THE JOURNAL OF EXPERIMENTAL ZOOLOGY 258:240-245 (1991)
Reversal of Postmortem Degeneration of Mouse Oocytes During Meiotic Maturation In Vitro ALLEN C. SCHROEDER, DANIEL JOHNSTON, AND JOHN J . EPPIG The Jackson Laboratory, Bar Harbor, Maine 04609 The developmental capacity of oocytes matured in vitro following isolation at the ABSTRACT germinal vesicle stage from freshly killed mice (control)was compared with that of oocytes isolated from the carcasses of mice killed 3,6,9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3,6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.
The mammalian ovary contains many more immature oocytes, and hence more potential eggs, than could ever be presented for fertilization by normal ovulatory or superovulatory mechanisms. Ovarian oocytes, therefore, constitute a vast, mostly unused, resource of female gametes that could be harvested to increase the numbers of rare or endangered animals. Mammalian ovarian germinal vesicle (GV)-stage oocytes will, following isolation from the follicular environment, undergo maturation in culture producing eggs with full developmental potential t o live young (Schroeder and Eppig, '84, '89; VanBlerkom and McGaughey, '78; Shalgi et al., '79; Staigmiller and Moor, '84; Goto et al., '88; Sirard and First, '88; Vanderhyden and Armstrong, '89). Unanticipated death of rare or endangered animals and the resultant postmortem degeneration of the oocytes, however, could prevent the utilization of the oocytes for animal propagation. The experiments reported here were conducted to determine the impact of postmortem degenerative changes on the developmental capacity of mouse oocytes matured in vitro. Oocytes retrieved from animals deceased for several hours demonstrate a full developmental capacity even after pronounced post0 1991 WILEY-LISS, INC.
mortem degenerative changes have occurred. The culture system allows the oocytes to reverse these degenerative changes during maturation, and the eggs produced are capable of fertilization and development to live young after transfer t o foster mothers. These techniques may be important for the rescue of oocytes from recently deceased valuable zoological, agricultural, and endangered mammals.
MATERIALS AND METHODS Oocyte maturation Immature germinal vesicle stage cumulus cellenclosed ovarian oocytes were obtained from groups of 22-24-day-old (C57BL/6J x SJL/J)F1 juvenile mice that had received 5 IU of pregnant mares' serum gonadotropin (Dyosynth, Chicago, IL) 44-52 hr before death induced by cervical dislocation. Oocytes were isolated from groups of control mice immediately after death and from Received April 11, 1990; revision accepted August 24, 1990. Address reprint requests to John J . Eppig, The Jackson Laboratory, Bar Harbor, ME 04609. Allen C . Schroeder is a Visiting Investigator from Gettysburg College, Gettysburg, PA 17325.
REVERSAL OF POSTMORTEM CHANGES IN OOCYTES
the carcasses of experimental mice killed 3, 6, 9, and 12 hr earlier then held at room temperature. As described previously (Schroeder and Eppig, '84), oocyte-cumulus cell complexes were isolated and matured for 15-16 hr in 35 x 10 Corning tissue culture dishes containing Eagles minimum essential medium supplemented with Earle's salts, 0.23 mM pyruvate, 75 mglliter penicillin G potassium salt, 50 mg/liter streptomycin sulfate, 5%fetal bovine serum (Sterile Systems) (MEM t FBS), and follicle-stimulating hormone (FSH 1 pg/ml; oFSH-17, NIADDK). Medium was continuously gassed with a humidified mixture of 5% C02, 5% 02,and 90% N2. Cultures were carried out at 37°C in a gassed modular incubator chamber (Billups Rothenberg).
