Am J Hum Genet 31:446-457, 1979
Late Replicating X Chromosomes in Human Triploidy PATRICIA A. JACOBS,1 AILEEN M. MATSUYAMA, IRENE M. BUCHANAN, AND CAROLA WILSON
SUMMARY The status of X-chromosome replication was studied in twenty-seven 69,XXY and nine 69,XXX human triploids in which the parental origin of the additional haploid set was known from the study of chromosome heteromorphisms. Among the 69,XXY triploids, fourteen had no late replicating X, two had one late replicating X in all cells examined, and eleven had two populations of cells, one with one late replicating X chromosome, and one without any. Among the 69,XXX triploids, four had a single late replicating X, and five had two populations of cells, one with one late replicating X, and one with two late replicating X chromosomes. There was no correlation between the parental origin of the triploidy and the type of X-chromosome inactivation. However the number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue when compared with those grown from extra-embryonic tissue. In cultures derived from extra-embryonic tissue there was a significant correlation between the gestational age of the sample and the proportion of late replicating X chromosomes. The older the specimen, the greater the number of late replicating X chromosomes.
INTRODUCTION
Triploidy is a common condition in man, occurring in approximately 2% of all clinically recognized conceptions [1]. Triploidy is of considerable interest in any investigation of the mechanism of X-chromosome inactivation. As first pointed out by Received January 1, 1979; revised February 27, 1979. This work was supported by a grant from the Spencer Foundation, by grant HD-07879 from the National Institutes of Health, and by grant 6-47 from the National Foundation. I All authors: Department of Anatomy and Reproductive Biology, John A. Bums School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. © 1979 by the American Society of Human Genetics. 0002-9297/79/3104-0012$01.00
446
X CHROMOSOME REPLICATION IN TRIPLOIDY
447
Hamden [2], it is the only situation for which the simple formula for predicting the number of X chromatin bodies, and thus presumed inactive X chromosomes, does not apply. This formula, B = x - p12, where B is the number of X chromatin bodies or inactive X chromosomes, x is the number of X chromosomes, and p the ploidy of the cell, gives the value of 0.5 for 69,XXY triploids and 1.5 for 69,XXX triploids. Such fractions have no biological reality in terms of the Lyon hypothesis. In practice sex chromatin observations on triploids have shown that 69,XXY triploids may be either chromatin negative or positive, while 69,XXX triploids may have a maximum of either one or two sex chromatin bodies [3]. However, sex chromatin is far from an ideal indicator of the presence or absence of an inactive X chromosome or chromosomes. The frequency with which it is seen varies from tissue to tissue and is affected by a number of factors including age, density of cells, the stage of the cell cycle, and staining techniques used [4, 5]. Even in favorable material from normal females, the percentage of X chromatin positive cells varies between 20 and 90. Identification of the late replicating or allocyclic X chromosome at metaphase, rather than its condensed state in interphase nuclei, provides a much more direct method for determining the number of presumptively inactive X chromosomes. The recently developed techniques utilizing BrdU labeling provide a reliable method, with excellent resolution, for studying DNA replication [6]. By studying the pattern of late replicating X chromosomes in triploids in which the parental origin of the additional haploid set is known from examination of chromosome heteromorphisms [7] or HLA haplotypes [8], it can be determined whether the parental origin of the X chromosome affects the pattern of inactivation. Recently it has been demonstrated in both the laboratory mouse and rat that, while the fetus shows random inactivation of the X chromosomes, in certain extra-embryonic tissues the paternally derived X chromosome is always inactivated [9-11]. Therefore in any study of X-chromosome inactivation in embryonic material, it is of interest to determine the status of inactivation in tissue derived both from the fetus itself and from the extra-embryonic component of the conceptus. This paper reports our observations on thirty-six spontaneously aborted human triploid conceptuses. We have investigated: (1) the number of presumptively inactive X chromosomes, utilizing a BrdU pulse technique to identify the allocyclic X chromosomes; (2) the relationship of the number of late replicating X chromosomes to the parental origin of the X chromosomes; and (3) whether the number of allocyclic X chromosomes differs in tissues derived from the fetus proper and from the fetal membranes. METHODS
Material grossly identified as originating from the conceptus was set up in culture. Where more than one type of tissue was available, different tissues were cultured separately. The basic methods of tissue culture and cytogenetic procedures have been described previously [12]. In each instance where a triploid conceptus was observed, every effort was made to obtain a blood sample from the parents and, by comparing Q- and C-band heteromorphisms of the parents and the conceptus, to determine the origin of the additional haploid complement in the conceptus. In the great majority the extra chromosomes were found to be paternal in origin, usually as the
result of dispermy [7].
