Mouse gonadal differentiation in vitro in the presence of fetal calf serum

Cell Differentiation and Development, 21 (1989) 19-28

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Elsevier Scientific Publishers Ireland, Ltd. CDF 00589

Mouse gonadal differentiation in vitro in the presence of fetal calf serum Sarah Mackay

and Robert

A. Smith

Department of Anatomy, University of Glasgow, Scotland

(Accepted 6 March 1989)

Other workers have shown that feud calf serum (F.C.S.) inhibits the differentiation of the rat testis, and disrupts established testicular cords, in vitro. To investigate the possibility of a serum effect in the mouse, indifferent urogenital complexes and differentiated gonads were removed from sexed fetuses and cultured for 7 days in medium with or without the addition of fetal calf serum. Cultures were assessed by light and electron microscopy. Testicular and ovarian differentiation occurred in the presence of fetal calf serum. Serum did not prevent basal lamina development in testicular cords of explants cultured to the equivalent of day-17 control testes. Ovarian differentiation;

Testicular differentiation;

Serum effect; Mouse; Ultrastructure

Introduction

The mouse gonad forms from the indifferent blastema in two main stages, a stage of differentiation when the two sexes first become distinguishable, with the establishment of testicular or ovigerous cords, followed by a stage of development during which the differentiated gonad becomes organised, with the appearance of structures characteristic of the mature ovary or testis (for review see Byskov, 1986). Both these critical periods have been investigated by culture in vitro, which allows the examination of such problems as the origin of the gonadal blastema, the mechanisms of cell differentiation within it, the role of the mesonephros and the regulation of meiosis. Previous studies of the first stage of differentiation

Correspondence address: Dr. S. Mackay, Department of Anatomy, University of Glasgow, Glasgow G12 SQQ, Scotland.

include those of Byskov and Sax& (1976) and Taketo and Koide (1981) while other workers have examined the second stage (Odor and Blandau, 1971; Baker and Neal, 1973; Evans et al., 1982). In all cases fetal calf or horse serum was used as a component of the culture medium. Recently attention has been drawn to possible detrimental effects of serum in vitro. Agelopoulou et al. (1984) have shown an inhibitory action of fetal calf serum on the differentiation of the rat testis: serum prevented the formation of testicular cords and also disrupted newly formed cords. However, the authors also reported that serum improved the general appearance, growth and number of mitoses seen in cultures. If cultured mouse gonads could be shown to benefit from these advantages of serum without suffering any inhibitory effects, then the mouse would be the more favourable species to use in studies of gonadal morphogenesis in vitro. Agelopoulou and co-workers point out that previous results from

0922-3371/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland, Ltd.

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cultures of mouse gonads in media containing serum are equivocal, since indifferent complexes were not always sexed at the time of explantation; they also point out that some studies showed serum to be a retarding factor. Taketo et al. (1986), by contrast, have shown that mouse gonadal primordia explanted on day 11 of gestation will only differentiate in vitro when serum is added to the culture medium. We now present results of a study which extends our previous findings (Mackay and Smith, 1986) by examining the effect of serum on the differentiation and subsequent development of mouse gonads. Care has been taken to sex and stage fetuses at the time of explantation. Both testes and ovaries have been investigated and electron microscopy has been used to assess possible ultrastructural effects which may have been overlooked in studies limited to light microscopical evaluation of cultures.

Materials and Methods

An inbred colony of CBA mice maintained on a light reversal regime was used for matings, which took place between 12.00 noon and 15.00 h at the start of the 9 h dark period. The day of finding a vaginal plug was designated day 0 of pregnancy. Animals were killed by cervical dislocation, uterine horns dissected and fetuses removed with amnions and collected in Hank’s buffer. Further microdissections were carried out in a laminar-flow cabinet. Two sets of experiments were carried out. In the first group of experiments only those fetuses of chronological age 11.5 and 12 days post coitum (p.c.), assessed on morphological criteria as stages 19 and 20 of Theiler (1972) (i.e. before testicular cord formation in the male), were used. A piece of amnion was removed and stained by the chromatin test of Farias et al. (1967); this enabled the genetic sex of fetuses to be determined by the presence of X-chromatin in females. Each fetus provided an explant consisting of the paired (right and left) urogenital complexes (mesonephroi and genital ridges) with a small piece of intervening dorsal mesentery. In the second set of experiments, fetuses removed on day 14 p.c., and assessed as stage 22 of