241
total of 200 embryos were transferred. Also, to determine whether developmentally competent oocytes could be retrieved from mice which were not primed with PMSG, 60 embryos derived from unprimed mice were transferred as described above. Blood was drawn from live born offspring at the age of 4 weeks and hemoglobin profiles determined to insure that offspring were derived from transferred embryos (Downs et al., '86). His tolog y Ovaries and oocytes were fixed in a solution of 3% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, at room temperature for 3 and 1 hr, respectively. After dehydration in ethanol, the ovaries were embedded in JB-4 resin, sectioned at 2 pm, and stained with hematoxylin and eosin. The complexes were washed in cacodylate buffer overnight and postfixed for 0.5 hr in 1%Os04 in the same buffer at 4°C.After dehydration in a graded ethanol series and propylene oxide, the oocytes were embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1OOCX electron microscope.
Fertilization and embryonic development in vitro The medium used for fertilization and embryo culture in vitro was a modified Whitten's medium (WM; Hoppe, '85), supplemented with 3 mg/ml crystallized lyophilized bovine serum albumin (ICN). After maturation in MEM + FBS, oocytecumulus cell complexes were washed through three 2.5 ml changes of WM and transferred t o a 0.5 ml drop of WM overlayed with paraffin oil. RESULTS Fertilization was carried out with 10 pl of a sperm Developmental capacity of cumulus suspension prepared from both cauda epididycell-enclosed oocytes matured in vitro mides of a 4 month-old B6SJLF1 male housed infollowing isolation from carcasses of mice dividually from the time of weaning. The cauda epididymides were excised into a 0.9 ml drop of Cumulus cell-enclosed oocytes were isolated WM under oil at 37"C, aseptically minced, and from mice immediately after sacrifice (control) allowed to disperse for 15 min before insemina- and from the carcasses of mice sacrificed 3, 6, 9, tion. The insemination dishes were incubated for and 12 hr earlier then kept at room temperature. 6-8 hr then all ova were washed with two 2.5 ml As shown in Figure 1, the yield of cumulus cellchanges of WM and cultured at 37°C in 12 x 75 enclosed germinal vesicle-stage oocyte cumulus mm stoppered and gassed borosilicate glass cul- cell complexes decreased with increasing time ture tubes containing l ml of WM. Frequency of postmortem. Compared with controls, the yield of development to the 2-cell stage was assessed after intact germinal vesicle-stage complexes 3 hr after 18 hr. Two-cell stage embryos were cultured in death was approximately half. Nevertheless, even WM for an additional 4 days to determine the after 12 hr postmortem, about 4 intact complexes frequency of development to expanded blasto- per mouse were retrieved. After culture for 15-16 cysts. Data from four experiments were pooled, hr in the presence of FSH, more than 98% of ooand statistical comparisons between groups made cytes from both control and postmortem groups of by x2 analysis. oocytes underwent germinal vesicle breakdown (GVB) in vitro, produced a polar body, and underEmbryo transfer went mucification and expansion of the cumulus Ten 2-cell stage embryos derived from addi- oophorus. There was no difference in the kinetics tional groups of 6 hr postmortem oocytes that had of GVB between oocytes from control and 6 hr matured in vitro were transferred to each oviduct postmortem mice; GVB was completed within 90 ampullae of 8 week old B6SJLF1 foster mothers min after oocyte isolation (data not shown). on the morning of vaginal plug after mating with As shown in Figure 2, following insemination BALB/cJ vasectomized males (Pease et al., '89). A in vitro, the percentage of ova derived from 3 and
242
A.C. SCHROEDER ET AL. 50
a v)
3 40 L
a n v)
-a f 8 X
30
20
c
0 L
0
f z
10
0
0
3
6
9
12
Time Postmortem (h) Fig. 1. Number of intact oocyte-cumulus cell complexes isolated from mice at various times postmortem. Points indicate the mean and SEM of data from four experiments. Different letters indicate a significant difference (P < 0.01) using the Student-Newman-Keuls Multiple Range Test.
