Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos

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1 /. Embryol. exp. Morph. Vol. 64, pp , Printed in Great Britain Company of Biologists Limited 1981 Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos P. P. L. TAM AND M. H. L. SNOW 1 From the MRC Mammalian Development Unit, University College London SUMMARY Primitive-streak-stage mouse embryos were treated with Mitomycin C injected intraperitoneally into pregnant females at days post coitum. The newborn mice developed poorly and mortality was high during the suckling period. Many weaned survivors showed impaired fertility and poor breeding performance. Histological examination revealed a paucity of germ cells in the adult gonads. The deficiency was mainly caused by a severe reduction of the primordial germ cell population in early embryonic life, which was not fully compensated for during the compensatory growth phase of the Mitomycin C-treated embryo. Also contributing to such impaired fertility were retarded migration of the primordial germ cells into the genital ridges, poor development of the foetal gonad and secondary loss of the germ cells during gametogenesis in males. INTRODUCTION We have recently shown that a single intraperitoneal injection of 1-12/*g Mitomycin C () into pregnant mice does very extensive damage to primitive-streak-stage embryos, reducing cell number to about 15% of normal values, and resulting in severe developmental disturbance in the ensuing 48 h of embryogenesis. Subsequently, accelerated growth and morphogenesis restores gross morphology to normal by mid-organogenesis stages 3-4 days later. Although embryonic mortality is low, post-natal development is poor and fertility among offspring surviving to breeding age is low (Snow & Tam, 1979). This report concerns the developmental events underlying the reduced fertility following -treatment and describes the origin, proliferation and migration of primordial germ cells, and the formation of the foetal gonads in normal and -treated mice. MATERIALS AND METHODS Pregnant Q-strain mice received a single intraperitoneal injection of 1 ii% Mitomycin C (Sigma, London) in -25 ml -9 % NaCl between 6-75 and 7- days 1 Authors'" address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson Way, London NW1 2HE, U.K.

2 134 P. P. L. TAM AND M. H. L. SNOW Table 1. The viability and breeding performance of the offspring of Mitomycin C-treated pregnant females No. litters No. newborn Birth Viability No. alive at 7d 14 d 28 d No. weaned Breeding performance Class of mating Sex No. No. mated Fertile Fertile and sterile Sterile only <J post coitum (p.c.) Embryos are in early- and mid-primitive-streak stages at this time. Initial observations were made on the offspring of such mice that survived to breeding age. Each mouse was test-mated to a normal Q mouse of proven fertility for sufficient time to allow the production of several litters. Subsequently the animals were killed, both gonads removed and fixed in Bouin's fluid and examined histologically. The formation of gonads was studied in embryos between 8-5 day p.c. when primordial germ cells (PGCs) are observable in the developing hindgut, and 13-5 days when colonisation of the genital ridge is complete. Embryos were fixed in cold 8 % ethanol, dehydrated in absolute ethanol, cleared in chloroform and embedded in a low melting-point (54 C) wax. Serial sections were made at 8 or 1 ju,m and stained with Fast Red TR salt to detect alkaline phosphatase according to the azo-dye coupling method of Gomori (Gabe, 1975). They were mounted in glycerine. Complete undamaged and properly stained serial sections were obtained from 57 normal embryos and 17 -treated embryos, from 24 litters. PGCs were identified by the high content of alkaline phosphatase in their cytoplasm and on their membranes (Chiquoine, 1954; Ozdzenski, 1967; Jeon& Kennedy, 1973). PGCs were scored on every section of 8-5 to 11-5-day embryos. Abercrombie's formula (Abercrombie, 1946) was used to correct for cells registered in both of two adjacent sections, thus giving a better estimate of cell number. In 13-5-day embryos the number of PGCs in the genital ridges was computed from the size of the ridge (gonadal volume) which was derived from measurement of camera-lucida drawings and the number of PGCs per unit tissue volume (cell density) determined from at least four sections per gonad. PGCs lying outside the genital ridges were counted as in younger embryos.

