Mapping of genes for inhibin subunits α, βA, and βB on human and mouse chromosomes and studies of jsd mice

CENOMICS

5,91-99

(1989)

Mapping of Genes for lnhibin Subunits CY,,&, and & on Human and Mouse Chromosomes and Studies of jsd Mice D. E. BARTON,*,’ *Department

T. L. YANG-FENG,*

A. J. MASON,t

P. H. SEEBuRG,t AND U. FRANCKE*~~

of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, and tDepartment Developmental Biology, Genentech Inc., South San Francisco, California 94080 Received

January

24, 1989;

revised

March

of

6, 1989

for this substance. However, it was not until a reliable bioassay for the suppression of basal pituitary FSH secretion had been developed (Vale et al., 1972) that the true nature of inhibin began to emerge. Inhibin exists in two forms (A and B), each a dimer containing the same 18-kDA a! subunit and one of two j3 subunits, PA or @a(Ling et al., 1985). Inhibins A and B appear to have very similar biological activities. An additional activity, observed during the purification of inhibin from porcine follicular fluid, was that of stimulating FSH production in the pituitary cell bioassay. This substance, termed actiuin (Burger et al., 1988), consists of dimers of the p subunits of inhibin, either /3&A or PA& (Vale et al., 1986; Ling et al., 1986). Cloning and sequencing of cDNAs for the three subunits of inhibin revealed these polypeptides to be highly sequence-conserved and structurally related to transforming growth factor b (TGFB) (Mason et al., 1985). The inhibin genes are expressed in many tissues, including placenta (Mayo et al., 1986), but the ratio of inhibin a to @ mRNA differs widely and may determine the relative levels of inhibin and activin produced (Meunier et al., 1988). The widespread synthesis of inhibin and activin suggeststhat these proteins may have diverse functions in different tissues. Thus, recent work has shown that PAdimers also act as an erythroid differentiation factor (Yu et al., 1987; Murata et al., 1988). We have used the cloned human cDNAs for inhibins a, @A,and /3s (Mason et al., 1986) to determine the chromosomal location of these genes in the human and murine species. The inhibin CYand pa genes were mapped to human chromosome 2, regions q33 --f qter and ten + 913, respectively, and to mouse chromosome 1. The locus for inhibin DAwas mapped to human chromosome 7, region ~15 --* ~14, and to mouse chromosome 13. The inhibin genes are, therefore, widely dispersed, as are other members of the TGF/3 family (Barton et al., 1988). The mapping of the genesfor inhibin a and /3achains to human chromosome 2q and mouse chromosome 1 led US to examine the integrity of these genes in mice

Inhibin (INH) is a gonadal glycoprotein hormone that regulates pituitary FSH secretion and may also play a role in the regulation of androgen biosynthesis. There are two forms of inhibin that strongly inhibit pituitary FSH secretion. These share the same a subunit that is covalently linked to one of two distinct fi subunits (BA or @a). However, dimers of two fl subunits are potent stimulators of FSH synthesis and release in vitro. The /3 subunits share extensive sequence similarity with transforming growth factor 8. Recently isolated cDNAs for all three inhibin subunits have been used to map their cognate loci on human and mouse chromosomes by Southern blot analysis of somatic cell hybrid DNAs and by in situ hybridization. INHa and INH& genes were assigned to human chromosome 2, regions q33 + qter and ten + 913, respectively, and to mouse chromosome 1. The INH@* locus was mapped to human.chromosome 7~16 + p14 and mouse chromosome 13. The region of mouse chromosome 1 that carries other genes known to have homologs on human chromosome 2q includes the jsd locus (for juvenile spermatogonial depletion). Adult jsd/jsd mice have elevated levels of serum FSH and their testes are devoid of spermatogonial cells. The possibility that the mutation in jsd involves the INHa or INH& gene was investigated by Southern blotting of DNA from jsdf jsd mice, and no major deletions or rearrangements were detected. o io8s Academic PWW, lno.

INTRODUCTION

The idea that the gonads produce a water-soluble substance that can act on the pituitary gland to suppress gonadotropin production was first put forward in 1932 by McCullagh, who proposed the name inhibin 1 Present address: Department of Pathology, Cambridge University, Cambridge, UK. ’ To whom correspondence should be addressed at Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA 94305-5425. 91

o&XL7543/&x9

Copyright All

rights

0

1989 of reproduction

hy

$3.00

Academic

Press, Inc.

in any form

reserved.