6 h r postmortem mice that cleaved to the 2-cell stage was equivalent to the control group. When the interval between death and removal of the ovary for oocyte isolation was increased to 9 and 12 hr, the frequency of cleavage to the 2-cell stage was decreased. When 2-cell stage embryos
derived from the 3,6, and 9 hr postmortem groups were cultured an additional 4 days, the frequency of 2-cell embryos which developed to blastocysts was not significantly different from controls. Nevertheless, the number of blastocysts that developed from ova in the 9 hr postmortem group was
H Control
69
c Q)
3h
0 6h 9h
El
30
12h
20 10 0
2-Cell Embryos
Blastocysts from 2-Cell Embryos
Fig. 2. Developmental capacity of embryos derived from oocytes isolated from mice at various times postmortem. Different letters within a group of embryos indicate a significant difference (P < 0.05) using x2 analysis. Bars indicate either the percentage of inseminated ova that cleave to the 2-cell stage, the percentage of 2-cell stage embryos that develop to the blastocyst stage, or the percentage of ova that develop to
Blastocysts from Ova
the blastocyst stage. The figure shows the pooled results of four independent experiments wherein the total number of animals donating oocytes was 10 in the control group and 16 in all of the other groups. The total number of oocytes included was as follows: control, 480; 3 hr, 362; 6 hr, 209; 9 hr, 101; 12 hr, 67.
REVERSAL OF POSTMORTEM CHANGES IN OOCYTES
less because fewer ova cleaved t o the 2-cell stage. The 2-cell stage embryos derived from the 12 hr postmortem group did not develop to blastocysts. Oviductal transfer of 200 2-cell stage embryos derived from 6 hr postmortem mice produced 71 genetically marked live young (36%). Also, 18% (11 of 60) of 2-cell stage embryos derived from 6 hr postmortem mice which had not received exogenous gonadotropin priming developed to live young. Structure of 6 hr postmortem follicles and oocytes Control and exDerimenta1 cumulus cell-enclosed oocytes couli not be readily distinguished from one another using a dissecting microscope. In contrast, histological examination of fixed and stained control (Fig. 3A) and 6 hr postmortem ovaries (Fig. 3B) revealed large irregularly
243
shaped dark-staining inclusions randomly dispersed within the ooplasm of experimental oocytes and many pyknotic cumulus and mural granulosa cells throughout the follicle. An invagination of the follicle wall was also observed frequently. Furthermore, electron microscopy of oocytes isolated from postmortem mice (Fig. 4) showed that the inclusions were crystalline in nature. Microscopy of oocytes fixed at increasing intervals after death, beginning at 30 min, showed that small crystals were present by 1hr postmortem and grew larger and more numerous with increasing time after death. Six hour postmortem cumulus cell-enclosed oocytes were cultured and then fixed for electron microscopy at increasing intervals beginning at 30 min from the start of culture. The inclusions began t o disappear within 30 min in culture and were completely gone after 60 min.
Fig. 3. Photomicrographs of ovarian follicles from mice immediately after death (A) and 6 hr postmortem (B). Although the oocyte in the 6 hr postmortem follicle remains in the germinal vesicle stage (arrow), the cytoplasm contains many densely staining granules (arrowheads). The membrana granulosa appears degenerated in that many of the granulosa cells are pyknotic, more loosely adherent to each other, and there are invaginations of the follicle wall (X). x 240.
A.C. SCHROEDER ET AL.
