Influence of the mesonephros on the development of fetal mouse ovaries following transplantation into adult male and female mice

DEVELOPMENTAL

BIOLOGY

124,423-430(1987)

Influence of the Mesonephros on the Development of Fetal Mouse Ovaries following Transplantation into Adult Male and Female Mice TERUKO TAKETO-HOSOTANI~ AND ELAINE SINCLAIR-THOMPSON The Population

Council, Center for Biomedical

Research, 1230 York Avenue, New York, New York 10021

Received October 7, 1986; accepted in revised form July 2.4, 1987 We previously reported that fetal mouse ovaries frequently develop testicular structures following transplantation into adult male mice. The mechanism involved in gonadal sex reversal of ovarian grafts is not known. In the present study, we examined the influence of the adjacent mesonephros on development of the ovarian grafts. The results show that (1) when fetal ovaries were transplanted with the attached mesonephros, the frequency of ovotestis development was higher in male hosts than in female hosts, (2) the fetal ovaries that had been separated from the mesonephros developed testicular structures more frequently than those with the mesonephros, and the incidence of ovotestis development was comparable in male and femal hosts, (3) removal of the cranial or caudal half of the mesonephros resulted in a similar frequency of ovotestis development, and (4) when fetal ovaries were separated and reattached to the mesonephros, they developed testicular structures at a frequency similar to that of ovaries left attached to the mesonephros, and the sex of mesonephroi reattached to ovarian grafts did not influence the incidence of ovotestis development. These findings suggest that fetal ovaries can develop testicular structures after transplantation regardless of the sex of host, and that the adjacent mesonephros protects ovarian grafts from masculinizing stimuli more efficiently in female hosts than male hosts. o 1987 Academic PRSS. IX

INTRODUCTION

In contrast, MacIntyre (1956) and Turner and Asakawa (1964) have obtained ovotestes from fetal rat and mouse Ovotestis development from fetal ovaries following transplantation has been reported in the rat (Buyse, ovaries which had been cotransplanted with fetal testes into castrated male hosts but not from those ovaries 1935; Moore and Price, 1942; Holyoke, 1949; Torrey, 1950; MacIntyre, 1956; Mangoushi, 1975) and the mouse which had been transplanted alone. We have previously (Turner and Asakawa, 1964; Taketo et al., 1984; Taketo- reported that mouse fetal ovaries develop testicular structures when transplanted into male hosts, but not Hosotani et ab, 1985). Electron microscopic examination of the testicular structures of mouse ovotestes has re- into female hosts (Taketo et al, 1984). However, in later vealed all types of testicular somatic cells, i.e., Sertoli studies, we obtained ovotestes in female hosts, although cells, peritubular myoid cells, and Leydic cells, but not at a much lower frequency than in male hosts. Ultrastructures of the ovotestes that developed in female spermatogenic cells (Taketo et al., 1984; Taketo-Hosohosts (H. Merchant-Larios and T. Taketo, unpublished tani et ah, 1985). Furthermore, the amount of testosterobservation) were comparable to those developed in one produced from ovotestes is positively correlated male hosts (Taketo et al., 1984; Taketo-Hosotani et aZ., with the development of testis cords (Taketo-Hosotani 1985). Can these discrepancies be explained by differet ab, 1985). These findings suggest that mouse ovarian ences other than grafting sites, species, or strains? In primordia are bipotential and their development into the present study, we report that the mesonephros adtestes or ovaries can be influenced by environmental jacent to fetal ovaries influences gonadal sex determifactors. nation of ovarian grafts. The effect of the host’s sex on ovotestis development was contradictory in the previous transplantation studies (see the review, Taketo et al., 1985). Moore and Price MATERIALS AND METHODS (1942) and Torrey (1950) have reported an equal freThe Rockefeller University NCS(S) strain of mice was quency of ovotestis development from fetal rat ovaries used in this study. Urogenital complexes were dissected in male and female hosts. Buyse (1935) and Holyoke (1949) have also observed ovotestis development in fe- from fetuses between the 11th and 16th day of gestation male hostt.. out at a lower frequency than in male hosts. (d.g.; 0 d.g. = day of copulation). After various manipulations as described below, fetal gonads were trans1 Present address: McGill University, Royal Victoria Hospital, 687 planted beneath the kidney capsule of adult male or Pine Avenut -vest, Montreal, Quebec, Canada H3A 1Al. female mice (50-200 days old). Experimental designs 423