92

BARTON

carrying the jsd (juvenile spermatogonial depletion) mutation which maps to mouse chromosome 1 in a region of conserved synteny with human chromosome 2q. Homozygous jscZ/jsd male mice develop hypoplastic testes, azoospermia, and elevated levels of serum FSH (Beamer et aZ., 1988). Our studies have revealed no deletions or rearrangements in the inhibin (Y or pa genes in jsd homozygotes, but we have not ruled out the possibility that jsd results from a point mutation. MATERIALS

AND

METHODS

Probes The probes for inhibins a, PA, and Ps used for Southern blot analysis and in situ hybridization were gel-purified fragments, 1198,1251, and 1100 bp in size, derived from human inhibin cDNA clones (Mason et al., 1986). For Southern blot analysis, 20-30 ng of each fragment was labeled with 32P by random oligomer priming (Feinberg and Vogelstein, 1983). Somatic Cell Hybrids Chromosomal assignments in the human were carried out with a total of 19 hybrid clones derived from nine independent fusion experiments between rodent cell lines and human diploid fibroblasts or lymphocytes. The origin and characterization of the hybrids used for primary assignments have recently been summarized (Francke et al, 1986). The hybrids used for regional mapping on chromosome 2 have also been described (Francke, 1975; Alonso et al, 1988). To map the genes in the mouse, 18 Chinese hamster X mouse hybrid clones or subclones derived from four different series of hybrids (Francke et al., 1977; Francke and Taggart, 1979) and one mouse X rat hybrid (Joyner et al., 1985) were used. Methods DNA extraction and Southern blotting were carried out as summarized by Barton et al. (1987). Chromosome preparations from synchronized blood lymphocytes, in situ hybridization of tritium-labeled probe, and chromosome banding were carried out as described by Harper and Saunders (1981) with modifications (Francke et al., 1986). Mice Mice carrying the jsd mutation on a C57BL/J6 background were bred by Dr. W. Beamer at The Jackson Laboratory, Bar Harbor, Maine. The jsd/jsd homozygotes were identified (Beamer et al., 1988) and tissues were removed for DNA extraction. Normal C57BL/J6 mice and unaffected littermates, (+/+) or (jsd/+), were used as controls.

ET

AL.

RESULTS

Mapping Inhibin Subunit Genes in Man Inhibin a (INHA) was initially assigned to human chromosome 2 by analysis of EcoRI-digested DNA from 12 hybrids. A single 9-kb EcoRI fragment was detected in human DNA and in all hybrids that had retained human chromosome 2 (Table 1A). Confirmation of this result and regional localization was achieved by analysis of six additional hybrids, three of which contained rearrangements of human chromosome 2 (Fig. 1). A 12.5-kb BglII fragment was detected in human DNA and in hybrids containing region 2q13 + qter (Fig. 1, lane 4) or region 2q33 + qter in the presence (lane 8) or absence (lane 9) of 2p, indicating that INHA lies on the distal long arm (region 2q33 + qter). The same primary panel was used to map an 8.2-kb EcoRI fragment containing the major part of the inhibin PA gene (INHBA) to chromosome 7 (Fig. 2; Table 1B). Since the majority of hybrids used contained translocation-derived partial human X chromosomes, they had to be excluded from the analysis in Table 1B. In the five informative hybrids left, the INHBA signal was concordant with the human X chromosome. The excluded hybrids, however, exhibited discordancies for each segment of the X chromosome (data not shown). A 15-kb Hind111 fragment encoding the inhibin Pa gene (INHBB) was mapped to chromosome 2 (Table 1C). Regional assignment of INHBB on chromosome 2 was achieved by analysis of two hybrid lines containing rearrangements of chromosome 2 (Fig. 3). The 15-kb human HindIII fragment detected by the inhibin /3sprobe was present in a hybrid containing the entire long arm of chromosome 2 (Fig. 3, lane 10) but absent from a hybrid that contained only region 2q13 + qter (lane 6), placing the INHBB locus in the region 2cen -+ q13. These data and the data for inhibin a are presented diagramatically in Fig. 4. The inhibin PA gene was more precisely mapped on chromosome 7 by in situ hybridization. Analysis of 77 metaphase spreads from a normal 46,XY male revealed 106 grains on chromosomes, 24 of which were over 7p with a peak at 7~15 + p14 (Fig. 5). Sixteen (15%) of all labeled sites were at this specific region and the rest were randomly distributed over all chromosomes. Only one grain was present on the X chromosome. We therefore assign INHBA to chromosome 7, region p15 + p14. Mapping Inhibin Genes in Mouse A well-characterized hybrid cell panel, consisting of 11 mouse X Chinese hamster and one mouse X rat line that both segregate mouse chromosomes, was used to map the locus for the inhibin (Ysubunit in the mouse. The human inhibin a probe detected a 13-kb EcoRI

MAPPING

OF

INHIBIN

SUBUNIT

TABLE Correlation

of Human

Inhibin

Sequences

with

Human

1

Chromosomes

in Human

Human Hybridixation/ chromosome

1

2

3

4

5

6

7

8

9

A. Inhibin Concordant hybrids Discordant hybrids

+/+ -/+/-/+

Discordant hybrids Informative hybrids

Discordant

+/+ -/+/-/+

Discordant hybrids Informative hybrids

Discordant Discordant hybrids Informative hybrids

11

Somatic

Cell Hybrids

chromosome 12

13

14

15

16

17

18

19

20

21

22

X

cy

4

0

7

6

8

5

7

10

6

8

6

6

9

8

11

8

7

10

9

10

7

10

6

16

14

16

15

17

17

16

18

17

18

16

18

18

16

18

17

18

17

18

18

16

16

9

3

2

3

2

2

2

2

12

12

101101 5 7 3 4 3 1

2 3 6

4 2 2

@A

22211122301221133 6384675543337246332 3 302232 2 504112344541542650

114

00

65945806344665684764750 12

12

12

9

11

12

10

12

11

C. Inhibin Concordant

10

X Rodent

3431522314324322331331 3 9 11 5 6 8 7 7 6896974479467632 1 0 0 3 5 044352342344335223 30733536334356843765583