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DISCUSSION The present study has shown that developmentally competent oocytes can be retrieved from the ovaries of mice that have been deceased for a number of hours. Although pronounced postmortem degenerative changes occurred within the oocyte and follicle cells shortly after death, the culture system allowed some degenerative changes to be reversed and apparently normal maturation to occur. The principal reversible manifestation of degeneration was the presence of large irregularly shaped crystalline inclusions within the oocyte. The structure of the crystalline inclusions appeared similar t o those seen in the
oocytes of Mus cervicolor poppaeus (Szollosi and Claussen '78)' cytochalasin B-treated mouse ova (Moskalewski et al., '72), and within the blastomeres of Mus musculus embryos (Enders and Schlafke, '65; Calarco and Brown, '69). Following culture for 15-16 hr, oocytes from 6 hr postmortem mice had undergone germinal vesicle breakdown and extrusion of a polar body with a frequency equivalent to control complexes. Moreover, the crystalline structures had disappeared so that the ooplasm appeared indistinguishable from controls, and the remaining cumulus cells appeared normal. These results suggest that the culture system allowed the oocytes t o reverse degenerative changes. A similar
Fig. 4. Electron micrographs of an oocyte-cumulus cell complex isolated from a 6 hr postmortem mouse ovary illustrating the ultrastructure of the crystalline inclusion granules (arrows). The inset is a higher magnification of the crystalline structure. The cumulus cells show various degenerative changes including disrupted plasma membranes and cytoplasmic vesiculation. x 4,500. Inset x 36,000.
REVERSAL OF POSTMORTEM CHANGES IN OOCYTES
phenomenon had previously been observed during maturation in vitro of cumulus cell-enclosed oocytes from mice homozygous for the Snell dwarf mutation (Schroeder and Eppig, '89). An increasing number of pyknotic mural and cumulus granulosa cells were seen in the follicle with increasing time postmortem. The cumulus cells were less adherent t o each other and to the oocyte which resulted in a greater percentage of completely and partially denuded oocytes following isolation from the follicle. Thus, even with great care, the yield of intact oocyte-cumulus cell complexes from ovaries of the postmortem groups of mice was reduced when compared t o control ovaries. Live young were produced from oocytes retrieved from the carcasses of mice regardless of whether the mice had been primed with gonadotropin. This shows that oocytes with full developmental potential may be rescued from deceased animals even when death was unanticipated. This study, therefore, demonstrates the feasibility of recovering developmentally competent oocytes from the carcasses of valuable recently deceased zoological, agricultural, and endangered mammals. Refined species-specific techniques of oocyte maturation in vitro together with freezing of in vitro matured ova (Schroeder et al., 'go), could provide a way to salvage and preserve gametes that would otherwise be lost.
ACKNOWLEDGMENTS This research was supported by the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development funded by the National Institute of Child Health and Human Development NIH through cooperative agreement HD 21970. The technical assistance of Marilyn O'Brien and Suzanne Taylor is greatly appreciated. LITERATURE CITED Calarco, P.G., and E.H. Brown (1969) An ultrastructural and cytological study of preimplantation development of the mouse. J . Exp. Zool., 171:253-284.
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Downs, S.M., A.C. Schroeder, and J.J. Eppig (1986) The developmental capacity of mouse oocytes following maintenance of meiotic arrest in vitro. Gamete Res., 15:305-316. Enders, A.C., and S.J. Schlafke (1965) The fine structure of the blastocyst: Some comparative studies. In: Preimplantation Stages of Pregnancy. G.E.W. Wolstenholme and M. OConnor, eds. Little, Brown and Co., Boston, Ciba Foundation Symposium, pp. 29-59. Goto, K., Y. Kajihara, S. Kosaka, M. Koba, Y. Nakanishi, and K. Ogawa (1988) Pregnancies after co-culture of cumulus cells with bovine embryos derived from in vitro fertilization of in vitro matured follicular oocytes. J. Reprod. Fertil., 83:753-758. Hoppe, P.C. (1985) Techniques of fertilization in vitro. In: Reproductive Toxicology. R.L. Dixon, ed. Raven Press, New York, pp. 191-199. Moskalewski, S., W. Sawicki, B. Gabara, and H. Koprowski (1972) Crystalloid formation in unfertilized mouse ova under influence of cytochalasin B. J. Exp. Zool., 18O:l-12. Pease, S., A.C. Schroeder, and G.H. Schmidt (1989) Production of transgenic mice: Acupuncture needle-facilitated embryo transfer t o oviduct ampulla. Trends Genet. 