Theiler, were used. By this stage, gonads could be sexed morphologically by the presence of testicular cords in the male; culture from this stage was designed to test possible effects of serum on established testicular cords and on further differentiation of the ovary. After incubation for 20 min in buffered 0.125% type IV collagenase (Sigma Chemical Co., Poole, U.K.), which slightly loosened, but did not disrupt, the tissue, male and female explants were cultured separately, approximately 5 to a dish. Williams E medium (Gibco, Scotland) supplemented with 2 mM glutamine and 0.5% gentamytin was used; to test the effects of serum, 10% fetal calf serum (Flow Laboratories, Irvine, Scotland) was added to some cultures. Cultures were incubated in a humidified atmosphere of 5% CO,/95% air at 37’C for 7 days, during which their condition was assessed by phase contrast microscopy. Explants were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer with 3% glucose for 30 min. After a wash in buffer they were postfixed in 1% osmium tetroxide in cacodylate buffer and dehydrated through a graded ethanol series. Material was embedded in Spurr’s epoxy resin polymerised at 70 o C. For light microscopy 1 pm sections were cut on a Reichert Ultracut ultramicrotome (ReichertJung, F.R.G.) and stained with toluidine blue. Representative sections were studied from an interrupted series cut through each block, and the stage of morphological development compared with that of control gonads in vivo. Two to 4 gonads of each sex of stages 18, 20, 22, 23, 24, 25 and 27 (of chronological ages day 11, 12, 14, 15, 16, 17 and 19 (i.e. newborn), respectively) were examined by light microscopy; from these, selected stages were examined by transmission electron microscopy. 60-80 run sections were stained with uranyl acetate and lead citrate and examined on a JEM 100s electron microscope (JEOL, Tokyo).

ReSUltS Control gonadal morphology

In vivo control morphology was examined to establish a baseline for comparative assessment of

21 TABLE I Number of paired, day-11.5-12 complexes, sexed by X-chromatin stage of development reached at end of culture period Number of complexes

Culture conditions

26 27

+ F.C.S. -F.C.S.

Stage of development

analysis,

and maintained

for 7 days in vitro, with the equivalent

reached

Indifferent gonad

ovary (days) 14

15

17

14

15

17

6 5

1 1

3 3

3 5

5 6

5 6

3 1

Testis (days)

Necrotic explants have been omitted from the table: one explant from each group.

the degree of development of cultures in vitro. We have previously published illustrations of stages 18, 22, 23 and 25 (Mackay and Smith, 1986). At stage 22 (day 14) the testis showed definite testicular cords of pre-Sertoli cells arranged around central germ cells, some of which were in mitosis. Between cords there was a cellular interstitium. By stage 23 (day 15) a tunica albuginea could be identified deep to the coelomic epithelium; cords were larger with pale staining centrally placed germ cells. The testis at stage 25 (day 17) showed an increase in interstitial tissue including groups of newly differentiated Leydig cells (Fig. 3a), darkly stained flattened peritubular cells were arranged around the cords, and fewer mitotic figures were seen. The ovary at stage 22 (day 14) showed clusters of germ cells arranged in indistinct cords; by stage 23 (day 15) irregular ovigerous cords were more obvious, separated by blood vessels and stromal connective tissue. Processes, from somatic cells supported by developing basal laminae, were seen separating the cells of the cords on day 17 (stage 25) (Fig. 2a,b), and synaptonemal complexes in nuclei of germ cells provided evidence of meiosis at this stage. Effects of serum on differentiation in vitro

As detailed in Table I, explants from 26 sexed day-11.5-12 fetuses were cultured for 7 days in medium containing fetal calf serum (F.C.S.). Three genetically male and 3 genetically female explants remained at the indifferent gonad stage. Twentyseven explants were cultured for 7 days in the absence of F.C.S. Three genetically male and 2 genetically female explants remained at the indifferent gonad stage.