0012-1606/W$3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

424

DEVELOPMENTAL BIOLOGY

are outlined in Table 1. Each kidney of the host received one graft (or two grafts per host). Previous studies showed that the kidney side used for transplantation does not influence the incidence of ovotestis development (data not shown). Furthermore, the development of ovarian grafts on one side of the host is independent of that on the other side. Accordingly, the side of the host or combination of ovarian grafts in a host was not considered in the analysis of the present results unless otherwise stated. Gonadal grafts were removed from hosts between the 13th and the 16th days after transplantation. Tissues were fixed in Bouin’s solution, embedded in paraffin, sectioned at a 6-pm thickness, and stained with hematoxylin and eosin. Serial sections were examined with light microscopy for the presence of follicles and testis cords. Details of these structures have been reported previously (Taketo et aZ., 1984; Taketo-Hosotani et al., 1985) and are briefly summarized under Results. Ovarian grafts were grouped into “ovary” (containing only follicles) or “ovotestis” (containing both follicles and testis cords or testis cords only). The x2 test was used for statistical comparison of experimental groups (P < 0.05 was considered to be significant).

VOLUME 124, 1987

d.g. was cultured in vitro in order to identify the gonadal sex, while the contralateral gonad was used for tansplantation experiments. The culture conditions have been described previously (Taketo and Koide, 1981). All chemicals were purchased from GIBCO (New York). After 7 days of culture, the explants were observed under a dissecting microscope, and processed for histological examinations by light microscopy. Of the total 47 explants 19 differentiated into ovaries and the rest into testes. For transplantation experiments, gonadal primordia were either left attached to or separated from the adjacent mesonephros and then grafted into female hosts. Of the 19 grafts that were found to be ovarian primordia, 10 were transplanted with mesonephroi, and the remaining 9 were transplanted without mesonephroi. Six ovarian grafts with mesonephroi were transplanted into hosts which received testicular primordia on the opposite kidney side. The frequency of ovotestis development from these ovarian grafts was the same as that for those transplanted into hosts which received only ovarian grafts. The results were combined for statistical analysis. Experiment

Experiment

1

On the 11th d.g., gonadal primordia are sexually undifferentiated, and therefore their gonadal sex cannot be morphologically identified before transplantation. Hence, one of each pair of gonadal primorida of the 11th

2

Fetal testes of the 12th d.g. or older have started testicular organization. The testis cords can easily be recognized with a transmitting light under a dissecting microscope. Therefore, fetal ovaries of these ages can be identified by the absence of testicular structures. Fetal

TABLE 1 THE EXPERIMENTAL DESIGN

Expt no.

Developmental age of fetal ovaries (d.g.)

Manipulations of left and right fetal ovaries

Sex of hosts

Results included

1

11 11

+Ms, C +Ms, C

+Ms, T -MS, T

2

12 12

+Ms, T +Ms, T

-MS, T -MS, T

f”

Fig. 9 Figs. 9, 10

3

12 12

+Ms, T +Ms, T

+CrMs, T +CdMs, T

f f

Table 1 Table 1

4

12 12 12

+Ms, C, T +Ms, C, T *fMs, C, T

*fMs, C, T *mMs, C, T *mMs, C, T

f f f

Table 2 Table 2 Table 2

5

13 14 16

+Ms, T +Ms, T +Ms, T

-MS, T -MS, T -MS, T

f f f

Fig. 10 Fig. 10 Fig. 10

f f

Fig. 10 Fig. 10

Note. +Ms, with the adjacent mesonephros (intact); -MS, separated from the mesonephros; +CrMs, with the cranial half of the mesonephros; +CdMs, with the caudal half of the mesonephros; *fMs, reattached to a female mesonephros; *mMs, reattached to a male mesonephros; C, cultured in vitro; T, transplanted beneath the kidney capsule; m, male; f, female.