5:293. Schroeder, A.C., A.K. Champlin, L.E. Mobraaten, and J.J. Eppig (1990) Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J . Reprod. Fertil., 89:43-50. Schroeder, A.C., and J.J. Eppig (1984) The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev. Biol., 102:493-497. Schroeder, A.C., and J.J. Eppig (1989) Developmental capacity of mouse oocytes that undergo maturation in vitro: Effect of the hormonal state of the oocyte donor. Gamete Res., 24:81-92. Shalgi, R., N. Dekel, and P.F. Kraicer (1979) The effect of LH on the fertilizability and developmental capacity of rat oocytes matured in vitro. J. Reprod. Fertil., 55:429-435. Sirard, M.A., and N.L. First (1988) In vitro inhibition of oocyte nuclear maturation in the bovine. Biol. Reprod., 39: 229-234. Staigmiller, R.B., and R.M. Moor (1984) Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res., 9:221229. Szollosi, D., and P. Claussen (1978) Paracrystalline structures in the oocytes of a mouse, Mus cervi colorpopaeus. 9th International Congress on Electron Microscopy, Toronto, Vol. 11, 546-547. Van Blerkom, J., and R.W. McGaughey (1978) Molecular differentiation of the rabbit ovum. 11. During the preimplantation development of in vivo and in vitro matured oocytes. Dev. Biol., 63:151-164. Vanderhyden, B.C., and D.T. Armstrong (1989) Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes. Biol. Reprod., 40:720-728.
Reversal of Postmortem Degeneration of Mouse Oocytes During Meiotic Maturation In Vitro ALLEN C. SCHROEDER, DANIEL JOHNSTON, AND JOHN J . EPPIG The Jackson Laboratory, Bar Harbor, Maine 04609 The developmental capacity of oocytes matured in vitro following isolation at the ABSTRACT germinal vesicle stage from freshly killed mice (control)was compared with that of oocytes isolated from the carcasses of mice killed 3,6,9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3,6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.
The mammalian ovary contains many more immature oocytes, and hence more potential eggs, than could ever be presented for fertilization by normal ovulatory or superovulatory mechanisms. Ovarian oocytes, therefore, constitute a vast, mostly unused, resource of female gametes that could be harvested to increase the numbers of rare or endangered animals. Mammalian ovarian germinal vesicle (GV)-stage oocytes will, following isolation from the follicular environment, undergo maturation in culture producing eggs with full developmental potential t o live young (Schroeder and Eppig, '84, '89; VanBlerkom and McGaughey, '78; Shalgi et al., '79; Staigmiller and Moor, '84; Goto et al., '88; Sirard and First, '88; Vanderhyden and Armstrong, '89). Unanticipated death of rare or endangered animals and the resultant postmortem degeneration of the oocytes, however, could prevent the utilization of the oocytes for animal propagation. The experiments reported here were conducted to determine the impact of postmortem degenerative changes on the developmental capacity of mouse oocytes matured in vitro. Oocytes retrieved from animals deceased for several hours demonstrate a full developmental capacity even after pronounced post0 1991 WILEY-LISS, INC.
mortem degenerative changes have occurred. The culture system allows the oocytes to reverse these degenerative changes during maturation, and the eggs produced are capable of fertilization and development to live young after transfer t o foster mothers. These techniques may be important for the rescue of oocytes from recently deceased valuable zoological, agricultural, and endangered mammals.
MATERIALS AND METHODS Oocyte maturation Immature germinal vesicle stage cumulus cellenclosed ovarian oocytes were obtained from groups of 22-24-day-old (C57BL/6J x SJL/J)F1 juvenile mice that had received 5 IU of pregnant mares' serum gonadotropin (Dyosynth, Chicago, IL) 44-52 hr before death induced by cervical dislocation. Oocytes were isolated from groups of control mice immediately after death and from Received April 11, 1990; revision accepted August 24, 1990. Address reprint requests to John J . Eppig, The Jackson Laboratory, Bar Harbor, ME 04609. Allen C . Schroeder is a Visiting Investigator from Gettysburg College, Gettysburg, PA 17325.