B. Inhibin Concordant

93

GENES

+/+ -/+/-/+

12

10

10

12

11

12

11

12

11

11

11

5

5

11

6

9

8

6

11

6

9

3

16

18

17

18

17

18

18

17

17

8

@s

5 4452524313346340551542 8 11 4 68686895865479476643 10114142253320326115111 3 0 8 3 3 626335465843756582 4

0

9

4

7

7

17

15

17

15

17

18

6858878 16

18

16

18

16

18

Note. The numbers of hybrids that are concordant (+/+ or -/-) and discordant given for each chromosome. Hybrids in which a particular chromosome was present were excluded from the analysis.

fragment in mouse DNA (Fig. 6, lane 2) and in all hybrids containing mouse chromosome 1 (Fig. 6, lanes 3 and 4; Table 2A). Extended panels of 18 and 19 mouse X Chinese hamster hybrids were used to map inhibins @Aand @ain the mouse (Fig. 7). In P&I-digested mouse DNA, the inhibin /3~probe detected a 2.2-kb fragment that was present in all hybrids containing mouse chromosome 13 (Fig. 7; Table 2B), including the rat X mouse hybrid RTM9 that has retained a single mouse chromosome Rb(l1; 13) that consists of intact chromosomes 11 and 13 fused at the centromeres (Joyner et al., 1985; Miinke and Francke, 1987). In the same restriction digests, the inhibin @aprobe detected a 3.1kb fragment which co-segregated with mouse chromosome 1 (Fig. 7; Table 2C). Thus the (Yand ,& subunits, both of which map to the long arm of human chromosome 2, are encoded by mouse chromosome 1,

18

(+/and -/+) with the human inhibin sequences are in 10% or less of the cells or was structurally rearranged

while the gene for the /3~subunit, which maps to human chromosome 7p, is assigned to mouse chromosome 13.

Structure

of Inhibin

Genes in jsd Mutation

Several genes on the distal long arm of human chromosome 2 have homologous loci on the proximal half of mouse chromosome 1. Since inhibin (Y(and possibly inhibin pa) may also belong to this conserved syntenic group, other loci in this region were considered with regard to the possibility that any of them could be an inhibin locus. One locus, jsd, was identified as a candidate (Beamer et al., 1988). Male mice homozygous for this mutation develop elevated levels of serum FSH between 4 and 20 weeks of age (as would be expected in the case of an inhibin deficiency) and become sterile due to a disruption of spermatogenesis.

94

BARTON

ET

AL.

2

1

123456789

3

4

5

6

7

8-9

10

_

12.5 kb-

FIG. 1. Southern analysis of &W-digested DNA from somatic cell hybrids and controls with inhibin a cDNA probe. Lane 1, Chinese hamster (CH); lane 2, rat (R); lane 3, human (H); lane 4, CH X H hybrid containing region 2q13 + qter; lanes 5 and 6, CH X H hybrids with an intact human chromosome 2; lane 7, CH X H hybrid lacking chromosome 2; lane 8, R X H hybrid .containing 2p and 2q33 + qter; lane 9, R X H hybrid containing region 2q33 + qter only. The combined results assign the 12.5-kb fragment containing the ZNHA locus to region 2q33 + qter.

FIG. 3. Regional mapping of inhibin 0s on HSA2 by Southern analysis of HindIII-digested DNAs. Lane 1, 3T3 mouse (M); lane 2, Chinese hamster (CH); lane 3, rat; lane 4, human (H); lane 5, M X H hybrid containing HSAB; lane 6, CH X H hybrid containing HSA2q13 -t qter; lanes 7 and 8, CH X H hybrids containing HSAP; lane 9, CH X H hybrid lacking HSAP; lane 10, CH X H hybrid containing the entire long arm of HSA2. Hybrids in lanes 5, 7, 8, and 10 are positive for the human 15-kb fragment. The combined results assign the ZNHBB locus to region 2cen + q13. +

+

NT

NT

-

+

+ INHa NT lNHP8

1

signal

25

Testes and other organs were obtained from jsd/jsd mice and their phenotypically normal littermates. Their DNA was examined for deletions or rearrangements in the inhibin (Y and fin genes. As shown in Fig. 8, no differences were observed on Southern blots of BgZII-, P&I-, or HindIII-digested jsd/jsd and control DNA with either the inhibin CY(Fig. 8A) or the inhibin 6s (Fig. 8B) cDNA probe. Attempts at Northern blot analysis were hampered by the very low levels of the inhibin transcripts found even in normal testes. We