Testicular differentiation

Cultures were assessed as equivalent to day-14 controls if testicular cords were apparent. Therefore 13 out of 16 genetically male indifferent complexes developed testicular cords in the presence of F.C.S., with 3 reaching day-17 equivalent (Fig. la). Thirteen out of 16 genetically male indifferent complexes developed testicular cords in its absence, including 1 to a stage 25 testis (Fig. lb). Explants studied by electron microscopy included 4 which were equivalent to day-17 (stage 25) controls (Fig. le) (3 cultured with F.C.S. Fig. lc and f; and 1 without F.C.S. - Fig. lg), and 6 which were equivalent to day-15 controls (3 with F.C.S., and 3 without - Fig. Id). Typical basal laminae were seen in all cases. Vesicles containing flocculent material were commonly seen near to, or opening at, the basal surface of pre-Sertoli cells. Similar vesicles were also seen at the surface of the peritubular cells (Fig. lc,d). Junctions were seen between adjacent pre-Sertoli cells in day-17 equivalents (Fig. If). Ovarian differentiation

Cultures were assessed as equivalent to control ovaries on day-14 if clusters of germ cells were seen forming indistinct ovigerous cords. Day-15 control equivalents showed definite ovigerous cords separated by stromal cells, whilst those explants assessed as day-17 equivalents showed the first indication of follicle formation (see Fig. 2c,e). Synaptonemal complexes seen in germ cells provided evidence that meiosis was underway in day-17 equivalents (see Fig. 2d). Occasionally cytoplasmic bridges connected adjacent germ cells.

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23 TABLE II Number of day 14-14.5 Number of gonads

14 16 24 19

Ovaries Testes Ovaries Testes

p.c. gonads cultured for 7 days in vitro and equivalent stage of development

Culture conditions

+ F.C.S. + F.C.S. -F.C.S. -F.C.S.

Stage of development Ovary (days) 19

reached at end of culture period

reached Testis Disorganized

Static

Developed

(day 14)

(day 17)

4

9

2

8

7

2

14 23

Necrotic explants have been omitted from the table: one male cultured with F.C.S., two males cultured without F.C.S., one female cultured without F.C.S.

Delicate processes of somatic cells could be seen extending between germ cells (Fig. 2a). Effects of serum on maintenance and further development of differentiated gona& The results are summarised in Table II. Eleven out of 16 explanted testes cultured in the presence of serum remained static or developed to the equivalent of day-17 controls (Fig. 3a,b,c). Four became disorganized, and 1 was necrotic. Nine out of 19 explanted testes cultured without serum remained static or developed to day-17 equivalents (Figs. 3d, 3e); 8 became disorganized, and 2 were necrotic. Control ovaries at day 19 p.c. were examined for comparison with cultured material. These controls showed flattened somatic cells arranged around oocytes to form primordial follicles, delimited by a basal lamina (Fig. 4a,b). All 14 explanted ovaries cultured in the presence of serum developed to the equivalent of day-19 control ovaries (Fig. 4c,d). Twenty three out of 24 ovaries

cultured without serum developed to day-19 equivalents (Figs. 4e,f), 1 was necrotic. In some cultures, oocytes at the periphery of the explant seemed to be escaping from their follicles; this was rarely seen in explants cultured in the presence of serum.

Discussion

Agelopoulou et al. (1984) cultured indifferent urogenital complexes of rats for 4 days and found that addition of 15% F.C.S. to the culture medium prevented the differentiation of testicular cords, although studies which used human serum had even reported the effect at concentrations as low as 0.5% (Chartrain et al., 1984). These workers reviewed comparable experiments using the mouse and concluded that either serum effects could not be recognized since complexes were not sexed at the time of explant removal (Byskov ‘and Sax&, 1976) or that the serum effect was expressed as a

Fig. 1. (a) Light micrograph of day-11.5 urogenital complex cultured to the equivalent of a day-17 control testis in the presence of F.C.S. Cords with germ cells (GC) are delimited by darkly staining flattened peritubular cells (arrows). Note presence of interstitial tissue between cords. Bar = 25 pm. (b) Day-12 explant cultured to a similar stage in the absence of F.C.S. Note similar features: tunica albuginea (TA), germ cells (GC) and flattened peritubular cells (arrows) around cords. Bar = 25 pm. (c) Electron micrograph of the same specimen as in Fig. la. Vesicles (arrows) in peritubular cells (P) adjacent to basal lamina (BL). Note pre-Sertoli cell (S). Bar = 0.5 pm. (d) Electron micrograph of a day-12 urogenital complex cultured to the equivalent of a day-15 control testis in the absence of F.C.S. Note vesicles (arrows) near basal lamina. Bar = 0.5 pm. (e) Basal lamina (BL), pre-Sertoli (S) and peritubular (P) cells seen in day-17 control testis. Note vesicle (arrow) in peritubular cell adjacent to basal lamina. Bar = 1 pm. (f) Detail of basal lamina from an explant cultured to day-17 equivalent with F.C.S. In this, junctions (arrows) have formed between pre-Sertoli cells. Bar = 0.5 pm. (g) Detail of tubule base from explant cultured to the equivalent of day-17 control testis without F.C.S. Notice basal lamina (arrow) between pre-Sertoli (S) and peritubular (P) cells. Bar = 0.5 pm.