448
JACOBS ET AL.
Replication status of the X chromosome was determined utilizing a modification of the BrdU pulse labeling technique of Willard and Latt [6]. One day after routine subculture, the media was changed from FIO containing 10% fetal calf serum to FlO containing 20% fetal calf serum. Seven hours later I ml of a solution containing 276.7 ,ug of BrdU, 0.98 Ag of FUdr, and 14.65 ,ug of uridine was added to a 10 ml culture. The culture was harvested about 11 hrs later, colcemid having been added for the last 3-4 hrs. Cytogenetic preparations were made in the usual way, and the slides were either (1) stained in acridine orange at a concentration of I mg in 50 ml Sorenson's buffer for approximately 40 seconds, rinsed and mounted in buffer, and examined with a fluorescent microscope, or (2) stained in a 0.2 ug/ml solution of Hoechst in Sorenson's buffer for 45 min, mounted in buffer, and exposed to a 275 watt sun lamp about 30 cm away for 8-15 min. Subsequently the Hoechst-stained slides were placed in 2XSSC at 60'C for 4 hrs, stained in 4% Giemsa for 8 min, rinsed in buffer, dried and mounted in DePex (Gurr, Essex, England). In this procedure virtually all late replicating DNA synthesis takes place in the presence of BrdU. Therefore BrdU is incorporated into the newly synthesized DNA strand of the late replicating X chromosomes. When stained with acridine orange and examined under UV light, the chromosome regions that have incorporated BrdU appear red, while the remaining chromosome regions appear yellow-green. When stained by Hoechst, exposed to UV light, and subsequently stained with Giemsa, the late synthesizing regions containing BrdU appear much paler than the remainder of the chromosomes (figs. I and 2). Late replicating X chromosomes were scored by direct microscopic examination in both the temporary preparations stained with acridine orange and the permanent preparation stained with Giemsa. The preparations were scored by two or more observers who had no prior knowledge of the sex chromosome constitution or of the mechanism of the origin of the triploidy. The criteria used in scoring a cell for the presence or absence of allocyclic X chromosomes were: (1) that the cell appeared intact and the chromosomes reasonably well-spread; and (2) that the cell showed unequivocal R-banding in chromosomes 13, indicating that the cell had incorporated BrdU at the end of the
S-peiod. RESULTS
Preparations giving satisfactory results with respect to late replicating X chromosomes were obtained from a total of 36 triploids, 27 with an XXY, and nine with an XXX sex chromosome constitution. The menstrual age of the abortuses, the tissue cultured, and the origin of the < dditional haploid set are shown in table 1, together with the number of cells having 0, 1, or 2 allocyclic X chromosomes. As can be seen from table 1, the XXY triploids fall into three classes: those where virtually all the cells have no allocyclic X chromosome, those where virtually all cells have a single allocyclic X, and those where there are two distinct cell lines, one with no allocyclic X and one with one allocyclic X chromosome (fig. la and b). Among the XXX triploids there are two distinct classes: those where virtually all cells have a single allocyclic X, and those where there are two cell lines, one with one and one with two allocyclic X chromosomes (fig. 2a and b). No XXX triploids were seen in which all cells had two allocyclic X chromosomes. In two XXX triploids there appears to be a minor cell line with no late replicating X chromosome (fig. 3). We do not yet know whether these are cells in which all three X chromosomes are active, or technical artifacts from our failing to identify a late replicating X chromosome. The data in table 1 were examined by step-wise regression analysis to determine whether there was a correlation between the mechanism of origin of the triploidy, the pattern of X inactivation, the tissue examined, or the menstrual age of the fetus. For
X CHROMOSOME REPLICATION IN TRIPLOIDY
449
FIG. 1. -Triploid cells pulse labeled with BrdU to show late replicating X chromosome (arrow). a, 69,XXY cell with no late replicating X chromosome. b, 69,XXY cell with one late replicating X chromosome.
