suppiied by the Upjohn Company, Kalamazoo, Michilayer, Hartman ('25) believed that the gan. coating was a primitive, non-functional 2 Present address: Department of Gynecology and University of Kansas Medical Center, Kancharacter, shared in common with mono- Obstetrics, sas City, Kansas. 407 408 GILBERT S. GREENWALD plantation experiments. The experimental procedure is illustrated in figure 1. In the control series of transfers, donor females were mated to males of proven fertility and the future recipients were then injected intravenously with 100 i. u. of human chorionic gonadotrophin (APLxx). At 70 hours post coitum the donors were killed by the intravenous injection of an overdose of nembutal and the oviducts were removed and flushed with 5 ml of freshly prepared normal saline to which penicillin (20,000 i. u. per ml) had been added. The eggs were flushed into sterile 40 mm square staining dishes and examined at a magnification of 25 X with a dissecting microscope. The diameter of each ovum including the mucin layer was measured with an ocular micrometer. It had been previously determined that in this strain of rabbits the diameter of the eggs to the outer limit of the zona pellucida was relatively constant at 172.0 v. By subtracting this figure from the total diameter, the I DONORS I diameter of the mucin layer could be determined; the thickness of the mucin layer was then calculated by dividing by two. Before killing the donor animal, the recipient was anesthetized with nembutal (i.v. injection of .66 ml of standard veterinary nembutal per kg of body weight) and the cornua exposed by a midline abdominal incision. The ova were transferred to the uterine horns of the recipient by a braking pipette (Holter, '43) and ordinarily, not more than 15 to 20 minutes elapsed between the death of the donor and the transfer of ova to the host. A week after transplantation, the recipient was laparotomized and the number of implantation sites counted and compared with the number of ova that had been transferred. To produce eggs deficient in the mucin layer another series of animals was injected intramuscularly immediately after mating with 250 pg of estradiol cyclopentyl- I RECIPIENT I Induce ovulation with 70 hours later 70 hours PC. Flush tubes Measure ova Mote: Inject immediately with 250 pg of Depo- est rodiol Laparotomy at 10 days F! C. Fig. 1 Experimental design of the transplantation experiments. Depo-estradiol is the trade name for estradiol cyclopentylpropionate (ECP). MUCIN LAYER EXPERIMENTS 409 propionate (ECP). Ovulation was then induced in the recipients and the transfer procedure outlined for the control group was carried out. A total of 17 transfers were made with normal ova and 40 transfers of ova with a reduced layer of mucin (reduced mucin ova). In an additional series of transfers with reduced mucin or normal ova, the recipients were killed one, two or three days after transplantation and the blastocysts flushed from the uterus. The diameters of the blastocysts were determined with an ocular micrometer and compared with blastocysts recovered from untreated animals killed on days 4, 5 or 6 post coitum (P.c.). Twenty-five animals were used in these experiments. RESULTS At 70 hours P.c., there was a marked difference in the thickness of the mucin layer around ova recovered from control donors and from animals that had been injected with ECP (figs. 2, 3). However, ova of both groups had cleaved to the same extent and there was no difference in the percentage of unfertilized or abnormally developed eggs. The total diameter of ova was usually constant within any one rabbit in the control transfers, but there was considerable variation from animal to animal. Ova of different animals ranged in total diameter from 301 to 430 Fig. 2 A normal rabbit egg recovered 70 hours post coitum. x 100. Fig. 3 A rabbit egg recovered 70 hours post coitum from an animal injected immediately after mating with 250 pg of estradiol cyclopentylpropionate. Compare the thickness of the mucin layer of both ova. ~ 1 0 0 . p or expressed in terms of mucin thickness from 65 to 129 p. The injection of 250 clg of ECP immediately after mating resulted in a considerable reduction of the imucin layer but did not