XY follicle cells in ovaries of XX XY female mouse chimaeras

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1 Development 4, 63-6 () Printed in Great ritain The Company of iologists Limited 63 XY follicle cells in ovaries of XX XY female mouse PAUL S. URGOYNE, MIA UEHR and ANNE McLAREN MRC Mammalian Development Unit, Wolfson House, 4 Stephenson Way, London NW HE, UK Summary Oocytes with adhering follicle cells were sampled from ovaries obtained from GPI-IA-^GPI-I, comprising females and hermaphrodite. GPI analysis of individual oocytes revealed a marked bias towards the GPI- component in the germ line of this chimaeric combination. GPI-I XY oocytes were identified in the ovary from the hermaphrodite, the bias towards the GPI- germ line perhaps helping to counterbalance the normally severe selection against XY oocytes. GPI analysis of follicle cells revealed a much more balanced contribution of the two components to this ovarian cell type. Importantly, GPI-A follicle cells were identified in more than half the follicles from an XX<^XY female in which the GPI-A component was XY, supporting an earlier conclusion of Ford et al. (74) that XY cells can contribute to the follicles of XX++XY female mice. It is suggested that XY cells can be recruited to form follicle cells in XX<-»XY when there is a developmental mismatch between the two components, such that an ovary-determining signal produced by the XX component pre-empts the testisdetermining action of the Y. Key words: follicle cells, mouse, ovary, XX<-+XY females. Introduction We have recently shown that the Sertoli cells of adult XX<->XY male mouse are exclusively XY (urgoyne et al. ). This finding provides support for the view expressed by urgoyne et al. (6) that the Y chromosome acts cell autonomously in bringing about Sertoli cell differentiation. It is generally accepted that ovarian follicle cells and testicular Sertoli cells are derived from the same gonadal cell lineage (the 'supporting cell' lineage). If the presence of a Y chromosome in a cell of this lineage is both necessary and sufficient to ensure its development into a Sertoli cell, fetal XX*-»XY gonads would be expected to contain patches of XY Sertoli cells together with patches of XX prefollicular cells, so that the gonads should manifest as fetal ovotestes. That they are indeed ovotestes has recently been claimed by radbury (7). We have argued (urgoyne et al. ) that in most XX-wXY there is subsequent regression of the ovarian component because the Miillerian duct inhibitor (AMH) produced by the Sertoli cells also inhibits the formation and survival of oocytes (Vigier et al. 7) and, without oocytes, differentiation of prefollicular cells into follicle cells does not occur. XX<->XY gonads should only continue as ovotestes if there are too few Sertoli cells, and consequently insufficient AMH, to bring about oocyte failure. In rare cases, chance exclusion of XY cells from the supporting cell lineage might allow XX<-»XY gonads to develop as ovaries, but we would expect all the follicle cells to be XX. However, Ford et al. (74) reported finding XY mitotic cells (presumed to be follicle cells) in the ovaries of two XX<->XY female mouse. Although other sources of abundant mitoses in the ovary are difficult to envisage, the lack of direct proof that these XY cells were follicle cells prompted us to reinvestigate the origin of follicle cells in XX<-OCY chimaeric ovaries. Materials and methods (A) Mice and tissue sampling Aggregation were produced by standard techniques. One component was AL/c. The embryos giving rise to the other component were produced by mating males from a CA strain that was homozygous for the T6 translocation marker (Ford et al. 56) and in which the normal mouse Y had been replaced by a metacentric

2 64 P. S. urgoyne, M. uehr and A. McLaren 4 A Fig.. one marrow metaphases from the two XX<-»XY females. (A) XX metaphase from female Q. () XY metaphase from female Q showing the metacentric Y (thick arrow) and two T6 marker chromosomes (thin arrows). (C) XX metaphase from female D with a single T6 marker chromosome (arrow). (D) XY metaphase from female D showing a normal Y chromosome (arrow). ar, /an. variant (Winking, 7), with either () females from the same CA stock, or () C57L/6Mcl females, or (3) females from an F] cross between CA/Ca and C57L/6Mcl. AL/c expresses the glucose phosphate isomerase electrophoretic variant GPI-A while the CA crosses express GPI-. 