450
JACOBS ET AL.
FIG. 2. -Triploid cells pulse labeled with BrdU to show late replicating X chromosomes (arrows). a, 69,XXX cell with one late replicating X chromosome. b, 69,XXX cell with two late replicating X chromosomes.
451
X CHROMOSOME REPLICATION IN TRIPLOIDY TABLE 1 DATA ON THIRTY-SIX HUMAN TRIPLOIDS No. ALLOCYCLIC
MENS.
ORIGINt
X CHS.
AGE
(DAYS)
TIssUE*
............. .............
136 104
............. ............. ............. ............. ............. ............. ............. ............. ............. .............
137 130 126 117 171 105 123 112 130 144
a.c.v. a.c.v. a.c.v.
No.
XXY: K179 K288 K379 K440 K563 K603 K610 K619 K626 K635 K735 K322
,
....
K539 K736 K632 K778
74 165 51 220 81 121 187 . 82 169 63 54
.
. . .
dII
fetus fetus a.c.v. a.c.v. a.c.v. a.c.v. a.c.v. a.c.v. a.c.v.
+
+
+
+
+
+
yI
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
1
2
-
0 34 38 24 36 33 25 39 10 0 1 12 36 85 69 53 13 3 86 39 37 34 4 38 50 5 20 165 33 34 34
45 0 0 8 0 0 57 1 32 30 30 6 4 0 0 0 15 38 7 0 0 0 61 0 0 0 0 9 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
a.c.v.
fetus a.c.v. a.c.v. a.c.v. a.c.v.
fetus cord fetus
+
+
+
+
+
+
+
+
______-
0 0 0 0 4 5 70 3 9
-
+ +
a.c.v. a.c.v.
cord a.c.v.
....
K418 ............. K649 ............. K627 ............. K795 .............
cfI
0
fetus
....
K356 ............. K364 ............. K369 ............. K502 ............. K507 ............. K797 ............. K803 .............
26
yII
134 82 110 119
cord a.c.v. a.c.v.
cord
No parental blood No parental blood No parental blood
a.c.v.
XXX: K93
.............. .............
K189
K429 K536 K716 K732 K762 K411 K612
............. ............. ............. ............. ............. ............. .............
133 141 105 198 91 96 120 76 65
a.c.v.
1
55
fetus
0
40 33 81 92 45 28 25 48
a.c.v.
1
12 9
fetus a.c.v. a.c.v.
0
a.c.v. a.c.v. a.c.v.
* a.c.v. amnion, chorion, villi. t.Dispermy = 26; failure of first male meiotic division.
1
0
1
=
=
d1, second male
=
dII, first female
purposes of analysis, the tissue examined was considered as cord) or fetal membranes (amnion; chorion; villi).
=
YI,
or
second female
fetal (fetus
=
proper
QII
and
There was no correlation between the origin of the triploidy and the type of X-chromosome inactivation. Thus among the XPXMY triploids, there are abortuses with no late replicating X chromosomes and abortuses in which one X chromosome is late replicating. Among the four XMXMY triploids, there are three where neither X is
452
JACOBS ET AL.