24 23 22

P

21 16 15 14 13 12

11.2

INHBB

12

1

8.2 kb-

2

3

4

5

6

“1 i : ‘; ii ^( >+“^ / ,” ,,I

7

8

9

10

11

12

----

a---

::.I 14.2 14.3

13

-l

21

I

22 4

23 24 31

32

----

i

FIG. 2. Southern analysis of EcoRI-digested DNA from somatic cell hybrids and controls with the inhibin fla cDNA. Lane 1, Chinese hamster (CH); lane 2, human (H); lanes 3-13, CH X H hybrids. The hybrids in lanes 8 and 9 contain human chromosome 7 and are scored positive for the human-specific 8.2-kb fragment. The hybrid cell line in lane 11 retains chromosome 7 in only 10% of cells and generated a weak signal at 6.2 kb. This hybrid was excluded from Table 1. Since the smaller human fragment (lane 2) corn&rated with the larger CH fragment, it could not be scored reliably in the hybrid lanes. Comparison of relative fragment intensities in lanes 9 and 10 suggests that the smaller human fragment is present in lane 9 but not in lane 10, concordantly with the major 8.2-kb human fragment and with human chromosome 7.

-

33 34 35 36 37

--

2

---_I-,

FIG. 4. Schematic representation of regional mapping presented in Figs. 1 and 3. The solid bars represent portions of chromosome 2 present in four hybrid lines. The presence absence (--) of hybridization with the inhibin (Y and 0s cDNA and their regional assignments are indicated. NT, not tested.

results human (+) or probes

MAPPING

22

15

INHIBIN

n

21

P

OF

a.

l ooooe l ooooooooo

H

14

0.0

13 12

l

eo

‘1::: a 11.2

21

0 Y

q

10

2’21

32

3”: 35

36 7 FIG. 6. In situ hybridization of ‘H-labeled inhibin @A cDNA to human chromosome 7. Each dot represents a separate site labeled by autoradiographic silver grains. In 77 cells, 19 of all 106 grains (18%) were clustered at region 7~15 --* ~13.

conclude that the jsd mutation is not caused by a major deletion or rearrangement in either the inhibin cr or the inhibin /3n gene. DISCUSSION

The techniques of somatic cell hybridization, Southern blotting, and in situ hybridization have been used to map the loci for the three subunits of inhibin: INHA (a) to human (HSA) chromosome 2q33 + qter/ mouse (MMU) chromosome 1, INHBA (@A) to HSA7p15 --f p14/MMU13, and INHBB (&) to HSA2cen + ql3/MMUl. Apart from their function in inhibiting FSH production in pituitary cells, it is now clear that inhibin (a@ dimers) and activin (BP dimers) have wide-ranging roles as regulators of growth and differentiation (Yu et al., 1987; Meunier et al., 1988). When examining the human gene map, we note that none of the inhibin genes is syntenic with the locus for any other member of the TGFP family of growth factors. The gene for TGFPl (TGFB) has been mapped to chromosome 19, region q13.1 + q13.3 (Fujii et al.,

SUBUNIT

GENES

95

1986), that for TGF/32 (TGFBB) to chromosome band lq41, that for TGF/33 (TGFBS) to chromosome band 14q24 (Barton et al., 1988), and that for anti-Miillerian hormone (AMH) to chromosome 19p13.2 --* ~13.3 (Cohen-Haguenauer et al., 1987). This wide dispersal is not surprising in such an ancient family of genes, whose members include the decapentaplegic gene in Drosophila (Padgett et al., 1987) and the Vgl gene in Xenopus (Weeks and Melton, 1987), and may indeed have allowed the individual members of the family to develop their diverse tissue-specific functions, removed from the constraints of a coordinately regulated gene cluster. Widely dispersed members of ancient, highly conserved gene families provide excellent material for interspecies comparative mapping studies, and the inhibins are no exception. INHBA (HSA7p15 + p14/ MMU13) joins TCRG, the T-cell receptor y-chain locus, mapped to HSA7p15 and MMUl3 (Murre et al., 1985; Kranz et al., 1985). An interesting developmental gene that causesdominant mutations in humans (Greig cephalopolysyndactyly syndrome) was mapped to 7~13 by chromosomal rearrangements and by linkage studies (Brueton et al., 1988). It may be a homolog of the mouse mutation Xt (extra toes) on MMU13 for which many allelic forms have been described (Lyon et al., 1967; Johnson, 1969). Interestingly, the Hl histone gene cluster, HlFl, mapped to HSA7q22 or 7q32 + q36 (Chandler et al., 1979), has also been mapped to MMU13 (Graves et al., 1985). While no gene except INHBB has been mapped to proximal HSA2q and MMUl, the INHA locus on distal HSA2q and MMUl joins a group of seven other genes that all have closely linked homologs in the proximal half of MMUl. In addition, a region of conserved synteny exists between HSA2q and MMU2 (Table 3). Comparative mapping data can also suggest previously described mutant phenotypes as potential candidates that could result from lesions in the gene being

FIG. 6. Mapping of inhibin (Y in the mouse by Southern analysis of Chinese hamster (CH) (lane l), mouse (M) (lane 2), and CH X M hybrid cell DNAs (lanes 3-6) digested with EcoRI. The mousespecific 13-kb fragment is present in two hybrids that contain mouse chromosome 1 (lanes 3 and 4) and absent in a hybrid lacking this chromosome (lane 6).