TAKETO-HOSOTANI

AND SINCLAIR-THOMPSON

ovaries thus identified were either left attached to or separated from the adjacent mesonephros, and transplanted into male or female hosts. Since fetal ovaries of the 12th d.g. have been routinely used in our laboratory for transplantation studies, all data from previous and present works were pooled and analyzed (a part of these data has been published in Taketo et al., 1984). Experiment

3

The cranial or caudal half of the mesonephros was removed from one of each pair of fetal ovaries of the 12th d.g. The contralateral ovaries were left attached to the mesonephros (control). Each pair of ovaries was then transplanted into a female host. Experiment

6

Both sides of the fetal ovaries of the 12th d.g. (eight pairs) were separated from the mesonephros. Male mesonephroi were equally separated from the fetal testes of the 12th d.g. One of each pair of the fetal ovaries was reattached to a male mesonephros while the contralatera1 ovary was reattached to a female mesonephros. One of each pair of fetal ovaries (nine pairs) was separated from the mesonephros while the contralatera1 ovary was left attached to the mesonephros. Then, each fetal ovary without a mesonephros was reattached to either a male or a female mosonephros. All reconstructed or intact urogenital complexes were cultured for 1 day as described before, and transplanted into female hosts. Results from all experiments were combined for statistical analysis. Experiment

5

One of each pair of fetal ovaries of the 13th, 14th, or 16th d.g. was separated from the mesonephros, and the contralateral ovary was left attached to the mesonephros. Subsequently, each pair of ovaries was transplanted into a female host. RESULTS

Histological

Observations

Representative structures of ovarian grafts are shown in Figs. l-6. Follicles were composed of large oocytes enveloped in two to five layers of pregranulosa cells, which contained dark nuclei and scant cytoplasm (Figs. 2 and 6). Testis cords were composed of Sertoli cells, which contained nuclei near the basement membrane and abundant cytoplasm in the center (Figs. 4 and 6). These structures were comparable with the testis cords that had developed in testicular grafts (Figs. 7 and 8) except for the absence of spermatogenic

Mouse Ovary Developwwnt

425

cells. In addition to the testis cords, two types of distinct cord structures were seen in ovarian grafts. The sex cord was packed with several oocytes and pregranulosa cells (data not shown; see Taketo-Hosotani et al., 1985). The rete cord was composed of epithelial cells, which contained scant cytoplasm and often formed lumens (Fig. 2). The rete structures were not seen in the ovarian grafts that had been separated from the mesonephros. Follicles, testis cords, and rete cords occasionally formed continuous or transitional structures (Taketo-Hosotani et ah, 1985). These mixed structures were not considered in the following results. Removal of the Whole Mesonephros As summarized in Fig. 9, when fetal ovaries were transplanted on the 12th d.g. with intact mesonephroi, they developed testicular structures more frequently in male hosts than in female hosts (P < 0.05). When fetal ovaries were separated from the mesonephros, the frequency of ovotestis development was higher than when mesonephroi were left intact for both male and female hosts (P < 0.005). The sex difference of the host effect was absent. When fetal ovaries with attached mesonephroi were transplanted into female hosts, the incidence of testicular development decreased as the developmental age of ovarian grafts advanced, as summarized in Fig. 10. The ovarian grafts on the 11th d.g. yielded the highest percentage of ovotestes, whereas none of the fetal ovaries of the 16th d.g. developed testicular structures. Removal of the mesonephros increased the incidence of ovotestis development over the period examined, except that it did not induce any testicular development from fetal ovaries of the 16th d.g. Remcwal of Half of the Mesonephros To examine whether the function of the mesonephros is localized with respect to development of ovarian grafts, only the cranial or caudal half of the mesonephros was removed from each fetal ovary. As summarized in Table 2, the incidence of testicular development following transplantation into female hosts was not affected differentially by the particular portion of the mesonephros. Efect

of Male and Female Mesonephroi

Fetal ovaries were separated from the mesonephros, reattached to male or female mesonephroi, and cultured in vitro for 1 day. After culture, reconstituted or intact urogenital complexes were transplanted into adult female mice. As summarized in Table 3, culture for 1 day did not change the capability of fetal ovaries to develop