FIG. 3.-A 69,XXX triploid cell pulse labeled with BrdU showing no evidence of a late replicating X chromosome.
late replicating, and one where the majority of cells have a late replicating X. The majority of XPXPXM triploids have a single late replicating X, but in two XPXPXM triploids, there is a minor cell line with two late replicating X chromosomes and in one XPXPXM triploid, the majority of cells have two late replicating X chromosomes. A minor cell line with no late replicating X chromosome was seen in two specimens. In the two XPXMXM triploids, there are cells with one and cells with two late replicating X chromosomes. Therefore, the parental origin of the X chromosome does not appear to affect the pattern of X-chromosome replication nor, by extension, X-chromosome inactivation. The only significant finding with respect to the origin of the triploidy was that of reduced menstrual age in the triploids that resulted from failure of the second maternal meiotic division. While there are only three such triploids, the early abortion of this class is highly significant (P < 0.01). The fact that these conceptions are homozygous for the maternal genes between the centromere and the most proximal chiasma may be a factor relating to early termination of these pregnancies. Homozygosity for so large a number of genes might be deleterious even in the presence of a single dose of the homologous genes contributed by the father. However, the only other reported case of triploidy caused by the failure of the second maternal meiotic division was a live-born XXX [ 13]. Therefore, if our explanation for the unusually early demise of our triploids resulting from this mechanism is valid, it clearly is not universally applicable. This is
X CHROMOSOME REPLICATION IN TRIPLOIDY
453
not surprising as not every such triploid would be expected to receive genes that were lethal during early gestation in the homozygous maternal contribution. There is a significant difference between the menstrual age of the specimens from which we obtained fetal tissue (134.5 days) and those from which we obtained only 0.05). It is known from other tissue from the fetal membranes (111.0 days) (P studies that triploidy can be associated with a whole variety of phenotypes ranging from empty gestational sacs with no evidence of fetal development, to stillbirths, and very rarely live-born infants [14]. Therefore, it is not surprising that in our series the specimens from which we obtained fetal tissue aborted later than those from which we obtained only tissue from the fetal membranes. The number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue by comparison with those grown from extra-embryonic tissue (P
Late Replicating X Chromosomes in Human Triploidy PATRICIA A. JACOBS,1 AILEEN M. MATSUYAMA, IRENE M. BUCHANAN, AND CAROLA WILSON
SUMMARY The status of X-chromosome replication was studied in twenty-seven 69,XXY and nine 69,XXX human triploids in which the parental origin of the additional haploid set was known from the study of chromosome heteromorphisms. Among the 69,XXY triploids, fourteen had no late replicating X, two had one late replicating X in all cells examined, and eleven had two populations of cells, one with one late replicating X chromosome, and one without any. Among the 69,XXX triploids, four had a single late replicating X, and five had two populations of cells, one with one late replicating X, and one with two late replicating X chromosomes. There was no correlation between the parental origin of the triploidy and the type of X-chromosome inactivation. However the number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue when compared with those grown from extra-embryonic tissue. In cultures derived from extra-embryonic tissue there was a significant correlation between the gestational age of the sample and the proportion of late replicating X chromosomes. The older the specimen, the greater the number of late replicating X chromosomes.