96

BARTON

ET

TABLE Correlation

of Mouse

Inhibin

Sequences

with

Mouse

AL.

2

Chromosomes

in Mouse

Human Hybridization/ chromosome

12

34

5

6

7

A. Inhibin Concordant

hybrids

Discordant

hybrids

Discordant Informative

hybrids hybrids

+/+ -/-

65 63

212343240413434244 345424555434214443

+/-/+

01 03

444213426243222321 321142011221452223

04

12

+I+ -/+/-/+

Discordant Discordant Informative

hybrids hybrids

Discordant Discordant Informative

hybrids hybrids

chromosome

8

9

10

11

12

13

14

15

16

17

18

19

X

12

11

12

10

11

12

11

12

12

12

10

11

12

11

12

11

12

11

9

11

9

14

8

12

8

5

4

6

9

6

2

7

11

6

5

7

3

7

0

9

10

11

9

5

18

17

17

18

18

16

18

18

17

18

16

8 17

14 17

6

3

4

0

7

3

5

7

5

8

5

8

6

5

10

3

6

5

3

4

2

3

2

2

6

11 19

6 19

8 17

7 18

9 19

11 18

6 19

5 17

5 18

7 17

flA

23320233111341323231 54

98

865

21123211333103121113 710

5556852406069984

911 16 18

+/+ -/-+-I-/+

Cell Hybrids

765355437464674544

12

67 18 17

8

8

16

16

9 17

C. Inhibin Concordant

Somatic

(Y

B. Inhibin Concordant

X Rodent

10

9

74

5

5

3

7

@s 7

566646788666325754

015462247 05432253111322674235 069784778

17

19

Note. The numbers of hybrids that are concordant for each chromosome. Hybrids in which a particular were excluded from the analysis.

19

18

17

(+/+ or -/-) chromosome

17

18

19

18

18

and discordant (+/and -/+) with the mouse inhibin sequences are given was present in fewer than 10% of the cells or was structurally rearranged

mapped. The jsd locus is 4 + 2 CM proximal of Idh-1 on MMUl. jsd is a recessive mutation that is characterized in homozygous male mice by small testes and azoospermia. In serum, testosterone and LH levels are normal but FSH levels are elevated (Beamer et al., 1988).

123456 123456 kb

.23 -12

44.

kb

FIG. 7. Southern filter of P&I-digested Chinese hamster (CH) (lane 1). mouse (M) (lane 2), and CH X M hybrid cell DNAs (lanes 3-10) hybridized simultaneously with @A and 0s probes. The 3.1-kb mouse fragment (8s) is present in lanes 4,5,6, and 10 and absent in all the others, concordantly with mouse chromosome 1. The 2.2kb mouse fragment (DA) is present only in the hybrid in lane 9, which is the only one in this figure that contains mouse chromosome 13. This fragment was also present in the rat X mouse hybrid RTM9 (not shown).

FIG. 8. Genomic analysis of (A) inhibin a and (B) inhibin 0s in jsd mice. DNA from a jsd/jsd mouse (lanes 2,4, and 8) and DNA from a normal littermate (lanes 1, 3, and 5) were digested with BglII (lanes 1 and 2), PstI (lanes 3 and 4), and HindI11 (lanes 5 and 6). No differences in fragment sizes or intensities were observed.

MAPPING

OF INHIBIN

SUBUNIT

97

GENES

TABLE 3 The Map Locations in the Mouse of Loci on Human Chromosome 2q Human chromosome region

Human locus symbol

2q12+q21 2q134q21 2q32+qter 2q32+qter 2q32+qter 2q32-tqter 2q33+q35 2q34+q36 2q35+q36 2q36+q37 2q33-rqter 2cen+q13

ILlA ILlB ACHRD ACHRG IDHl MYLl CRYGl FNl VL GCG INHA INHBB

Gene name

Mouse locus symbol

Mouse chromosome region

Interleukin-1, alpha Interleukin-2, beta Acetylcholine receptor, delta peptide Acetylcholine receptor, gamma peptide Isocitrate dehydrogenase 1, soluble Myosin light polypeptide 1, alkali; skeletal, fast Crystallin, gamma polypeptide 1 Fibronectin 1 Villin Glucagon Inhibin, (Ysubunit Inhibin, @ssubunit

Il-la Il-lb Acrd Acrg Idh-1 Mylf cwg-1 Fn-1 Vi1 Gcg Inha Inhbb

2 2 1 proximal 1 proximal 1 proximal 1 proximal 1 proximal 1 proximal 1 proximal 2