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DEVELOPMENTAL BIOLOGY

VOLUME 124, 1987

FIG. 1. A fetal ovary with the mesonephros (12 d.g.) transplanted into a female host. Many follicles (f) of various developmental stages were seen throughout the graft. A rete cord (r) was present near the follicles. The mesonephric duct (m) was seen at the bottom. (X125). FIG. 2. A part of the ovarian graft shown in Fig. 1. Oocytes (0) were enveloped in layers of pregranulosa cells (p). The rete cord was corn1losed of epithelial cells (e), which formed a lumen (1) in the center. (X500) FIG. 3. A fetal ovary (12 d.g.) separated from the mesonephros and transplanted into a female host. Numerous testis cords (t) developed i n the peripheral area of the graft. Several follicles (f) were seen in the center. The scale bar indicates 100 pm in Figs. 1 and 3. (X125) FIG. 4. A part of the ovarian graft shown in Fig. 3. The testis cords were composed of Sertoli cells (s), the nuclei of which were arranged near the basement membrane and the abundant cytoplasm toward the center of the cord. The scale bar indicates 100 pm in Figs. 2 and 4. (x! 500)

TAKETO-HOSOTANI AND SINCLAIR-THOMPSON

Mouse Ovary Development

427

into a female host. One testis cord was seen in the FIG. 5. A fetal ovary with the caudal half of the mesonephros (12 d.g.) transplanted periph lery while several follicles (f) developed near the center. A rete cord (r) was seen close to the follicles and the testis cord. (X125) FIG. 6. A part of the ovarian graft shown in Fig. 5. The nuclei of Sertoli cells (s) were larger and stained lighter than those of pregranul osa cells (13). 0, oocytes. (X500) FIG. 7. A fetal testis with mesonephros (m) (12 d.g.) transplanted into a femal host. Testis cords (t) developed throughout the graft. The SC:ale bar in dicates 100 pm in Figs. 5 and 7. (X125) FIG .8. A part of the testicular graft shown in Fig. 7. Germ cells (g) in meiotic prophase (zygotene and pachytene stages) were surroundec dby Serto: Ii cells (s) forming the testis cords. The scale bar indicates 100 grn in Figs. 6 and 8. (X500)

428

DEVELOPMENTAL BIOLOGY

VOLUME 124, 1987 TABLE 2 DEVELOPMENT OF TESTICULAR STRUCTURES IN OVARIAN GRAFTS WITH HALF MESONEPHROI FOLLOWING TRANSPLANTATION INTO ADULT FEMALE HOSTS

Mesonedhros

+

+

-

-

Sex

F

M

F

M

of

host

FIG. 9. Influence of the mesonephros on the development of ovarian grafts. Open and hatched columns show the frequency of ovotestis development from fetal ovaries of the 12th d.g. after transplantation into female (F) and male (M) hosts, respectively. Number of grafts examined in each group is shown in parentheses. x2 test: a/b, P < 0.05; a/c, b/d, P < 0.005.

testicular structures. In fact, the incidence of ovotestis development was higher than in the ovarian grafts that had been transplanted immediately after dissection. The effect of reattached mesonephroi was similar to that of intact mesonephroi. The sex of the reattached mesonephros had no effect on development of ovarian grafts. DISCUSSION

The present study shows that the presence of the adjacent mesonephros influences sex determination of ovarian grafts. When fetal ovaries with attached mesonephroi were transplanted on the 12th d.g., paralleling normal development in fetuses, a certain percentage developed testicular structures dependent on the sex of