INTRODUCTION
Triploidy is a common condition in man, occurring in approximately 2% of all clinically recognized conceptions [1]. Triploidy is of considerable interest in any investigation of the mechanism of X-chromosome inactivation. As first pointed out by Received January 1, 1979; revised February 27, 1979. This work was supported by a grant from the Spencer Foundation, by grant HD-07879 from the National Institutes of Health, and by grant 6-47 from the National Foundation. I All authors: Department of Anatomy and Reproductive Biology, John A. Bums School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. © 1979 by the American Society of Human Genetics. 0002-9297/79/3104-0012$01.00
446
X CHROMOSOME REPLICATION IN TRIPLOIDY
447
Hamden [2], it is the only situation for which the simple formula for predicting the number of X chromatin bodies, and thus presumed inactive X chromosomes, does not apply. This formula, B = x - p12, where B is the number of X chromatin bodies or inactive X chromosomes, x is the number of X chromosomes, and p the ploidy of the cell, gives the value of 0.5 for 69,XXY triploids and 1.5 for 69,XXX triploids. Such fractions have no biological reality in terms of the Lyon hypothesis. In practice sex chromatin observations on triploids have shown that 69,XXY triploids may be either chromatin negative or positive, while 69,XXX triploids may have a maximum of either one or two sex chromatin bodies [3]. However, sex chromatin is far from an ideal indicator of the presence or absence of an inactive X chromosome or chromosomes. The frequency with which it is seen varies from tissue to tissue and is affected by a number of factors including age, density of cells, the stage of the cell cycle, and staining techniques used [4, 5]. Even in favorable material from normal females, the percentage of X chromatin positive cells varies between 20 and 90. Identification of the late replicating or allocyclic X chromosome at metaphase, rather than its condensed state in interphase nuclei, provides a much more direct method for determining the number of presumptively inactive X chromosomes. The recently developed techniques utilizing BrdU labeling provide a reliable method, with excellent resolution, for studying DNA replication [6]. By studying the pattern of late replicating X chromosomes in triploids in which the parental origin of the additional haploid set is known from examination of chromosome heteromorphisms [7] or HLA haplotypes [8], it can be determined whether the parental origin of the X chromosome affects the pattern of inactivation. Recently it has been demonstrated in both the laboratory mouse and rat that, while the fetus shows random inactivation of the X chromosomes, in certain extra-embryonic tissues the paternally derived X chromosome is always inactivated [9-11]. Therefore in any study of X-chromosome inactivation in embryonic material, it is of interest to determine the status of inactivation in tissue derived both from the fetus itself and from the extra-embryonic component of the conceptus. This paper reports our observations on thirty-six spontaneously aborted human triploid conceptuses. We have investigated: (1) the number of presumptively inactive X chromosomes, utilizing a BrdU pulse technique to identify the allocyclic X chromosomes; (2) the relationship of the number of late replicating X chromosomes to the parental origin of the X chromosomes; and (3) whether the number of allocyclic X chromosomes differs in tissues derived from the fetus proper and from the fetal membranes. METHODS
Material grossly identified as originating from the conceptus was set up in culture. Where more than one type of tissue was available, different tissues were cultured separately. The basic methods of tissue culture and cytogenetic procedures have been described previously [12]. In each instance where a triploid conceptus was observed, every effort was made to obtain a blood sample from the parents and, by comparing Q- and C-band heteromorphisms of the parents and the conceptus, to determine the origin of the additional haploid complement in the conceptus. In the great majority the extra chromosomes were found to be paternal in origin, usually as the
result of dispermy [7].
448
JACOBS ET AL.