Serial histologic studies of testes in jsd/jsd mice between 3 and 10 weeks of age have provided evidence for an early wave of spermatogenesis. However, spermatogonial cells are not replaced by mitotic divisions and only Sertoli cells remain in the adult testicular tubules. Female jsd/jsd mice are morphologically and functionally normal, and their reproductive life span is not reduced (Beamer et aZ., 1988). These findings and the position of the jsd locus within the HSABq/ MMUl conserved syntenic group of loci suggestedthat the jsd locus might encode inhibin (Yor inhibin 0s. The loss of inhibin production could lead to a lack of inhibition of FSH secretion, which could result in overstimulation of spermatogonial cell division and early depletion of spermatogonial stem cells, a scenario resembling the course of events in jsd/jsd males. The sexual dimorphism in the phenotypic effects of this mutation suggests that the /3 subunit gene is the more likely candidate, since expression of Pa is higher than that ofpA in the rat testis and the reverse of this situation exists in the ovary (Meunier et al, 1988). To test this hypothesis, we have examined the inhibin LYand /3sgenes in jsd mice by Southern blot analysis. While the data presented demonstrate that the inhibin (Yand @sgenes are not involved in major deletions or rearrangements in jsd/jsd mice, lesions of this kind are known to be the exception rather than the rule as causesof mutations. The possibility that a point mutation in an inhibin gene is responsible for the jsd phenotype will be investigated by further studies. ACKNOWLEDGMENTS We are grateful to Dr. Wes Beamer, supported by NIH Grant DK17947, for his generosity in providing mouse tissues and helpful comments on the manuscript. This work was supported by NIH Research Grant GM26105 (to U.F.).

Refs. (47,9) (47,9) (37,18) (37, 18) (11, 36, 20) (39) (42,43) (1% 44) (40) (41,24) This report This report

REFERENCES 1. ALONSO, M. A., BARTON, D. E., AND FRANCKE, U. (1988). Assignment of the T-cell differentiation gene MAL to human chromosome 2, region ten -+ q13. Immunogenetics 27: 91-95. 2. BARTON, D. E., ARQUINT, M., RODER, J., DUNN, R., AND FRANCKE, U. (1987). The myelin-associated glycoprotein gene: Mapping to human chromosome 19 and mouse chromosome 7 and expression in quivering mice. Genomics 1: 107-112. 3. BARTON, D. E., FOELLMER, B. E., Du, J., TAMM, J., DERYNCK, R., AND FRANCKE, U. (1988). Chromosomal mapping of genes for transforming growth factors 82 and 83 in man and mouse: Dispersion of TGF-p gene family. Oncogene Res. 3: 323-331. 4. BEAMER, W. G., CUNLIFFE-BEAMER, T. L., SHUL~, K. L., LANGLEY, S. H., AND RODERICK, T. H. (1988). Juvenile spermatogonial depletion (jsd): A genetic defect of germ cell proliferation of male mice. Biol. Reprod. 38: 899-908. 5. BRUEMN, L., HUSON, S. M., WINTER, R. M., AND WILLIAMSON, R. (1988). Chromosomal localisation of a developmental gene in man: Direct DNA analysis demonstrates that Greig cephalopolysyndactyly maps to 7~13. Amer. J. Med. Genet. 31: 799804. 6. BURGER, H. G., IGARASHI, M., BAIRD, D., MASON, T., BARDIN, W., MCLACHLAN, R., CHAPPEL, S., MIYAMOTO, K., DE JONG, F., MOUDGAL, A., DEMOULIN, A., NIESCHLAG, E., DE KRETSER, D., ROBERTSON, D., FINDLAY, J., SASAMOTO, S., FORAGE, R., SCHWARTZ, N., FUKUDA, M., STEINBERGER, A., HASEGAWA, Y., TANABE, K., LING, N., AND YING, S.-Y. (1988). Inhibin: Definition and nomenclature, including related substances. J. Clin. Endocrirwl.

Metab.

66: 885-886.

7. CHANDLER, M. E., KEDES, L. H., COHN, R. H., AND YUNIS, J. J. (1979). Genes coding for histone proteins in man are located on the distal end of the long arm of chromosome 7. Science 205: 908-910. 8. COHEN-HAGUENAUER, O., PICARD, J. Y., MATTEI, M.-G., SERERO, S., VAN CONG, N., DE TAND, M. F., GUERRIER, D., HORSCAYLA, M. C., JOSSO, N., AND FREZAL, J. (1987). Mapping of the gene for anti-MtilIerian hormone to the short arm of human chromosome 19. Cytogenet. Cell Genet. 44: 2-6. 9. D’EUSTACHIO, P., JADIDI, S., FUHLEIRIGGE,R. C., GRAY, P. W., AND CHAPLIN, D. D. (1987). Interleukin-1 alpha and beta genes: Linkage on chromosome 2 in the mouse. Immunogenetics 26: 339-343.