35 overt were produced: 7$, l ^ a n d 7d". The female were mated to AL/c males and the genotype (deduced from coat colour) and sex of the progeny was recorded. The final objective was to obtain information on the sex chromosomal constitution of the follicle cells and oocytes of XX<H>XY female. Since offspring are rarely obtained from the XY component of XX<-»XY females (Ford et al. 75), only females with progeny predominantly or exclusively of one genotype were selected for analysis, but a strong bias in favour of GPI- progeny rendered this criterion of little value. In order to circumvent this problem some of the later females were karyotyped from spleen biopsies. The females selected for analysis were pruned with 5i.u. PMSG (Folligon, Intervet Laboratories) and killed 44 h later. Samples of liver, kidney, spleen and adrenal were frozen in liquid nitrogen and stored at -7 C for subsequent GPI analysis; a sample of bone marrow was taken for subsequent chromosome analysis. The ovaries were removed and teased apart in M medium (Whittingham, 7). The follicles released were ruptured and the oocytes with adhering follicle cells ('cumulus') were washed individually three times in M, and then were pipetted up and down in a drawn pipette to detach the follicle cells. The oocytes and follicle cell samples were collected and frozen in less than jtl of M (uehr & McLaren, 5) and stored for less than week at - C for subsequent GPI analysis. () GPI- analysis The somatic tissue samples were homogenized in approximately three times their volume of phosphate-buffered saline (Dulbecco A: Oxoid), centrifuged briefly and the supernatant applied to the gel. Oocytes and granulosa cell

3 XY follicle cells in XX**XY ovaries Fig.. GPI- gel for five of the follicles from XX<-XY female D. The follicles (from left to right) were scored as follows: >A, >A, >A, >>>A, only. The GPIA component in this chimaera was XY. samples were freeze-thawed three times and applied directly to the gel from the pipette in which they had been collected. Electrophoresis on cellulose acetate plates ('Helena' Titan-III Iso-Viz) was carried out as previously described (uehr & McLaren, 5). After the stain was applied to the gels they were kept at room temperature in the dark until stain intensity was optimal (5- min). The relative intensity of the two GPI bands (A and ) was then judged by eye according to the following code: A>>> A>> A> A= Sample nearly all A A considerably stronger than A perceptibly stronger than Two bands of equal intensity etc. The number of cells collected in the cumulus samples varied, but we estimate that at least 5 cells were necessary to produce clear bands on the gel. Results The use of the T6 translocation marker and the metacentric Y chromosome marker enabled both sex chromosome complements to be identified in all but one of the overt aggregation. Metaphases from the two XX<H>XY females are illustrated in Fig.. The two XX-wXY females and the XX-wXY hermaphrodite identified in the present study, together with ten XX<->XY males from the same series of, identified during the study of urgoyne et al. (), are listed in Table. As might be expected, the two females had a relatively minor XY component. Table gives the results of the GPI typing of follicles and oocytes for the XX<-*XX and XX-^XY mice we have analysed, together with all the breeding data. The following points should be noted. First, the oocyte and breeding data are in very good agreement. Second, there is a strong bias in favour of GPI- oocytes and GPI-l-derived offspring. Of the two 65 females with a majority of GPI-lA-derived offspring, $Q has a GPI- XY cell line which is expected to be at a selective disadvantage in the germ line; indeed, the presence of an XY-derived offspring from Q and of XY-derived oocytes in c^l may in part be due to the selection in favour of GPI- in these. Third, in contrast to the oocytes, the GPI-A component is well represented in the follicles. Importantly, this holds true for XX<->XY$D (Fig. ) where the GPI-A component is XY. In Table 3, the GPI- A contribution to the follicles and oocytes is compared with the proportion of GPIA in non-ovarian tissues. In addition to the rarity of GPI-A oocytes, the GPI-A component is markedly under-represented in the coat of the crossbred<-nnbred mice, and to a lesser extent in the inbred<^>inbred combination. Comparing the GPI-A contribution to the follicles in XX<H>XY$D with that in the XX<-»XX females does not indicate any marked deficiency of XY cells. Apparently the presence of a Y chromosome interfered very little with the formation of follicle cells in this chimaera. In "L, the only follicle analysed was GPI- A (XX). Discussion The present results support the conclusion of Ford et al. (74) that XY cells can contribute to the follicles of XX<-»XY female mouse. How can the formation of XY follicle cells be reconciled with our earlier observation that Sertoli cells are exclusively XY in XX«-»XY male mouse? The dilemma can be stated as follows: if follicle cells and Sertoli cells are derived from the same cell lineage, and the Y acts cell autonomously within this lineage to bring about Sertoli cell differentiation, how can XY supporting cells avoid commitment to the