3 Proliferation and migration of mouse primordial germ cells 135 Table 2. The reduction in germ cells in gonads of offspring from -treated mice Sex Class of mating No. Empty seminiferous tubules (%) Mean 6 1 Fertile only 1 (4), 2, 4, 5, 18, 22, 3 8 Sterile only 44 22, 71, 1, 1 73 Both types 29, 4, 54,61 46? Fertile No litter 9 2 follicles (%) 24, 42, 53, 77, 86, 86, 1 (2) 47,5 The number of seminiferous tubules (fertile and 'empty') and the number of follicles of all stages of development were scored in 4-5 good sections of the testis and ovary respectively RESULTS Table 1 shows the viability of offspring from -treated mice and the breeding performance of successfully weaned young. Two females never mated; one developed ataxia which probably impaired her mating behaviour. Histological analysis of gonads shows a reduction in gonad size and of the number of germ cells (Table 2, Figs. 1, 2), particularly in the sub-fertile and sterile males where seminiferous tubules completely devoid of germ cells and containing only Sertoli cells were found (Fig. 1 b, c). In two of the sterile males the testes were completely devoid of germ cells (Fig. 1 d). In the females no ovary was found to be devoid of follicles, the smallest having about 24 % of the normal number of oocytes. There are more atretic follicles in -treated mice. Embryonic development Figure 3 illustrates alkaline-phosphatase-positive PGCs in various sites in their migration pathway. In Fig. 3 a PGCs are at the posterior end of the primitive streak at the base of the allantois. This example is from an 8-5-day embryo which is retarded in development. In normal embryos this developmental stage occurs at daysp.c. In a normal 8-5-day embryo, PGCs are found in the primary endoderm and early hindgut (Fig. 3b), by 9-5 days in the hindgut and just entering the mesentery (Fig. 3 c) and enter the genital ridges at days (Fig. 3d). The genital ridges are fully colonized by 13-5 days (Fig. 4). PGC number. Table 3 and Fig. 5 show the numbers of PGCs in normal and embryos according to gestational age. The lower PGC numbers in embryos do not simply reflect the retardation in overall development. Figure 6 illustrates graphically the relative development of the embryos with respect to PGC number, somite number and size, presomitic mesoderm length,

4 136 P. P. L. TAM AND M. H. L. SNOW (B) Fig. 1. (A) testis showing prolific spermatogenic activity. Bar = 2 fim. (B) Testis of sub-fertile male offspring from -treated mice showing empty seminiferous tubules. Bar = 2 fim. (C) Absence of germ cells in sterile tubules. Bar = 5 /im. (D) Testis of sterile male which is totally devoid of germ cells. Bar 2 /im.

5 Proliferation and migration of mouse primordial germ cells 137 (B) Fig. (2). (A) ovary showing follicles in various stages of development. (B) Ovary from an offspring of a -treated mouse showing many fewer follicles. Bar = 2/tm. axis length and foetal wet weight. Comparison of some of these growth parameters suggests they are under independent control (see later, and Snow, Tarn & McLaren, 1981). The PGC population doubling time in normal embryos is fairly uniform at about 16 h between 8-5 and 13-5 days. A similar value is found in embryos between 1-5 and 13-5 days, but at the beginning of their migration these PGCs divide very slowly (population doubling time 31 h), and between 9-5 and 1-5 days, very rapidly (doubling time 7 h) (Fig. 5). The period of rapid proliferation coincides with the period of maximum compensatory growth for other parts of the embryo but PGC number does not recover to normal in treated embryos and when genital ridge differentiation commences the gonads have about 5 % as many PGCs as normal (at days there were about 17% of normal values). There is considerable variation between embryos, even within a single litter, in the facility with which PGC number is restored. This variation is reflected in the very much larger range of PGC numbers observed in 11-5-day

6 138 P. P. L. TAM AND M. H. L. SNOW Fig. 3. The location of primordial germ cells (arrows) in mouse embryos between 8-5 and 13-5 days p.c. (A) In the primitive streak and base of the allantois. Bar = 5 /tin. (B) In the hind-gut endoderm. Bar = 2 /tin. (C) In the hind-gut and dorsal mesentery. Bar = 2/tin. (D) En route from mesentery to the genital ridges (GR). Bar = 2 /tm.