inhibit it completely. In 22 of 40 transfers, the ova had a mucin layer of 12 p while in the remainder it measured up to 43 p, The results of the transfer of ova from the control animals are summarized in table 1. Of a total of 148 normal eggs transferred at three days P.c., 102 were represented one week later by definite implantation sites (or 613.9% ). The success of implantation had no relation to the thickness of the mucin layer. At the time of laparotomy, only it few embryos were being resorbed but the close spacing of some implantation sites (e.g. transfers 14 and 16, table 1) probably would have resulted in further embryonic loss at later stages in pregnancy. Only 5 females were examined at term or shortly before delivery (table 1, transfers 1, 5,6, 11,12). In these animals, 23 of the original 31 implantation sites were represented by delivered young or living late term fetuses. Of 332 transplanted ova with a reduced coating of mucin, 97 eggs (or 29.2% ) implanted successfully (table 2). At 10 days p.c. considerably more embryos were be- 410 GILBERT S . GREENWALD TABLE 1 Results of the transfer of ova with a normal mucin thickness to recipient cornua at 3 days p.c. Ova arranged according to thickness of mucin layer Total Transfer diameter no. egg Mucin thickness No. eggs at transfer No. implantation sites (10 days Remarks P.C.) fi P 1 2 3 4 5 6 7 8 9 10 11 430 430 409 397 397 397 387 366 366 366 366 129 129 119 113 113 113 106 97 97 97 97 10 10 10 8 11 8 4 6 8 9 11 6 9 10 6 9 5 2 4 7 4 6 12 13 14 15 16 17 344 344 344 301 301 301 86 86 86 65 65 65 10 11 9 8 6 9 5 8 3 4 6 148 102 Total 8 ing resorbed than in the control series, but some fetuses were alive near term and several young were delivered (table 2, transfers 4, 5, 11, 19). Transplanted ova with a mucin thickness of from 22 to 43 p had a similar implantation rate (table 3). When ova with a minimal mucin coating (12 LI) were transplanted only 11 out of 22 recipients became pregnant although a number of the unsuccessful transfers appeared to be technically perfect. Despite this heavy loss of ova, in one transfer (table 2, no. 19) three normal living young were born at 34 days p.c. The size and number of preimplantation ova from one to three days after transfer is shown in figure 4. At 24 hours after transfer, the diameters of the normal ova (now 4 days old) fell within the size range of the control blastocysts (i.e. non-transferred group) while the reduced mucin ova were slightly smaller. However, by 5 days, there was no significant difference in the size of the blastocysts of the three groups. By 6 days p.c. there was considerable variation in the size of the control blastocysts. For instance, in female 807 blastocysts varied from 2.3 to 3.6 mm in diameter and in female 768 from 1.5 to 2.6 mm. 35 days p.c. - delivered 6 yg. 10 days p.c. -normal embryos 10 days p.c. - normal embryos 34 days p.c. - delivered 5 yg. 32 days p.c. - delivered 4 yg. 10 days p.c. -some adhesions 10 days p.c. - one embryo resorbing 10 days p.c. - some adhesions - 37 days - delivered 3 yg. Killed 28 days p.c. - 5 living Yg. 10 days p.c. - one embryo at cervix 10 days p.c. - 4 embryos crowded Poor transfer 10 days p.c. - 2 embryos crowded Ova transferred with a normal or diminished layer of mucin had reached the same size as the control eggs by day 6 (fig. 4). Of 47 normal ova that were transferred, 35 (74%) were flushed three days later from the cornua of the recipients while out of 52 reduced mucin ova 41 (79% ) were recovered. Thus, the recovery rate at day 6 was essentially the same for both groups. However, while 61.7% of the transferred control ova were large and viable blastocysts at day 6, only 30.7% of the blastocysts from the reduced mucin series were normal. The other reduced mucin ova were degenerating at day 6, apparently after first expanding to the size of normal day 5 ova and then collapsing (fig. 4). It should be stressed that some of the blastocysts developing from the reduced mucin ova were as well developed as control embryos although most had degenerated by day 6. For example, at the time of transfer the ova of female 843 had a mucin thickness of only 12 p, but 5 living blastocysts recovered from the recipient measured 2.3 to 2.7 mm in diameter. The recovery of blastocysts from the recipients before implantation permitted a comparison between the thickness of the 411 MUCIN LAYER EXPERIMENTS TABLE 2 . Results of the transfer o f ova w i t h a reduced mucin thickness to recipient Icornua at 3 days p.c. Total diameter egg thT$2:ss No. eggs at transfer _ No. implantation sites _ _ ~ -_____ Remarks (10 days P.C.