REVERSAL OF POSTMORTEM CHANGES IN OOCYTES
the carcasses of experimental mice killed 3, 6, 9, and 12 hr earlier then held at room temperature. As described previously (Schroeder and Eppig, '84), oocyte-cumulus cell complexes were isolated and matured for 15-16 hr in 35 x 10 Corning tissue culture dishes containing Eagles minimum essential medium supplemented with Earle's salts, 0.23 mM pyruvate, 75 mglliter penicillin G potassium salt, 50 mg/liter streptomycin sulfate, 5%fetal bovine serum (Sterile Systems) (MEM t FBS), and follicle-stimulating hormone (FSH 1 pg/ml; oFSH-17, NIADDK). Medium was continuously gassed with a humidified mixture of 5% C02, 5% 02,and 90% N2. Cultures were carried out at 37°C in a gassed modular incubator chamber (Billups Rothenberg).
241
total of 200 embryos were transferred. Also, to determine whether developmentally competent oocytes could be retrieved from mice which were not primed with PMSG, 60 embryos derived from unprimed mice were transferred as described above. Blood was drawn from live born offspring at the age of 4 weeks and hemoglobin profiles determined to insure that offspring were derived from transferred embryos (Downs et al., '86). His tolog y Ovaries and oocytes were fixed in a solution of 3% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, at room temperature for 3 and 1 hr, respectively. After dehydration in ethanol, the ovaries were embedded in JB-4 resin, sectioned at 2 pm, and stained with hematoxylin and eosin. The complexes were washed in cacodylate buffer overnight and postfixed for 0.5 hr in 1%Os04 in the same buffer at 4°C.After dehydration in a graded ethanol series and propylene oxide, the oocytes were embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1OOCX electron microscope.
Fertilization and embryonic development in vitro The medium used for fertilization and embryo culture in vitro was a modified Whitten's medium (WM; Hoppe, '85), supplemented with 3 mg/ml crystallized lyophilized bovine serum albumin (ICN). After maturation in MEM + FBS, oocytecumulus cell complexes were washed through three 2.5 ml changes of WM and transferred t o a 0.5 ml drop of WM overlayed with paraffin oil. RESULTS Fertilization was carried out with 10 pl of a sperm Developmental capacity of cumulus suspension prepared from both cauda epididycell-enclosed oocytes matured in vitro mides of a 4 month-old B6SJLF1 male housed infollowing isolation from carcasses of mice dividually from the time of weaning. The cauda epididymides were excised into a 0.9 ml drop of Cumulus cell-enclosed oocytes were isolated WM under oil at 37"C, aseptically minced, and from mice immediately after sacrifice (control) allowed to disperse for 15 min before insemina- and from the carcasses of mice sacrificed 3, 6, 9, tion. The insemination dishes were incubated for and 12 hr earlier then kept at room temperature. 6-8 hr then all ova were washed with two 2.5 ml As shown in Figure 1, the yield of cumulus cellchanges of WM and cultured at 37°C in 12 x 75 enclosed germinal vesicle-stage oocyte cumulus mm stoppered and gassed borosilicate glass cul- cell complexes decreased with increasing time ture tubes containing l ml of WM. Frequency of postmortem. Compared with controls, the yield of development to the 2-cell stage was assessed after intact germinal vesicle-stage complexes 3 hr after 18 hr. Two-cell stage embryos were cultured in death was approximately half. Nevertheless, even WM for an additional 4 days to determine the after 12 hr postmortem, about 4 intact complexes frequency of development to expanded blasto- per mouse were retrieved. After culture for 15-16 cysts. Data from four experiments were pooled, hr in the presence of FSH, more than 98% of ooand statistical comparisons between groups made cytes from both control and postmortem groups of by x2 analysis. oocytes underwent germinal vesicle breakdown (GVB) in vitro, produced a polar body, and underEmbryo transfer went mucification and expansion of the cumulus Ten 2-cell stage embryos derived from addi- oophorus. There was no difference in the kinetics tional groups of 6 hr postmortem oocytes that had of GVB between oocytes from control and 6 hr matured in vitro were transferred to each oviduct postmortem mice; GVB was completed within 90 ampullae of 8 week old B6SJLF1 foster mothers min after oocyte isolation (data not shown). on the morning of vaginal plug after mating with As shown in Figure 2, following insemination BALB/cJ vasectomized males (Pease et al., '89). A in vitro, the percentage of ova derived from 3 and