BARTON

98

10. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. B&hem. 132: 6-13. 11. FRANCKE, U. (1975). Regional localization of the human genes for malate dehydrogenase-1 and isocitrate dehydrogenase-1 on chromosome 2 by interspecific hybridization using human cells with the balanced reciprocal translocation t(1;2)(q32;q13). Cytogewt. CellGem% 14:308-312. 12. FRANCKE, U., AND TAGGART, R. T. (1979). Assignment of the gene for cytoplasmic superoxide dismutase (Sod-l) to a region of chromosome 16 and of Hprt to a region of the X chromosome in the mouse. Proc. Natl. Acad Sci. USA 76: 5230-5233. 13. FRANCKE, U., LALLEY, P. A., Moss, W., IVY, J., AND MINNA, J. D. (1977). Gene mapping in Mus musculus by interspecific cell hybridization: Assignment of the genes for tripeptidese-1 to chromosome 10, dipeptidese-2 to chromosome 18, acid phosphatase-1 to chromosome 12 and adenylate kinase-1 to chromosome 2. Cytogenet. Cell Genet. 19: 57-84. 14. FRANCKE, U., YANG-FENG, T. L., BRISSENDEN, J. E., AND ULLRICH, A. (1986). Chromosomal mapping of genes involved in growth control. Cold Spring Harbor Symp. Quant. Biol. 61: 855866. 15. FUJII, D., BRISSENDEN, J. E., DERYNCK, R., AND FRANCKE, U. (1986). Transforming growth factor p gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somat. CellMoLGenet. 12:281-288. 16. GRAVES, A., WELLMAN, S. E., CHIU, I.-M., AND MARZLUFF, W. F. (1985). Differential expression of two clusters of mouse histone genes. J. Mol. Biol. 183: 179-194. 17. HARPER, M. E., AND SAUNDERS, G. F. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. 18. HEIDMANN, O., BUONANNO, A., GEOFFROY, B., ROBERT, B., GUENET, J-L., MERILE, J. P., AND CHANGEUX, J-P. (1986). Chromosomal localization of muscle nicotinic acetylcholine receptor genes in the mouse. Science 234: 866-868. 19. HENRY, I., JEANPIERRE, M., BERNARD, M., WEIL, D., GREZESCHIK, K. H., RAMIREZ, F., CHU, M. L., AND JUNIEN, C. (1985). The structural gene for fibronectin (FN) maps to 2p323-qter. Cytogenet.

Cell Genet.

40: 650.

20. HUTTON, J. J., AND RODERICK, T. H. (1970). Linkage analyses using biochemical variants in mice. III. Linkage relationships of eleven biochemical markers. B&hem. Genet. 4: 339. 21. JOHNSON, D. R. (1969). Brachyphalangy, an allele of extra-toes in the mouse. Genet. Res. 13: 275-280. 22. JOYNER, A. L., LEBO, R. V., KAN, Y. W., TJIAN, R., Cox, D. R., AND MARTIN, G. R. (1985). Comparative chromosome mapping of a conserved homoeobox region in mouse and human. Nature (London) 314: 173-175. 23. KRANZ, D. M., SAITO, H., DISTECHE, C. M., SWISSHELM, K., PRAVTCHEVA, D., RUDDLE, F. H., EISEN, H. N., AND TONEGAWA, S. (1985). Chromosomal locations of the murine T-cell receptor alpha-chain gene and the T-cell gamma gene. Science 227: 941-944. 24. LALLEY, P. A., SAKAGUCHI, A. Y., EDDY, R. L., HONEY, N. H., BELL, G. I., SHEN, L.-P., RUTTER, W. J., JACOBS, J. W., HEINRICH, G., CHIN, W. W., AND NAYLOR, S. L. (1987). Mapping polypeptide hormone genes in the mouse: Somatostatin, glucagon, calcitonin and parathyroid hormone. Cytogenet. Cell Genet. 44: 92-97. 25. LING, N., YING, S-Y., UENO, N., ESCH, F., DENOROY, L., AND GUILLEMIN, R. (1985). Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc. Natl. Acad. Sci. USA 82: 7217-7221. 26. LING, N., YING, S-Y., UENO, N., SHIMASAKI, S., ESCH, F., HOTTA, M., AND GUILLEMIN, R. (1986). Pituitary FSH is re-

ET AL.

27.

28.

29.

30.

leased by a heterodimer of the @-subunits from the two forms of inhibin. Nature (London) 321: 779-782. LYON, M. F., MORRIS, T., SEARLE, A. G., AND BUTLER, J. (1967). Occurrences and linkage relations of the mutant “extra-toes” in the mouse. Genet. Res. 9: 383-385. MASON, A. J., HAYFLICK, J. S., LING, N., ESCH, F., UENO, N., YING, S-Y., GUILLEMIN, R., NIALL, H., AND SEEBURG, P. H. (1985). Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-p. Nature (London) 318: 659-663. MASON, A. J., NIALL, H. D., AND SEEBURG, P. H. (1986). &NCture of two human ovarian inhibins. B&hem. Biophys. Res. Commun. 135:957-964. MAYO, K. E., CERELLI, G. M., SPIESS,J., RIVIER, J., ROSENFELD, M. G., EVANS, R. M., AND VALE, W. (1986). Inhibin A-subunit cDNAs from porcine ovary and human placenta. Proc. Natl. Acad.

Sci. USA

83:

5849-5853.