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Fig. 2. (a) Electron micrograph of control ovary on day 17 p.c. Notice processes of somatic cells (arrows) beginning to separate oocytes in ovigerous cords. Bar = 2 pm. (b) Detail from Fig. a to show developing basal lamina (arrow) at outer surface of somatic cell. Bar = 0.25 pm. (c) Day-11.5 female indifferent gonad cultured to the equivalent of a day-17 ovary in the presence of F.C.S.. Note oocytes (arrows) in ovigerous cords separated by somatic cells (S). Bar = 10 pm. (d) Germ cell from (c) showing synaptonemal complex (arrow). Bar = 1 pm. (e) Day-12 female indifferent gonad cultured to the equivalent of a day-17 ovary in the absence of F.C.S.. Note oocytes (arrows). Bar = 10 pm.

retarding factor (Asayama and Furusawa, 1961). In the present study, urogenital complexes were from fetuses sexed at the time of explant removal; testicular cord formation occurred in the presence of 10% F.C.S., substantiating our previous study (Mackay et al., 1986). We found that cultures achieved approximately 5 days of development for 7 days in culture; but this retarding effect, presumably due to the trauma of explantation, was seen equally in cultures with and without serum. Indeed, Taketo et al. (1986) found a critical period, when serum components were essential for survival of mouse germ cells and for testicular cord organization, extending until the developmental stage of day 12 of gestation was reached. Female germ cells were found to be serum-dependent for a further 2 days. However, they assessed cultures only by light microscopy of paraffin sections,

which does not permit as critical an analysis as the present ultrastructural study. Further, in the rat the presence of F.C.S. in the culture medium caused the disintegration of testicular cords in testes explanted on days 14 and 15 p.c., though at later stages (day 16) cords were resistant to this effect (Agelopoulou et al., 1984). In our second set of experiments, disorganisation did occur in some male explants, but both in the presence and in the absence of F.C.S., and in fact was seen more often in cultures without serum. Some of this effect may be due to the enzymic loosening step on removal of explants, although ovaries excised on day 14 p.c. developed equally well both with and without serum, with no signs of similar disruption. The problem of successful culture of older male explants requires further consideration.

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Fig. 3. (a) Light micrograph of control testis on day 17 p.c. Notice tunica albuginea (TA), testicular cords with central germ cells (GC), peritubular cells (arrows) and interstitial tissue with Leydig cells (L). Bar = 25 11m. (b) Testis explanted on day 14 p.c. and cultured to the equivalent of Fig. a in the presence of F.e.S. Notice tunica albuginea (TA), germ cells (GC) and peritubular cells (arrows). Bar = 25 jjm. (c) Electron micrograph of specimen seen in Fig. b. Note basal lamina (arrows) delimiting pre-Sertoli cells (8), and germ cell (Ge). Bar = 0.5 p.m. (d) Testis explanted on day 14 p.c. and cultured to the equivalent of 3(a) in the absence of F.C.S. Note tunica albuginea (TA), germ cells (GC) and peritubular cells (arrows). Bar = 25 I-'m. (e) Electron micrograph of specimen seen in Fig. (d). Note germ cell (GC) and basal lamina (arrow) supporting pre-Sertoli cell (S). Bar = 2 p.m.