242
A.C. SCHROEDER ET AL. 50
a v)
3 40 L
a n v)
-a f 8 X
30
20
c
0 L
0
f z
10
0
0
3
6
9
12
Time Postmortem (h) Fig. 1. Number of intact oocyte-cumulus cell complexes isolated from mice at various times postmortem. Points indicate the mean and SEM of data from four experiments. Different letters indicate a significant difference (P < 0.01) using the Student-Newman-Keuls Multiple Range Test.
6 h r postmortem mice that cleaved to the 2-cell stage was equivalent to the control group. When the interval between death and removal of the ovary for oocyte isolation was increased to 9 and 12 hr, the frequency of cleavage to the 2-cell stage was decreased. When 2-cell stage embryos
derived from the 3,6, and 9 hr postmortem groups were cultured an additional 4 days, the frequency of 2-cell embryos which developed to blastocysts was not significantly different from controls. Nevertheless, the number of blastocysts that developed from ova in the 9 hr postmortem group was
H Control
69
c Q)
3h
0 6h 9h
El
30
12h
20 10 0
2-Cell Embryos
Blastocysts from 2-Cell Embryos
Fig. 2. Developmental capacity of embryos derived from oocytes isolated from mice at various times postmortem. Different letters within a group of embryos indicate a significant difference (P < 0.05) using x2 analysis. Bars indicate either the percentage of inseminated ova that cleave to the 2-cell stage, the percentage of 2-cell stage embryos that develop to the blastocyst stage, or the percentage of ova that develop to
Blastocysts from Ova
the blastocyst stage. The figure shows the pooled results of four independent experiments wherein the total number of animals donating oocytes was 10 in the control group and 16 in all of the other groups. The total number of oocytes included was as follows: control, 480; 3 hr, 362; 6 hr, 209; 9 hr, 101; 12 hr, 67.
REVERSAL OF POSTMORTEM CHANGES IN OOCYTES
less because fewer ova cleaved t o the 2-cell stage. The 2-cell stage embryos derived from the 12 hr postmortem group did not develop to blastocysts. Oviductal transfer of 200 2-cell stage embryos derived from 6 hr postmortem mice produced 71 genetically marked live young (36%). Also, 18% (11 of 60) of 2-cell stage embryos derived from 6 hr postmortem mice which had not received exogenous gonadotropin priming developed to live young. Structure of 6 hr postmortem follicles and oocytes Control and exDerimenta1 cumulus cell-enclosed oocytes couli not be readily distinguished from one another using a dissecting microscope. In contrast, histological examination of fixed and stained control (Fig. 3A) and 6 hr postmortem ovaries (Fig. 3B) revealed large irregularly
243
shaped dark-staining inclusions randomly dispersed within the ooplasm of experimental oocytes and many pyknotic cumulus and mural granulosa cells throughout the follicle. An invagination of the follicle wall was also observed frequently. Furthermore, electron microscopy of oocytes isolated from postmortem mice (Fig. 4) showed that the inclusions were crystalline in nature. Microscopy of oocytes fixed at increasing intervals after death, beginning at 30 min, showed that small crystals were present by 1hr postmortem and grew larger and more numerous with increasing time after death. Six hour postmortem cumulus cell-enclosed oocytes were cultured and then fixed for electron microscopy at increasing intervals beginning at 30 min from the start of culture. The inclusions began t o disappear within 30 min in culture and were completely gone after 60 min.