31. MCCULLAGH, D. R. (1932). Dual endocrine activity of the testes. Science 76: 19-20. 32. MEUNIER, H., RIVIER, C., EVANS, R. M., AND VALE, W. (1988). Gonadal and extragonadal expression of inhibin o(,PA, and j3B subunits in various tissues predicts diverse functions. Proc. Natl. Acad

Sci. USA

86:

247-251.

33. MUNKE, M., AND FRANCKE, U. (1987). The physical map of Mus musculus chromosome 11 reveals evolutionary relationships with different syntenic groups of genes in Homo sapiens. J. Mol. Euol. 25: 134-140. 34. MURATA, M., ETO, Y., SHIBAI, H., SAKAI, M., AND MURAMATSU, M. (1988). Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin PA chain. Proc. Natl. Acad. Sci. USA 86:

2434-2438.

35. MURRE, C., WALDMANN, R. A., MORTON, C. C., BONGIOVANNI, K. F., WALDMANN, T. A., SHOWS, T. B., AND SEIDMAN, J. G. (1985). Human y-chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7. Nature (Lendon) 316:549-552. 36. NARAHARA, K., KIMURA, S., KIDDAVA, Y., TAKAHASHI, Y., WAKITA, Y., KASAI, R., NAGAI, S., NISHIBAYSHI, Y., AND KIMOTO, H. (1985). Probable assignment of soluble isocitrate dehydrogenase (IDHl) to 2q33.3. Hum. Genet. 71: 37-40. 37. NEF, P., MAURON, A., STALDER, R., ALLIOD, C., AND BALLIVET, M. (1984). Structure, linkage, and sequence of the two genes encoding the 6 and y subunits of the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 81: 7975-7979. 38. PADGETT, R. W., ST. JOHNSTON, R. D., AND GELBART, W. M. (1987). A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-@ family. Nature (London) 325: 81-84. 39. ROBERT, B., BARTON, P., MINTY, A., DAUBAS, P., WEYDERT, A., BONHOMME, F., CATALAN, J., CHAZO~ES, D., GUENET, J. L., AND BUCKINGHAM, M. (1985). Investigation of genetic linkage between myosin and actin genes using an interspecific back-cross. Nature (London) 314: 181-183. 40. ROUSSEAU-MERCK, M. F., SIMON-CHAZOTTES, D., ARPIN, M., PRINGAULT, E., LOUVARD, D., GUENET, J. L., AND BERGER, R. (1988). Localization of the villin gene on human chromosome 2q35-q36 and on mouse chromosome 1. Hum. Genet. 78: 130133. 41. SCHROEDER,W. T., LOPEZ, L. C., HARPER, M. E., AND SAUNDERS, G. F. (1984). Localization of the human glucagon gene (GCG) to chromosome segment 2q36-37. Cytogenet. Cell Genet. 38:76-79. 42. SHILOH, Y., DONLON, T., BRUNS, G., BREITMAN, M. L., AND TSUI, L-C. (1986). Assignment of the human y-crystallin gene cluster (CRYG) to long arm of chromosome 2, region q33-36. Hum. Genet. 73: 17-19.

MAPPING 43.

44.

45.

46.

OF

INHIBIN

SKOW, L. C. (1982). Localization of a gene controlling electrophoretic variations in mouse -r-crystallin. Exp. Eye Res. 34: 509-516. SKOW, L. C., ADKISON, L., WOMACK, J. E., BEAMER, W. G., AND TAYLOR, B. A. (1987). Mapping of the mouse fibronectin gene (Fn-1) to chromosome 1: Conservation of the Idh-l-CrygFn-1 synteny group in mammals. Genomics 1: 283-286. VALE, W., GRANT, G., AMOSS, M., BLACKWELL, R., AND GUILLEMIN, R. (1972). Culture of enzymatically dispersed anterior pituitary cells: Functional validation of a method Endocrinology 91: 562-572. VALE, W., RIVIER, J., VAUGHAN, J., MCCLINTOCK, R., CORRIGAN, A., WOO, W., XARR, D., AND SPIESS, J. (1986). purification

SUBUNIT

99

GENES

and characterization ovarian follicular

of an FSH fluid. Nature

releasing

protein

(London) 321:

from porcine 776-782.

47.

WEBB, A. C., COLLINS, K. L., ALJRON, P. E., EDDY, R. L., NAKAI, H., BYERS, M., HALEY, L. L., HENRY, W. M., AND SHOWS, T. B. (1986). Interleukin-1 gene (ILl) assigned to long arm of human chromosome 2. Lympkokine Res. 5: 77-85.

48.

WEEKS, localized a growth

49.

Yu, J., SHAO, L., LEMAS, V., Yu, A. L., VAUGHAN, J., RIVIER, J., AND VALE, W. (1987). Importance of FSH-releasing protein and inhibin in erythrodifferentiation. Nature (London) 330: 765-767.

D. L., AND MELTON, D. A. (1987). A maternal mRNA to the vegetal hemisphere in Xenopus eggs codes for factor related to TGF-0. Cell 61: 861-867.