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30-40 P B 258 258 43 41 9 7 6 3 258 236 236 236 236 236 215 215 215 215 215 215 215 215 215 215 195 41 32 32 32 32 32 22 22 22 22 22 22 22 22 22 22 12 8 10 8 9 10 12 3 6 11 10 7 8 9 7 4 10 4 0 5 6 7 1 0 3 3 6 9 6 1 0 0 2 0 4 195 195 195 195 195 195 195 195 195 195 195 12 12 12 12 12 12 12 12 12 12 12 10 8 5 3 2 6 4 1 2 3 1 0 9 5 4 9 11 9 9 8 11 95 Total 332 10 days p.c. - adhesions - 35 days - delivered no yg. Perfect transfer 35 days p.c. - delivered 5 yg. 24 days p.c. - had 4 living fetuses 2 resorbing embryos at 10 days Embryo resorbing Perfect transfer 17 days p.c. -one resorbing Healthy embryos 10 days p.c. - 2 resorbing - 29 days - 3 living 1resorbing - 10 days Healthy embryos Some eggs unfertilized? Normal embryos 34 days - delivered 10 days p.c. - 1resorbing 3 Yg. 10 days p.c. - 2 resorbing Healthy embryos 2 resorbing 1resorbing 1resorbing Resorbing See text for details ~ _ _ _ _ _ 97 TABLE 3 Relationship between the mucin thickness o f the transferred reduced mucin ova and successful implantation ~ Total diameter of ova at transfer P 258 236 215 195 195 Mucin thickness No. of females No. of ~~~~~~t No. eggs transferred No. implanted 0 24 49 75 89 95 9 19 30 39 0 24 332 97 P 43 32 22 12 12 3 5 10 11 11 Total 40 2 4 7 11 % 37.5 38.8 40.0 37.1 - 29.2 412 GILBERT S . GREENWALD 0 0 A 000 Control ova Transferred normal ova Transferred reduced ova O O 0 A A ...HA AAA eeee AA AAA A 00 0000 eeee A 00 00 erne O 0000 ee AAAAA e* A A I 6 5 4 -- Age of ova (days post coitum) Fig. 4 The size of individual blastocysts at 4-6 days post coitum. The asterisks designate ova that were degenerating. TABLE 4 Mucin thickness at 3 days p.c. and the size of individual blastocysts at 6 days Total diameter of ova at transfer P < 195l 195' 215l 301 38 7 430 Diameter of recovered 6 day blastocysts Mucin thickness mm P < 12 3.3, 1.4 2.3,2.7,2.4,2.7,2.5 3.0, 2.1,2.5,2.2,3.0,2.8, 1.9 2.0,2.0,2.2,2.0 2.6,2.6,2.9,2.2,3.0,3.0,3.0,3.i 2.3,2.9, 3.5,3.4,3.4,4.0,2.0,3.6 12 22 65 106 129 'Donor animals had been injected with ECP. mucin layer of day three tuba1 ova and the ultimate size reached by the blastocysts. Six representative transfers involving ova ranging from 195 to 430 in total diameter are listed in table 4. The 5 largest blastocysts recovered corresponded to ova that were 430 in diameter at the time of transfer. With these exceptions, there appears to be no significant positive corre- lation between initial thickness of the mucin layer and the size of the blastocysts. DISCUSSION It is apparent that reduction of the mucin layer to a thickness of only 12 p is not incompatible with normal implantation and development of the rabbit egg. However, the rate of successful implantation was sharply reduced (tables 1, 2). MUCIN LAYER EXPERIMENTS After the present research was completed, Adams reported some preliminary experiments on the role of the mucin layer in development of the rabbit blastocyst ('60). Two-celled ova were recovered and cultured in vitro for 24 hours; 52 moruIae with a mucin layer of 10 M or less were then transferred to 7 recipients. At laparotomy at 10 days P.c., 44% of the eggs had implanted successfully in 5 recipients. These results are in substantial agreement with the present findings. The reduced mucin ova in the present experiments were produced by injecting rabbits immediately after mating with 250 ug of estradiol cyclopentylpropionate (ECP). Estrogens have a deleterious effect on cleaving sea urchin eggs by arresting mitoses (Agrell, '54) but there is no evidence that exogenous estrogens directly influence the development of mammalian ova. In the present experiments, ECP did not interfere with the normal cleavage pattern; the ova with a reduced mucin layer were identical at three days p.c. with normal untreated ova (figs. 2, 3). Similarly, rabbit ova