Fig. 3. Photomicrographs of ovarian follicles from mice immediately after death (A) and 6 hr postmortem (B). Although the oocyte in the 6 hr postmortem follicle remains in the germinal vesicle stage (arrow), the cytoplasm contains many densely staining granules (arrowheads). The membrana granulosa appears degenerated in that many of the granulosa cells are pyknotic, more loosely adherent to each other, and there are invaginations of the follicle wall (X). x 240.
A.C. SCHROEDER ET AL.
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DISCUSSION The present study has shown that developmentally competent oocytes can be retrieved from the ovaries of mice that have been deceased for a number of hours. Although pronounced postmortem degenerative changes occurred within the oocyte and follicle cells shortly after death, the culture system allowed some degenerative changes to be reversed and apparently normal maturation to occur. The principal reversible manifestation of degeneration was the presence of large irregularly shaped crystalline inclusions within the oocyte. The structure of the crystalline inclusions appeared similar t o those seen in the
oocytes of Mus cervicolor poppaeus (Szollosi and Claussen '78)' cytochalasin B-treated mouse ova (Moskalewski et al., '72), and within the blastomeres of Mus musculus embryos (Enders and Schlafke, '65; Calarco and Brown, '69). Following culture for 15-16 hr, oocytes from 6 hr postmortem mice had undergone germinal vesicle breakdown and extrusion of a polar body with a frequency equivalent to control complexes. Moreover, the crystalline structures had disappeared so that the ooplasm appeared indistinguishable from controls, and the remaining cumulus cells appeared normal. These results suggest that the culture system allowed the oocytes t o reverse degenerative changes. A similar
Fig. 4. Electron micrographs of an oocyte-cumulus cell complex isolated from a 6 hr postmortem mouse ovary illustrating the ultrastructure of the crystalline inclusion granules (arrows). The inset is a higher magnification of the crystalline structure. The cumulus cells show various degenerative changes including disrupted plasma membranes and cytoplasmic vesiculation. x 4,500. Inset x 36,000.
REVERSAL OF POSTMORTEM CHANGES IN OOCYTES
phenomenon had previously been observed during maturation in vitro of cumulus cell-enclosed oocytes from mice homozygous for the Snell dwarf mutation (Schroeder and Eppig, '89). An increasing number of pyknotic mural and cumulus granulosa cells were seen in the follicle with increasing time postmortem. The cumulus cells were less adherent t o each other and to the oocyte which resulted in a greater percentage of completely and partially denuded oocytes following isolation from the follicle. Thus, even with great care, the yield of intact oocyte-cumulus cell complexes from ovaries of the postmortem groups of mice was reduced when compared t o control ovaries. Live young were produced from oocytes retrieved from the carcasses of mice regardless of whether the mice had been primed with gonadotropin. This shows that oocytes with full developmental potential may be rescued from deceased animals even when death was unanticipated. This study, therefore, demonstrates the feasibility of recovering developmentally competent oocytes from the carcasses of valuable recently deceased zoological, agricultural, and endangered mammals. Refined species-specific techniques of oocyte maturation in vitro together with freezing of in vitro matured ova (Schroeder et al., 'go), could provide a way to salvage and preserve gametes that would otherwise be lost.
ACKNOWLEDGMENTS This research was supported by the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development funded by the National Institute of Child Health and Human Development NIH through cooperative agreement HD 21970. The technical assistance of Marilyn O'Brien and Suzanne Taylor is greatly appreciated. LITERATURE CITED Calarco, P.G., and E.H. Brown (1969) An ultrastructural and cytological study of preimplantation development of the mouse. J . Exp. Zool., 171:253-284.
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