E” g 5 cl D .I? ;; al z 2 z G 2 s 2 IL

0

With

Izd

Without

mesoneohros mesonephros

80

Region of the mesonephros attached to ovarian grafts

No. of grafts

Whole (control) Cranial half Caudal half

31 17 14

Development Ovaries

of

Ovotestes

19 12 9

12 5 5

Frequency of ovotestis development (%I 39 29 36

the host. In contrast, fetal ovaries without mesonephroi also transplanted on the 12th d.g. frequently developed testicular structures in both male and female hosts. Similar results were obtained in inbred strains SJL/J and C57BL/6J (data not shown). These observations suggest that fetal ovaries can develop testicular structures upon transplantation regardless of host’s sex, and that the adjacent mesonephros suppresses testicular development in ovarian grafts in female hosts. It has been reported that rat gonads with mesonephroi can develop into ovotestes after transplantation into female hosts (Buyse, 1935). Torrey (1950) transplanted rat gonadal ridges with or without mesonephroi and did not find any difference in their development. Therefore, the mesonephros appears not to be crucial for the development of rat ovarian grafts. However, it is not clear whether the mesonephros was maintained functional under the conditions used by these investigators. It is also conceivable that the effect of the mesonephros was not significant at the developmental ages of fetal rat ovaries used in these experiments. The effect of the mesonephros on ovotestis development was most prominent when fetal mouse ovaries were transplanted on the 12th or 13th d.g. Ovarian primordia without mesonephroi of the 11th d.g. developed into ovotestes at a frequency similar to those of the 12th or 13th d.g., while an attached mesonephros did not reduce the incidence of testicular development as much

60

TABLE 3 DEVELOPMENT OF TESTICULAR STRUCTURES IN OVARIAN GRAFTS WITH REATTACHED MESONEPHROI FOLLOWING CULTURE AND TRANSPLANTATION INTO FEMALE HOSTS

40

20

0 11

12 Developmental

13 stage

14

16

(d.g.)

FIG. 10. Influence of the mesonephros on the development of ovarian grafts transplanted at various developmental stages. * indicates a significant difference (P < 0.005) between ovarian grafts transplanted with (open columns) and without (hatched columns) the mesonephros.

Source of the mesonephros attached to ovarian grafts

No. of grafts

Ovaries

Self (intact) Female fetuses Male fetuses

27 16 19

15 8 10

Development

of

Ovotestes 12 8 9

Frequency of ovotestis development (W) 44 50 47

TAKETO-HOSOTANIANDSINCLAIR-THOMPSON

as that of older ovaries. This observation can be explained if the function of the mesonephros of the 11th d.g. is more easily disturbed during dissection or transplantation since the gonadal primordia is less developed and more fragile than older ovaries. An alternative explanation is that the mesonephros isolated from fetuses of the 11th d.g. cannot influence ovarian grafts to the same extent as can those from older fetuses. On the 14th d.g. or later, fetal ovaries showed reduced or lost ability to develop testicular structures, and the mesonephros did not have a clear influence on their development. Other investigators have reported the influence of the mesonephros on gonadal differentiation. According to Byskov and others, the mesonephros of either sex produces a factor which can induce meiosis of germ cells in fetal mouse ovaries and testes (Byskov, 1974; Byskov and Saxen, 1976). Contribution of mesonephric cells to follicular development has been suggested in the mouse and sheep (Byskov et ah, 1977; Byskov, 1978; Upadhyay et al., 1979; Zamboni et al., 1979). From these findings it is conceivable that removal of the mesonephros prevents ovarian development and thereby promotes testicular organization. However, we have observed that follicles do develop in ovarian grafts without mesonephroi while testicular development is absent when microencapsulated in semipermeable membranes and transplanted into male hosts (Taketo-Hosotani, 1987). It is therefore more likely that the mesonephros protects ovarian grafts from a masculinizing influence of the host. This protecting effect of the mesonephros would be more potent in female hosts than in male hosts, because either the function of the mesonephros depends on some factors in female hosts or, the male host has a higher capacity to promote testicular development and so overcomes the influence of the mesonephros. We have found that the amount of testosterone produced by ovotestes is positively correlated to the degree of testicular development (Taketo-Hosotani et ab, 1985) whereas an increase in testosterone levels in the host promotes testicular development in ovarian grafts (Taketo and Merchant-Larios, 1986). The mesonephros may counteract the effect of testosterone. It has been reported that the mesonephros of the rabbit fetus accumulates testosterone by binding to it (Wilson, 1973), and that the mesonephros prevents production of testosterone from fetal and neonatal rabbit testes (Grinsted et ah, 1982). To examine whether the function of the mesonephros is localized in the cranial or caudal region, we transplanted fetal ovaries with half of the mesonephros into female hosts. The results showed that either region of the mesonephros could prevent testicular development as the whole mesonephros. Furthermore, the mesonephros from male or female had a similar influence on