cultured in vitro with estrone or estradiol go through cleavage stages at the normal rate and diazo compounds coupled with estrone fail to penetrate and vitally stain such ova (Pincus and Kirsch, '36). The injection of 5 mg of stilbestrol at 5 days p.c. also does not alter the mitotic rate in the extra-embryonic layer of the rabbit blastocyst (Moog and Lutwak-Mann, '58). While there are no indications that rabbit ova are directly affected by estrogen and while there are no visible effects on the morula stage, a deleterious effect on the blastocysts at the time of the abrupt morphological and biochemical reorganization of the developing blastocyst (Brachet, '60) cannot be excluded. It is possible that the greater mortality of the reduced mucin ova is due in part to their development in an environment dominated by estrogen. It is apparent from figure 4 and table 4 that the thickness of the mucin layer at day three does not correlate with the ultimate size reached by the blastocyst. By day 6 P.c., some reduced mucin ova had expanded to the same size as normal blastocysts, This appears to rule out Gregory's 413 theory that the muciri layer is necessary for expansion of the blastocyst ('30). The degeneration of blastocysts developing from reduced mucin ova might indicate a protective role of the mucin layer in accordance with Assheton's hypothesis (1895), but a number of the reduced mucin ova grew into large and viable blastocysts at day 6. Thus, the results of the transplantation experiments do not provide clear-cut evidence for any specific protective role for the mucin layer. However, the most critical experiment has not yet been performed. Until ova completely devoid of any trace of mucin are transplanted, the possibility still exists that the blastocyst may require only a thin coating for its normal differentiation. Preliminary attempts to produce such mucinless ova have so far been unsuccessful. After increasing the single post-copulatory injection of ECP from 250 to 500 clg, the minimal thickness of mucin was unchanged at 12 CI (Greenwald, unpublished). This suggests that minimal amounts of mucin may always be covering the surface of the tubal epithelium and that exogenous estrogens prevent only the release of the stored intra-epithelial content. A n alternative explanation for the failure to inhibit mucin release completely with estrogens could be the rapid release of progesterone within 100 minutes after coitus (Forbes, '53). Progesterone is responsible for the normal secretion of mucin and exogenous estrogens prevent the discharge by competing with proeesterone (Greenwald, '58). The initial rapid release of progesterone could lead to a limited discharge of mucin before effective inhibitory levels of circulating estrogen are reached. To test this possibility, rabbits were injected two or three days before ovulation with 250 pg of ECP and ovulation was then induced with gonadotrophins. However, after this treatment the recovered tubal ova were still invested with a thin mucin layer. The mucin layer can be reduced markedly by injecting recently mated rabbits with estrogens (fig. 3) or by removing twocelled ova from the oviduct and culturing them in in vitro (Adams, '60). It is possible that the thin muciii layer surrounding such ova could be dissolved with proteo- 414 GILBERT S. GREENWALD lytic enzymes (Braden, ’52) without affecting the egg itself. The role, if any, of the rnucin layer cannot be established with certainty until ova without any trace of the coating are transferred to the cornua of pseudopregnant recipients. SUMMARY These experiments were designed to determine whether or not the mucin layer is essential for development and implantation of the rabbit blastocyst. Usually, the normal three day old morula is invested by a mucin layer varying in thickness from 65 to 129 w. In previous experiments this layer was greatly reduced after estrogen was injected into rabbits immediately after coitus. In order to circumvent the effect of estrogen on transport of ova and development of the endometrium, ova with a layer of mucin as thin as 12 p were produced by injecting does immediately post coitum with 250 ug of estradiol cyclopentylpropionate. The ova were transplanted at three days