7 Proliferation and migration of mouse primordial germ cells 139 Fig. 4. The genital ridges of 13-5-day mouse embryos. (A) male and (B) female. Bar = 5 /tm. embryos than at other times (Fig. 7). At 8-5 and 9-5 days the PGC population in embryos is fairly uniformly depleted and no embryo falls within the normal range; at 11-5 days however, while many embryos show severely reduced PGC numbers some 35 % could be classified as normal, and thus fully recovered. No embryos were found to be without germ cells but 5 (33 %) were recorded with less than 2 at 8-5 days. In some 13-5-day male genital ridges there were apparently germ-cell-free patches, suggesting incomplete or non-random colonization of the gonad. There was no difference observed between male and female embryos, either

8 14 P. P. L. TAM AND M. H. L. SNOW Table 3. The numbers of primordial germ cells (PGCs) in 8-5- to 13-5-day mouse embryos Age (day p.c.) Group No. litters No. embryos Mean PGC No. (±1 S.E.) ±17 34±6 364 ±32 58 ±8 112 ± ± ± ± ± E L Age of embryos (days p. c.) Fig. 5. The increase in number of PGCs in mouse embryos between 8-5 and 13-5 days p.c. =, O = -treated. normal or -treated, that could reasonably account for the more severe post-natal effect seen in males (Tables 1, 2). Table 4 shows PGC number in embryos of 1-5 to 13-5 days, and Table 5 gives the gonadal volume in 13-5-day embryos. (The 13-5-day embryos were sexed by gonad histology and younger embryos from chromosome preparations made from fetal membranes. Some of these preparations were inadequate for confident sexing and hence not all embryos are included in Table 4). Although there is a clear difference between

9 Proliferation and migration of mouse primordial germ cells J L Age (days/j.c. ) Fig. 6. The relative development of -treated embryos with respect to presomitic mesoderm length ( ), size of newly formed somite (+), somite number (#), axial length (A), foetal wet weight (O), and PGC number ( ). 8-5 d d. O, o d 8«,8X.ot.I d 8. 8.JlJ.ol.olJ, d o, o, 4 Number of PGCs Fig. 7. The range in numbers of PGCs in embryos of 8 5 to 13-5 days. =, O == -treated, Note the large variation in number in 11-5 day -embryos.

10 142 P. P. L. TAM AND M. H. L. SNOW Table 4. Comparison of PGC numbers between male and female mouse embryos Age (day p.c.) Group Statistics ; Student /-test. male vs. female A t \ 1-5 d n.s. n.s n.s. n.s n.s. n.s. Male (n) 174±12(4) 642 ±62 (8) 3316 ±279 (6) 1646 ±24 (1) ±32 (1) 1356±937 (13) Number of PGCs norma Male t (1) = 3.6, P < 1 t (14) = 4-9, P < 1 t (21) = 6-4, P < 1 n.s., no significant difference. Female (n) 977 ±163 (8) 569 ±93 (6) 2854 ±414 (2) 1538 ±223 (7) ±223 (4) 14556±174 (17) 1 vs. A Female t (8) = 2.4, P < 5 t (7, = 2-8, P < 5 t (l9 ) = 3-2, P < 1 Table 5. The size of the genital ridges of 13-5-day mouse embryos Gonadal volume (x Group Male (± S.E. («)) Female (± S.E. («)) 653 ±43 (1) 459 ±18 (13) * Means of the average volume of the two gonads Statistics; Student t-test. $ S t (12) = 3-2, P < 1? t U9) = 3-4, P < 1 42 ±34 (4) 329±1 (17) in each embryo. <? t(2d = 4-5, P < 1 t(28) = 6'6, P < 1 the sexes with respect to gonadal volume, and male gonads surfer a greater size reduction in response to treatment, the magnitude of the difference seems insufficient to account for, and difficult to relate to, the totally germ-cellfree testes found in sterile males. Examination of seven post-natal mice up to 5 days of age gives no further clue to the manner in which the 'empty' testes arise. All testes examined, although small, showed no evidence of the empty seminiferous tubules observed later. PGC migration. Figure 8 illustrates the proportions of PGCs found in various sites between 8-5 and 13-5 days and suggests migration is slightly retarded with respect to time in embryos. However, since the whole embryo is somewhat