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Fig. 4. (a) Control ovary on day of birth (day 19 p.c.). Notice oocytes (0) surrounded by follicular cells. Bar = 25 pm. (b) Electron micrograph of similar specimen to Fig. (a). Note oocyte (0) and surrounding follicular cells delimited by a basal lamina (arrow). Bar = 2 pm. (c) Ovary explanted on day 14 p.c. and cultured for 7 days in the presence of F.C.S. Note oocytes (0) surrounded by follicular cells. Bar = 25 pm. (d) Electron micrograph of specimen seen in Fig. c. Note basal lamina (arrows). Bar = 2 pm. (e) Ovary explanted on day 14 p.c. and cultured for 7 days in the absence of F.C.S. Some free oocytes are seen at edge of explant (arrows). Bar = 25 pm. (f) Electron micrograph of developing follicle from Fig. e. Note basal lamina (arrows). Bar = 2 pm.

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Investigations on the mechanism of the serum effect have suggested the involvement of alterations to basal lamina formation; for example, Magre (1985) concluded that, in the presence of serum, differentiating rat Sertoli cells were not polarised or delimited by a basal lamina except in rare cases. Similar studies had shown previously that serum prevented the morphogenesis of rat testis in vitro by impairing the aggregation of Sertoli cells into testicular cords (Chartrain et al., 1984). Pelliniemi et al. (1984) confirmed this inhibition of testicular cord formation by serum in rats; their controls cultured in the absence of serum developed cords consisting of fetal Sertoli cells surrounded by a basal lamina and some peritubular cells. Tung et al. (1984) suggested that rat Sertoli cells and peritubular myoid cells, in co-culture, interacted to form a basement membrane which neither could produce alone. Agelopoulou and Magre (1987) reported that laminin and fibronectin were present only in basement membranes of rat testes differentiating in vivo, whereas they were found both in basement membranes and among stromal cells when differentiation occurred in vitro. In the present study, basal laminae developed in explants of mouse gonads, cultured in the presence of serum. In day-14 ovaries that developed to the equivalent of day-19 controls in vitro, early follicular cells were supported on a basal lamina. Although in our earlier study (Mackay and Smith, 1986), we found little evidence of a basal lamina in the one male explant that reached a day-17 equivalent, in this larger study basal laminae were evident in all testes which developed to this stage. It is interesting that both pre-Sertoli and peritubular cells were also seen and both showed vesicles at their membranes adjacent to the basal lamina. It appears that, in both the rat and the mouse, the aggregation of Sertoli cells to form testicular cords (the first sign of testicular differentiation) is associated with the alignment of these cells on a basement membrane. Suarez-Quian et al. (1984) have shown that Sertoli cells in vitro most resembled their in vivo counterparts when plated on to type IV collagen plus laminin (constituents of basal laminae), displaying polarity and height. Furthermore, neonatal rat Sertoli cells grown in

reconstituted basement membrane gels have been shown to undergo morphogenesis into cords that provide an environment suitable for germ cell differentiation (Hadley et al., 1985). In the rat testis in vitro, the addition of serum may lead to disruption of normal basal lamina formation and consequent inhibition of testicular cords. However, Grund et al. (1986) have shown that the reaggregation of separated cells from neonatal rat testis cultured in vitro resembles the differentiation of embryonic male gonads, with fetal bovine serum necessary for proper testicular cord differentiation. The inhibitory effect of serum in the rat appears to be a transitory one. At the time of entry to the gonadal anlage, primordial germ cells are thought to undergo a major change in phenotype, after which they show little migratory activity and are no longer invasive on cellular substrates (Donovan et al., 1986). In our second set of experiments, some peripherally placed oocytes, mainly in ovaries cultured without F.C.S., reverted to an apparent migratory phenotype (Fig. 4e). Similar motile germ cells have been reported in cultures of day-16 mouse ovaries (Blandau et al., 1963). Clearly, the control of germ cell phenotype by its microenvironment (involving, for example, possible interactions between factors in F.C.S. and constituents of the extracellular matrix) warrants further study. Since in our experiments both male and female cultures differentiate equally well in the presence and in the absence of F.C.S., we propose that the mouse is a more favourable species for future studies and indeed may be preferable, as cultures benefit from growth effects of serum without the limitations of its detrimental effects upon differentiation.

Acknowledgements

We thank Professor R.J. Scothorne and Miss S.J. Tavendale for their critical reading of the manuscript. Miss C. Mackenzie assisted with some of the initial experimental procedures and Miss M. Hughes with photography, for which we are most grateful. An equipment grant from the Uni-

LO

versity of Glasgow Medical Research gratefully acknowledged.

Fund is

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