p.c. to the cornua of pseudopregnant recipients and the success of implant ation determined. Of a total of 332 ova transferred with a reduced layer of mucin, 97 (or 29.2%) implanted one week later. When 148 control ova were transferred similarly, 68.9% implanted successfully. Since reduction of the mucin layer was the only variable it appears that lack of sufficient mucin interfered with implantation. However, in one instance normal living young were delivered at term that had developed from eggs with an envelope of mucin as thin as 12 I-I. Furthermore, 6 day blastocysts recovered during the preimplantation period and derived from ova similarly deficient in the mucin layer were found to have expanded to the same size as normal embryos. By day 6 only 31% of such ova were large and viable blastocysts; the remainder were degenerating after developing to the same size as normal 5 day eggs. These experiments indicate that normal expansion and implantation of the rabbit blastocyst sometimes occurs despite a marked reduction in the thickness of the mucin layer. The mucin layer may not be essential, therefore, for normal development of the rabbit egg. However, it re- mains to be established whether ova completely devoid of any coating of mucin can undergo normal development. LITERATURE CITED Adams, C. E. 1958 Egg development i n the rabbit: the influence of post-coital ligation of the uterine tube and of ovariectomy. J. Endocrin., 16: 283-293. 1960 Development of the rabbit eggs with special reference to the mucin layer. Advance Abstracts of the First Intl. Congress of Endocrinol., Abstract No. 345, p. 687, Copenhagen. Agrell, I. 1954 Oestradiol and testosterone propionate as mitotic inhibitors during embryogenesis. Nature, 173: 172. Assheton, R. 1895 A reinvestigation into the early stages of development of the rabbit. Quart. J. Micr. Sci., 37: 113-164. Brachet, J. 1960 The biochemistry of development. Pergamon Press, New York. Braden, A. W. H. 1952 Properties of the membranes of rat and rabbit eggs. Aust. J. Sci. Res. Ser. B. BioI. Sci., 5: 460471. Cohnen, K. 1927 Uber den Mechanismus der Eiwanderung durch den Eileiter mit besonderer Beriicksichtigung der zyklischen Veranderungen am Eileiterepithel des Kaninchens. Ztschr. Mikroskop. Anat. Forsch., l l : 4 7 s 4 9 2 . Flerko, B. 1955 Die Epithelien des Eileiters und ihre hormonalen Reaktionen. Ibid., 61: 99-118. Forbes, T. R. 1953 Pre-ovulatory progesterone in the peripheral blood of the rabbit. Endocrinology, 53: 79-87. Fredricsson, B. 1959 Studies on the morphology and histochemistry of the fallopian tube epithelium. Acta Anat. Supp. 37, 38: 1-23. Greenwald, G. S. 1957 Interruption of pregnancy in the rabbit by the administration of estrogen. J. Exp. Zool., 135: 461-482. 1958 Endocrine regulation of the secretion of mucin in the tuba1 epithelium of the rabbit. Anat. Rec., 130: 477496. 1959 The comparative effectiveness of estrogens in interrupting pregnancy in the rabbit. Fertility and Sterility, 10: 155-161. Gregory, P. W. 1930 The early embryology of the rabbit. Carnegie Inst. Wash. Pub. 407, Contrib. to Embryol., 21: 141-168. Hartman, C. G. 1925 On some characters of taxonomic value appertaining to the egg and the ovary of rabbits. J. Mammalogy, 6: 114121. Hay, M., C. E. Adams and C. Lutwak-Mann 1959 The effect of certain agents upon the early rabbit embryo. J. Endocrinol., 20: ii-iii. Holter, H. 1943 Technique of the Cartesian diver. Compt. rend. Lab. Carlsberg, SBr. Chim., 24: 399478. Moog, F., and C. Lutwak-Mann 1958 Observations on rabbit blastocysts prepared as flat mounts. J. Embryol. Exp. Morph., 6: 57-67. Moreaux, R. 1913 Recherches sur la morphologie et la fonction glandulaire de 1’6pith6lium MUCIN LAYER EXPERIMENTS de la trompe uterine chez les mammiferes. Arch. d'Anat. Micr., 14: 515-576. Pincus, G., and R. E. Kirsch 1936 The sterility in rabbits produced by injections of oestrone and related compounds. Am. J. Physiol., 11 5: 219-228. 415 Westman, A. 1930 Studies of the function of the mucous membrane of the uterine tube. Acta Obstet. Gynec. Scand., 10: 288-298. Zachariae, F. 1959 Acid mucopolysaccharides in the female genital system and their role in the mechanism of ovulation. Doctoral Thesis. Periodica. Copenhagen, 1-63.