Sertoli cell pathway and become follicle cells? It is possible to escape this dilemma either by rejecting the assumption of a common cell lineage, or by rejecting our claim that the Y acts cell autonomously in this lineage. However, we feel that our conclusion that the Y acts cell autonomously is soundly based, and evidence from Taketo-Hosotani et al. (5) that follicle cells can 'transdifferentiate' (see urgoyne, ) into Sertoli cells confirms impressions from the study of T6/X Sxr ovotestes (Ward et al. ) and XO+XY mosaic ovotestes (P.S.., unpublished data) that Sertoli cells and follicle cells are derived from the same lineage. McLaren (7) has suggested an alternative solution whereby a product of the supporting cell lineage, produced as a consequence of Y chromosome activity, has to reach a certain threshold concentration in order to complete commitment of the

4 66 P. S. urgoyne, M. uehr and A. McLaren Table. Estimated proportions of XY cells in relation to sex for the XX++XY Chimaera type Mouse XY component Sex XX AL'wXY CA XX CA x C57<-»XY AL XX AL~XY CA XX AL<->XY CA XX AL<->XY CA XX AL<->XY CA XX AL*->XY CA x C57 XX CA^XY AL XX CA<-XY AL XX CA-wXY AL CT <S a O" " XX AL^XY CA XX CA x Fl-wXY AL XX AL<->XY CA L D Q <?? *Mean of bone marrow (karyotypes), adrenal, kidney, liver and spleen values. The visual GPI score for each organ sample was equated to approximate percentages: A>>>, 5:5; A», :; A>, 67:; A =, 5:5; etc. tright gonad was an ovary with associated oviduct and uterine horn. Left gonad was an ovotestis with associated caput epididymis. The ovotestis was incompletely descended. There was no vas deferens. The lower half of the left uterine horn was present. Table. The numbers of follicles" oocytes* and offspring by GPI type for the female and hermaphrodite GPI[type Inbred<->inbred (GPI-A) (GPI-) A Offspringt A Oocytes A Follicles A& XX<->XX$R XX~XX?G XX<->XX$H XX<->XX$J XX<->XX$K XX*->XX$M XX~XX$N XX<->XX?O XX<->XX$P XO«XX?F XX«->XY$Q XX<->XY^L Sterile Inbred*->crossbred (GPI-A) (GPI-) XX<->?$I XX<->XX$ XX<->XX$A XX«XX$E XX«-XX?C XY-wXXPD 6 6 *No ovarian tissues were sampled from seven females: $C because she was presumed to be XX<->XX from the breeding data, $'s M,N,O,P and F because they were shown not to be XX«->XY by spleen biopsy karyotyping, and $Q because she died when the anaesthetic was administered in preparation for spleen biopsy. $?Q proved to be XX«->XY on analysis of postmortem bone marrow metaphases. tthe offspring are classified according to the GPI-type of the oocytes from which they are derived.

5 XY follicle cells in XX-^XY ovaries 67 Table 3. Relative GPI-IA contributions to oocytes, follicles and non-ovarian tissues % GPI-IA type (GPI-IA) (GPI-) Oocytes Follicles* Organst one marrow Coat* XX-wXXjR XX<-O(X$G XX<-»XX$H XX<-XX$J XX<->XX$K XX<->XYc/L 53 > Inbred crossbred (GPI-IA) (GPI-) XX? $ I XX XX$ XX XX $ A XX XX 5 E XY XX.D <5 < Mean of individual follicle values. tmean of adrenal, kidney, liver and spleen values. $ Estimated by eye when the mouse was killed. supporting cells to the Sertoli cell pathway. With too few XY supporting cells this threshold is not reached, thus allowing the cells to differentiate as follicle cells. This explanation is compatible with our present result, since the XY component was smaller in female D, in which we identified XY follicle cells, than in any of the XX<-»XY male, in which the XY supporting cell lineage evidently gave rise to Sertoli cells. It cannot however account for the female chimaera described by Ford et al. (74) in which 7 % of the presumed follicle cells were XY, and the XY contribution to other somatic tissues was correspondingly large. It may be significant that both female XX<->XY reported by Ford et al. (74) had an XY component derived from the AKR strain. Washburn & Eicher (3) showed that the AKR Y chromosome on a C57L/6 background, in association with the T hp mutation, leads to the development of ovaries and ovotestes in which all the follicle cells must be XY. They suggest (Eicher & Washburn, 6) that the AKR Y chromosome carries a relatively lateacting allele at the testis-determining locus, Tdy, which fails partially or completely to pre-empt the early-acting C57L/6 programme for ovary determination. The same explanation has been advanced to account for the occurrence of XY females when a Mus domesticus Y *" chromosome is introduced into a C57L/6 background (Eicher & Washburn, 3, 6). We have observed that the Y^** chromosome is indeed later acting than the C57L Y, as judged by the timing of testis cord formation in C3H$ x C57L/6C? and C3H$ x C57L/6-Y pos d" crosses (S. J. Palmer and P.S.., unpublished data). urgoyne () suggested that a similar mismatch between the timing of testis determination and ovary determination could explain the presence of XY follicle cells in XX<H>XY female, if the XY