11 Proi Table 6. The location of primordial germ cells in mouse embryos at 1- to 36-somite stages, equivalent to days p.c. Somite no. of embryos Group No. of, embryos fa PS Distribution ALYS of PGCs: mean no (%) A HGN MCW GR Total PGC no PS, primitive streak; ALYS ridge (5-3) 22 (16-2) 8 6-1) 4(1-3) 1 (-2) 5(1-3) 1 (-3) 5' s 3 115(83-8) 39 (78-6) 261 (91-5) 58 (961) 273 (671) 47 (14-9) 69 (6-4) 3 (4-9) S" 21 (7-2) 2 (3-7) 122 (29-9) 266 (83-6) 934 (85-9) 554 (89) 7(17) 4(1-2) 83 (7-7) 38 (61), allantoic base and yolk-sac endoderm; HGN, hind-gut endoderm; MCW, mesentery and coelomic wall; GR, genital <^ -t " a' i Hi

12 144 P. P. L. TAM AND M. H. L. SNOW d 1 n rk 9-5 d CO t 1 o 1-5 d o " d 13-5 d n i k. n. Primitive Yolk sac Gut Mesentery Genital streak allantois ridges Fig. 8. The migration of PGCs from the primitive streak to the genital ridge, =, = -treated. retarded it would seem more meaningful to assess PGC migration with respect to developmental stage. Somite number can be used as an index of developmental status but may be misleading (Snow & Tarn, 1979; and in preparation). Nevertheless in Table 6 the distribution of germ cells is given with respect to somite number. Migration still appears retarded for early somite stages, but then appears to accelerate such that in embryos the PGCs seem further along their migration path than controls in embryos of 21-3 somites. Beyond 1-5 days (33 somites) there is no discrepancy in somite numbers between control and embryos but the entry of PGCs into the genital ridge is delayed in embryos (Table 7). DISCUSSION The PGC numbers reported here for normal embryos are in very close agreement with the figures given by Mintz & Russell (1957) in a study of 8- to

13 Proliferation and migration of mouse primordial germ cells 145 Table 7. The entry of primordial germ cells into genital ridges in normal and -treated embryos between 1-5 and 13-5 days p.c. Mean no. ofpgcsf%) Group Age (d) Sex No. of embryos Extragonadal sites 13 (95) 9 (92) 221 (7) 193 (7) 337(1) 332 (1) 611 (95) 542 (95) 641 (39) 64 (39) 12(1) 114(1) Genital ridge 44(5) 77(8) 396 (93) 2662 (93) (99) 2213 (99) 31(5) 27(5) 15 (61) 934(61) (99) (99) Total <J 9 <J 9 <J 9 c? day-old embryos with a C37BL/6 genetic background. Their study did not extend to full genital ridge colonization but the increase from 4 PGCs at 8 days to some 4 at 12 days represents a population doubling time of around 14 h (compared to our 16 h) and would suggest that by 13-5 days their mice should have about 24 PGCs in their genital ridges. It is clear that the reduced fertility in mice exposed to during primitivestreak-stages of embryogenesis is the result of germ cell deficiency in the gonads. In females the paucity of germ cells can be accounted for by a severe reduction in primordial germ cells early in embryonic life which is not wholly compensated for. In males the finding of sterile testes totally devoid of germinal tissue indicates a secondary loss of germ cells since no embryo was seen without substantial numbers of germ cells at the time of onset of gonadal differentiation. Even if it is assumed that the empty testes are derived from those genital ridges containing the fewest PGCs in 13-5-day embryos then a testis with some 3% of normal numbers of germ cells would be expected. No ovary entirely devoid of germ cells has been found so it would appear perhaps that in females a functional gonad results from a similar severely depleted 13-5-day genital ridge although it would perhaps be expected that such females would have a shorter reproductive life than normal mice. The mechanism of the secondary loss of germ cells in males is not known but it is probably brought about by degeneration of the tissue rather than loss by emigration from the testis or by differentiation of the entire population into sperm which were then shed. Firstly although emigration of germ cells from the testis tubule has been reported in the rabbit (Gould & Haddad, 1978) it is not