Replication status of the X chromosome was determined utilizing a modification of the BrdU pulse labeling technique of Willard and Latt [6]. One day after routine subculture, the media was changed from FIO containing 10% fetal calf serum to FlO containing 20% fetal calf serum. Seven hours later I ml of a solution containing 276.7 ,ug of BrdU, 0.98 Ag of FUdr, and 14.65 ,ug of uridine was added to a 10 ml culture. The culture was harvested about 11 hrs later, colcemid having been added for the last 3-4 hrs. Cytogenetic preparations were made in the usual way, and the slides were either (1) stained in acridine orange at a concentration of I mg in 50 ml Sorenson's buffer for approximately 40 seconds, rinsed and mounted in buffer, and examined with a fluorescent microscope, or (2) stained in a 0.2 ug/ml solution of Hoechst in Sorenson's buffer for 45 min, mounted in buffer, and exposed to a 275 watt sun lamp about 30 cm away for 8-15 min. Subsequently the Hoechst-stained slides were placed in 2XSSC at 60'C for 4 hrs, stained in 4% Giemsa for 8 min, rinsed in buffer, dried and mounted in DePex (Gurr, Essex, England). In this procedure virtually all late replicating DNA synthesis takes place in the presence of BrdU. Therefore BrdU is incorporated into the newly synthesized DNA strand of the late replicating X chromosomes. When stained with acridine orange and examined under UV light, the chromosome regions that have incorporated BrdU appear red, while the remaining chromosome regions appear yellow-green. When stained by Hoechst, exposed to UV light, and subsequently stained with Giemsa, the late synthesizing regions containing BrdU appear much paler than the remainder of the chromosomes (figs. I and 2). Late replicating X chromosomes were scored by direct microscopic examination in both the temporary preparations stained with acridine orange and the permanent preparation stained with Giemsa. The preparations were scored by two or more observers who had no prior knowledge of the sex chromosome constitution or of the mechanism of the origin of the triploidy. The criteria used in scoring a cell for the presence or absence of allocyclic X chromosomes were: (1) that the cell appeared intact and the chromosomes reasonably well-spread; and (2) that the cell showed unequivocal R-banding in chromosomes 13, indicating that the cell had incorporated BrdU at the end of the
S-peiod. RESULTS
Preparations giving satisfactory results with respect to late replicating X chromosomes were obtained from a total of 36 triploids, 27 with an XXY, and nine with an XXX sex chromosome constitution. The menstrual age of the abortuses, the tissue cultured, and the origin of the < dditional haploid set are shown in table 1, together with the number of cells having 0, 1, or 2 allocyclic X chromosomes. As can be seen from table 1, the XXY triploids fall into three classes: those where virtually all the cells have no allocyclic X chromosome, those where virtually all cells have a single allocyclic X, and those where there are two distinct cell lines, one with no allocyclic X and one with one allocyclic X chromosome (fig. la and b). Among the XXX triploids there are two distinct classes: those where virtually all cells have a single allocyclic X, and those where there are two cell lines, one with one and one with two allocyclic X chromosomes (fig. 2a and b). No XXX triploids were seen in which all cells had two allocyclic X chromosomes. In two XXX triploids there appears to be a minor cell line with no late replicating X chromosome (fig. 3). We do not yet know whether these are cells in which all three X chromosomes are active, or technical artifacts from our failing to identify a late replicating X chromosome. The data in table 1 were examined by step-wise regression analysis to determine whether there was a correlation between the mechanism of origin of the triploidy, the pattern of X inactivation, the tissue examined, or the menstrual age of the fetus. For
X CHROMOSOME REPLICATION IN TRIPLOIDY
449
FIG. 1. -Triploid cells pulse labeled with BrdU to show late replicating X chromosome (arrow). a, 69,XXY cell with no late replicating X chromosome. b, 69,XXY cell with one late replicating X chromosome.
450
JACOBS ET AL.
FIG. 2. -Triploid cells pulse labeled with BrdU to show late replicating X chromosomes (arrows). a, 69,XXX cell with one late replicating X chromosome. b, 69,XXX cell with two late replicating X chromosomes.
451
X CHROMOSOME REPLICATION IN TRIPLOIDY TABLE 1 DATA ON THIRTY-SIX HUMAN TRIPLOIDS No. ALLOCYCLIC
MENS.
ORIGINt
X CHS.
AGE
(DAYS)
TIssUE*
............. .............
136 104
............. ............. ............. ............. ............. ............. ............. ............. ............. .............
137 130 126 117 171 105 123 112 130 144
a.c.v. a.c.v. a.c.v.
No.
XXY: K179 K288 K379 K440 K563 K603 K610 K619 K626 K635 K735 K322
,
....
K539 K736 K632 K778
74 165 51 220 81 121 187 . 82 169 63 54
.
. . .
dII
fetus fetus a.c.v. a.c.v. a.c.v. a.c.v. a.c.v. a.c.v. a.c.v.