component acts too late to pre-empt the process of ovary determination initiated by the XX component. Unlike the McLaren (7) 'threshold' concept, the mismatch model can accommodate the female XX^-»XY of Ford et al. (74), in which the XY supporting cell population would have been recruited to form follicle cells before the late-acting AKR Y chromosome could initiate Sertoli cell development. In the present study, the XY follicle cells were found in an XX«XY female in which the XY component was inbred and the XX component was crossbred, a situation which would be expected to lead to the XY component being retarded relative to the XX component. If this 'mismatch' explanation for the formation of XY follicle cells in XX<-»XY is correct, then it requires that follicle cell development (unlike Sertoli cell development) is not cell autonomous, but involves cell-cell interaction. The inducing signal could derive from oocytes, since in the absence of oocytes no differentiation of follicle cells occurs. Macintyre, aker & Wykoff (5) reported the inhibition of Sertoli cell differentiation in XY embryonic rat gonads cografted with older ovaries: the time

6 6 P. S. urgoyne, M. uehr and A. McLaren frame within which the ovaries were effective coincided with the presence of early meiotic oocytes. In conclusion, we now envisage two routes by which XX<-»XY chimaeric mouse gonads can develop as ovaries. First, in XX<-»XY with a very minor XY component, change exclusion of XY cells from the supporting cell lineage will allow ovarian development. Second, genetic differences between the two components of the chimaera can result in a developmental mismatch, with the testis-determination process being pre-empted by the process of ovary determination initiated by the XX component. Clearly, we now need to find out whether XY follicle cells can also form in sex chromosome where there is no reason to expect a developmental mismatch between the two components. ecause the gonads of such mice may only rarely develop as ovaries, it will be important to include ovotestes in any future studies. We thank Robin Lovell-adge for carrying out some embryo transfers and, together with Peter Koopman, for suggested amendments to the manuscript. References RADURY, M. W. (7). Testes of XX^XY chimeric mice develop from fetal ovotestes. Dev. Genet., 7-. UEHR, M. & MCLAREN, A. (5). Expression of glucose-phosphate isomerase in relation to growth of the mouse oocyte in vivo and in vitro. Gamete Res., 7-. URGOYNE, P. S. (). Role of mammalian Y chromosome in sex determination. Phil. Trans. R. Soc. Lond. (in press). URGOYNE, P. S., ANSELL, J. D. & TOURNAY, A. (6). Can the indifferent mammalian XX gonad be sexreversed by interaction with testicular tissue? In Development and Function of the Reproductive Organs (ed. A. Eshkol,. Eckstein, N. Dekel, H. Peters & A. Tsafriri), pp Rome: Ares-Serono Symposia. URGOYNE, P. S., UEHR, M., KOOPMAN, P., ROSSANT, J. & MCLAREN, A. (). Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX<-*XY chimaeric mouse testes. Development, EICHER, E. M. & WASHURN, L. L. (3). Inherited sex reversal in mice: Identification of a new primary sexdetermining gene. J. exp. Zool., EICHER, E. M. & WASHURN, L. L. (6). Genetic control of primary sex determination in mice. A. Rev. Genet., FORD, C. E., EVANS, E. P., URTENSHAW, M. D., CLEGG, H., ARNES, R. D. & TUFFREY, M. (74). Marker chromosome analysis of tetraparental AKR<->CA-T6 mouse. Differentiation, 3-3. FORD, C. E., EVANS, E. P., URTENSHAW, M. D., CLEGG, H. M., TUFFREY, M. & ARNES, R. D. (75). A functional 'sex-reversed' oocyte in the mouse. Proc. R. Soc. Lond., 7-7. FORD, C. E., HAMERTON, J. L., ARNES, D. W. H. & LOUTIT, F. (56). Cytological identification of radiation. Nature, Lond. 77, MACINTYRE, M. N., AKER, L. & WYKOFF, T. W. (5). Effect of the ovary on testicular differentiation in heterosexual embryonic rat gonad transplants. Archs Anat. microsc. Morph. exp. 4, MCLAREN, A. (7). Sex determination and H-Y antigen in mice. In Genetic Markers of Sex Differentiation (ed. F. P. Haseltine, M. E. McLure & E. H. Goldberg), pp New York: Plenum. TAKETO-HOSOTANI, T., MERCHANT-LARIOS, H., THAU, R.. & KOIDE, S. S. (5). Testicular cell differentiation in fetal mouse ovaries following transplantation into adult male mice. J. exp. Zool. 36, -37. VIGIER,., WATRIN, F., MAGRE, S., TRAN, D. & Josso, N. (7). Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to it in vitro. Development, WARD, H.., AKER, T. G. & MCLAREN, A. (). A histological study of the gonads of T6H/XS*r hermaphrodite mice. /. Anat. 5, WASHURN, L. L. & EICHER, E. M. (3). Sex reversal in XY mice caused by dominant mutation on chromosome 7. Nature, Lond., -34. WHITTINGHAM, D. G. (7). Culture of mouse ova. /. Reprod. Fert. Suppl. 4, 7-. WINKING, H. (7). Marker Y chromosome. Mouse news Lett. 5, 53. (Accepted August )