Mouse Ovary Development

429

the development of ovarian grafts. This is probably because the mesonephros of the 12th d.g. is sexually undifferentiated (Dyche, 1979). It has been known that mesonephric development is regulated by hormones secreted from the fetal testes after gonadal sex differentiation begins (Jost et ah, 1973). Our present findings may suggest that the protection by the mesonephros can be rather physical in nature. On the other hand, the frequency of testicular development was lowest when fetal ovaries with mesonephroi were kept intact and transplanted soon after dissection, implying that the mesonephros has a biological function which is easily disturbed during dissection, separation, or culture. The ability to influence gonadal development appears to be distributed throughout the mesonephros. Our present observation refutes the claim that the tubular structures in ovarian grafts are not testis cords but the rete. According to Byskov, the rete system can be divided into three regions: extraovarian, connecting, and intraovarian (Byskov, 1978). In the present experiments, extraovarian and connecting rete components were removed by separating the mesonephroi from fetal ovaries, but the intraovarian rete may have been left in ovarian grafts. However, rete structures in ovarian grafts were distinct from the testis cord. The epithelial cells of the rete were similar to pregranulosa cells of follicles as reported previously (Odor and Blandau, 1969; Zamboni, 1974), and often formed lumens. These structures were seldom seen in the ovarian grafts that had been separated from the mesonephros. If the mesonephros contributes to the testicular development, removal of the mesonephros would decrease the incidence of testicular development. However, we have obtained the opposite results. Therefore, it is unlikely that the testis cords developed from the rete in ovarian grafts. It has been established that genes on the Y chromosome determine an indifferent gonad to develop into a testis in mammals (Stewart, 1983; Short, 1979). On the other hand, it has been suggested that XY cells produce a diffusible factor which can induce testicular differentiation of the XX cells. For example, in freemartins, i.e., heterosexual twins of cattle, sheep, and goats, which share the placenta and circulation, female fetuses develop testicular components (Lillie, 1916, 1917; Jost et al., 1973). A male-predominant, histocompatibility-Y (H-Y) or serologically detectable male (SDM) antigen was once proposed as a testis-determining gene product (Wachtel et al, 1975; Ohno et aZ., 1979). However, this hypothesis has been contradicted by recent results of other investigators (Haseltine et al., 1981; Either et aL, 1982; McLaren et al, 1984 Simpson et al, 1987). In our present study, testicular structures developed in ovarian grafts without the influence of the Y chromosome, i.e., in female hosts. This finding is inconsistent with

430

DEVELOPMENTALBIOLOGY

the role of the Y chromosome in testis determination. However, it is conceivable that genes on the Y chromosome control the structural genes on autosomes or the X chromosome for testicular organization, and that the host environment somehow induces the intermediate process leading to testicular organization. It is possible that the mechanism of ovotestis development following transplantation is different from that of testicular organization during normal development. Nevertheless, study of the gonadal sex reversal following transplantation should provide invaluable information toward the understanding of the complex process of gonadal sex differentiation. Present results suggest the importance of the adjacent mesonephros for gonadal development.

VOLUME124, 1987 tion in heterosexual embryonic rat gonad transplants. Anat. Rec. 124,27-41.

MANGOUSHI,M. A. (1975). Scrotal allografts of fetal ovaries. J. Anat. 120,595-599.

MCLAREN, A., SIMPSON,E., TOMONARI,K., CHANDLER,P., and HOGG, H. (1984). Male sexual differentiation in mice lacking H-Y antigen. Nature

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