+
+
+
+
+
+
yI
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
1
2
-
0 34 38 24 36 33 25 39 10 0 1 12 36 85 69 53 13 3 86 39 37 34 4 38 50 5 20 165 33 34 34
45 0 0 8 0 0 57 1 32 30 30 6 4 0 0 0 15 38 7 0 0 0 61 0 0 0 0 9 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
a.c.v.
fetus a.c.v. a.c.v. a.c.v. a.c.v.
fetus cord fetus
+
+
+
+
+
+
+
+
______-
0 0 0 0 4 5 70 3 9
-
+ +
a.c.v. a.c.v.
cord a.c.v.
....
K418 ............. K649 ............. K627 ............. K795 .............
cfI
0
fetus
....
K356 ............. K364 ............. K369 ............. K502 ............. K507 ............. K797 ............. K803 .............
26
yII
134 82 110 119
cord a.c.v. a.c.v.
cord
No parental blood No parental blood No parental blood
a.c.v.
XXX: K93
.............. .............
K189
K429 K536 K716 K732 K762 K411 K612
............. ............. ............. ............. ............. ............. .............
133 141 105 198 91 96 120 76 65
a.c.v.
1
55
fetus
0
40 33 81 92 45 28 25 48
a.c.v.
1
12 9
fetus a.c.v. a.c.v.
0
a.c.v. a.c.v. a.c.v.
* a.c.v. amnion, chorion, villi. t.Dispermy = 26; failure of first male meiotic division.
1
0
1
=
=
d1, second male
=
dII, first female
purposes of analysis, the tissue examined was considered as cord) or fetal membranes (amnion; chorion; villi).
=
YI,
or
second female
fetal (fetus
=
proper
QII
and
There was no correlation between the origin of the triploidy and the type of X-chromosome inactivation. Thus among the XPXMY triploids, there are abortuses with no late replicating X chromosomes and abortuses in which one X chromosome is late replicating. Among the four XMXMY triploids, there are three where neither X is
452
JACOBS ET AL.
FIG. 3.-A 69,XXX triploid cell pulse labeled with BrdU showing no evidence of a late replicating X chromosome.
late replicating, and one where the majority of cells have a late replicating X. The majority of XPXPXM triploids have a single late replicating X, but in two XPXPXM triploids, there is a minor cell line with two late replicating X chromosomes and in one XPXPXM triploid, the majority of cells have two late replicating X chromosomes. A minor cell line with no late replicating X chromosome was seen in two specimens. In the two XPXMXM triploids, there are cells with one and cells with two late replicating X chromosomes. Therefore, the parental origin of the X chromosome does not appear to affect the pattern of X-chromosome replication nor, by extension, X-chromosome inactivation. The only significant finding with respect to the origin of the triploidy was that of reduced menstrual age in the triploids that resulted from failure of the second maternal meiotic division. While there are only three such triploids, the early abortion of this class is highly significant (P < 0.01). The fact that these conceptions are homozygous for the maternal genes between the centromere and the most proximal chiasma may be a factor relating to early termination of these pregnancies. Homozygosity for so large a number of genes might be deleterious even in the presence of a single dose of the homologous genes contributed by the father. However, the only other reported case of triploidy caused by the failure of the second maternal meiotic division was a live-born XXX [ 13]. Therefore, if our explanation for the unusually early demise of our triploids resulting from this mechanism is valid, it clearly is not universally applicable. This is
X CHROMOSOME REPLICATION IN TRIPLOIDY
453
not surprising as not every such triploid would be expected to receive genes that were lethal during early gestation in the homozygous maternal contribution. There is a significant difference between the menstrual age of the specimens from which we obtained fetal tissue (134.5 days) and those from which we obtained only 0.05). It is known from other tissue from the fetal membranes (111.0 days) (P studies that triploidy can be associated with a whole variety of phenotypes ranging from empty gestational sacs with no evidence of fetal development, to stillbirths, and very rarely live-born infants [14]. Therefore, it is not surprising that in our series the specimens from which we obtained fetal tissue aborted later than those from which we obtained only tissue from the fetal membranes. The number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue by comparison with those grown from extra-embryonic tissue (P