14 146 P. P. L. TAM AND M. H. L. SNOW extensive and is probably rare. Secondly, the male PGCs are of proven mitotic competence and it seems improbable that all the cells of the mitotic stem line should embark upon terminal differentiation into sperm at an early age and thus deplete the entire germ cell population. It seemed likely that the loss would occur when mitotic proliferation resumed after the gonocyte growth phase, since extensive degeneration of germ cells is seen in the normal rat testis at this time (Roosen-Runge & Leik, 1968; Hilscher et al. 1974). In the rat the atresia is maximal at 4-6 days and declines rapidly thereafter (Beaumont & Mandl, 1962, 1963). Up to 5% of the gonocyte population may fail to resume mitosis and die (Clermont & Perey, 1957; Novi & Saba, 1968). In the mouse there is no evidence of degeneration in early postnatal males (Snow & Tarn, unpublished observations; P. S. Burgoyne, personal communication), but considerable atresia is seen in testes 1 or 2 days before birth (A. McLaren, personal communication). The healthy appearance of the testes in young males suggests that they survive the resumption of mitosis and that the secondary loss of germ cells occurs later than 7 days post partum. The PGC population in -treated embryos is only partially restored after the initial depletion but other tissues and organs appear to recover to full size by daysp.c. (Fig. 6; Snow & Tarn, 1979; Tarn, in preparation). The failure to restore full numbers of PGCs is due to the fact that a raised proliferation rate is only achieved between 9-5 and 1-5 daysp.c. rather than over the whole period of development from 7-5 to 13-5 days, as happens with other organ systems This fact has an important bearing on the assessment of the rate of migration of PGCs in -treated embryos. The results in Table 6 suggest a slightly retarded migration during early somite stages, more rapid passage through hindgut and the mesentery, but delayed entry into the genital ridge. Since the period of maximum proliferation of PGCs in -treated embryos, 9-5 to 1-5 days or 22- to 32- somite stage, coincides with the time the cells are in the mesentery, it seems more likely that the increased proportion of PGCs in the mesentery at this time (Fig. 8 and Table 6) is the result of a population increase by cell division rather than immigration from the hindgut. P. P. L. Tam was supported by a British Commonwealth Scholarship. REFERENCES ABERCROMBIE, M. (1946). Estimation of nuclear population from microtome sections. Anat. Rec. 94, BEAUMONT, H. M. & MANDL, A. M. (1962). A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat. Proc. Roy. Soc. Lond. B 155, BEAUMONT, H. M. & MANDL, A. M. (1963). A quantitative study of primordial germ cells in the male rat. /. EmbryoL exp. Morph. 11, CHIQUOINE, D. A. (1954). The identification, origin and migration of the primordial germ cells in the mouse embryos. Anat. Rec. 118, CLERMONT, Y. & PEREY, B. (1957). Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat. 1,

15 Proliferation and migration of mouse primordial germ cells 147 GABE, M. (1975). Histological Techniques. Detection of Phosphatase, pp Paris: Masson. GOULD, R. P. & HADDAD, F. (1978). Extratubular migration of gonocytes in the foetal rabbit testis. Nature Loud. 273, HILSCHER, B., BULTHOFF, B., KRAMER, U., BIRKE, A., PELZER, H. & GAUSS, G. (1974). Kinetics of gametogenesis. I. Comparative histological and autoradiographical studies of oocytes and transitional prospermatogonia during oogenesis and prospermatogenesis. Cell Tissue Res. 154, JEON, K. W. & KENNEDY, J. R. (1973). The primordial germ cells in early mouse embryos: light and electron microscopic studies. Devi Biol. 31, MINTZ, B. & RUSSELL, E. S. (1957). Gene induced embryological modifications of primordial germ cells in the mouse. /. exp. Zool. 134, Novi, A. M. & SABA, P. (1968). An electron microscopic study of the development of the rat testis in the first 1 postnatal days. Z. Zellforsch. mikrosk. Anat. 86, OZDZENSKI, W. (1967). Observations on the origin of primordial germ cells in the mouse. Zool. Pol. 17, ROOSEN-RUNGE, E. C. & LEIK, J. (1968). Gonocyte degeneration in the postnatal rat. Am. J. Anat. 122, SNOW, M. H. L. & TAM, P. P. L. (1979). Is compensatory growth a complicating factor in mouse teratology? Nature, Lond. 279, SNOW, M. H. L., TAM, P. P. L. & MCLAREN, A. (1981). On the control and regulation of size and morphogenesis in mammalian embryos. In Levels of Genetic Control in Development 39th Symp. Soc. Dev. Biol. (ed. S. Subtelny). (In Press). {Received 17 September 198, revised 1 March 1981)

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