Extraction of Mullerian inhibiting substance from newborn calf testis

DEVELOPMENTAL

BIOLOGY

Extraction

69,73-84

of Mullerian

(1979)

Inhibiting

Substance

from Newborn

Calf Testis

DAVID A. SWANN, PATRICIA K. DONAHOE, YASUO ITO, YASUHIDE MORIKAWA, AND W. HARDY HENDREN Departments

of Biological

Chemistry and Surgery, Harvard Medical School at the Massachusetts Hospital, Boston, Massachusetts 02114

General

Received July 17, 1978; accepted in revised form September 29, 1978

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel fdtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K,, values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-O.5 M column, and (4) in constituents eluted between 0.15 and 0.20 MNaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins. INTRODUCTION

1971). Using a semiquantitative modification of this test system (Donahoe et al., 1977b), MIS activity has been demonstrated in fetal and perinatal rat testis (Donahoe et al., 1976), in fetal, newborn, and prepubertal bovine testis (Donahoe et al., 1977a), and in human testis during the first 2 years of life (Donahoe et al,, 1977c). The in vitro organ culture assay system has also been used to investigate the nature of MIS. The results obtained indicated that MIS was not a steroid and that it was nondialyzable (Josso, 1972). Josso et al. (1975) showed that MIS activity was present in the media of fetal calf testes after 4 hr of incubation. Attempts to detect activity in fetal testicular homogenates, how-

Normal development in the male embryo requires the action of testosterone, which stimulates the differentiation of the Wolffian duct. Another process, however, is also essential, namely, the regression of the Mullerian duct. Based on embryonic castration experiments, Jost (1953) postulated the existence of a second testis hormone, Mullerian inhibiting substance (MIS), which was responsible for the regression of this duct in the male. Subsequent studies led to the development of an in vitro organ culture assay (Picon, 1969) for the detection of MIS and the finding that the testis-specific activity was present in the embryonic testes of a number of mammalian species (Josso. 73

0012lfi06/79/030073-12$02.00/O Copyright Q 1979 by Academic Press. Int All rights of reproduction in any form reservrd.

74

DEVELOPMENTAL BIOLOGY

VOLUME 69, 1979

ever, were unsuccessful (Josso et al., 1977). suspended in 250 ml 1 M guanidine hydroThe activity from incubation media of fetal chloride, 0.05 M Tris-HCl, pH 7.2, containcalf testes was enhanced when the media ing 0.005 M benzamidine. The tissue suswere concentrated 4x, and was again non- pended in the guanidine solution at 4°C dialyzable. Cycloheximide added to the tis- was transported to the laboratory and exsue culture media decreased the MIS activ- tracted for a further 48 hr at 4°C with ity. It was concluded that the presence of gentle stirring. The suspension was then activity in the media was due to the de centrifuged at 120,OOOgfor 1 hr at 4°C in novo synthesis of a macromolecule, proba- an ultracentrifuge. The tissue residue was bly a protein. After fractionation of the discarded and the supernatant was then incubation media by gel filtration chroma- used for various fractionation procedures. tography, the activity was located in a fracProcedures (Fig. 1) tion with an apparent molecular weight of Fractionation 200,000 to 295,000 (Picard and Josso, 1976). Density gradient sedimentation. The Our approach to the problem of identidensity of the supernatant was adjusted to fying the testicular substance responsible 1.50 g/ml by the addition of cesium chlofor MIS activity has employed a direct ex- ride. This solution was then centrifuged at traction procedure. This approach was 120,OOOg for 64 hr at 4’C. At the end of this made possible by the finding that newborn time, fractions were obtained via a capillary calf testis has high MIS activity (Donahoe tube inserted into the base of the centrifuge et al., 1977a). This finding, coupled with tube. The presence of nucleic acids and the fact that the freemartin calf, the female proteins in fractions was detected by mearunt of a heterosexual twinning with re- suring the absorbance at 260 and 280 nm, gressed Mullerian ducts (Lillie, 1916), an respectively. The density was determined “experiment in nature” which provides ev- by weighing known volumes of the fracidence that bovine MIS is capable of acting tions. at a distance in uivo, makes this species a Gel filtration chromatography. Aliquots particularly good choice for MIS character- of the protein fraction obtained by density ization. If MIS can act at a distance in uiuo, gradient fractionation were fractionated on it presumably must be secreted and there- a Bio-Gel A-0.5M column (200 x 2 cm) fore probably can be extracted. Direct ex- eluted with 1 M guanidine chloride, 0.05 M traction from whole testis avoids the prob- Tris-HCl, pH 7.2. The column was operated at a flow rate of 20 ml/hr, and 11-ml lems of (1) isolation of MIS from incubation fractions were collected. The column efmedia containing serum and (2) moditication of MIS by metabolism or interaction fluent was monitored for the presence of with other components during the in vitro protein constituents by measuring the abincubation procedure. For these reasons, sorbance at 280 nm. Chromatography on DEAE Bio-Gel. we carried out direct extraction using a dissociative solvent plus a protease inhibiFractionation of aliquots of the protein tor to eliminate the possibility that a small fraction obtained by density gradient sedimolecule with high MIS activity may be mentation or the fractions obtained by gel interacting with a larger carrier protein. filtration chromatography was carried out using a column (20 x 2 cm) packed with MATERIALS AND METHODS DEAE Bio-Gel A-50 ion exchanger. Prior Newborn testes (1.5 g per testis) were to application to the column, the sample obtained from animals after death and dis- was dialyzed overnight against water, for 8 sected free of adjacent tissues at the slaugh- hr against 1 M NaCl, overnight against 0.15 terhouse. This tissue (30 g) was diced rap- M NaCl, and for 48 hr against 0.05 M idly by an automatic tissue chopper and Tris-HCl, pH 7.2. The column was oper-

SWANN ET

MIS Extraction

AL.

NEWBORN (

CALF 30gm

1 5 gm / testis

from Whole Testes

75

TESTES

wet

wght

)

E-X T/i’A C T/ON

48 hrs , 4°C in Guonldlmum chloride ( 1M 1 w+h Benzam~dlne (0 005 M t

CENTRfFUGATlON 1 hr

I 20,000 x g 4OC

TISSUE RESIDUE

SUPERNATANT* YIELD-

490

mg

freeze

drred

(NoI

DENS/T Y GRAD/EN T SEDIMEN TA NON wth CsCI i 150 g / ml) 120,000

x g ,

64 hrs,

4°C

/

PROTEIN YIELD: Derwty

FRACTION 100 mg -

135g/ml

GEL CHROMA TOGRAPUY Bto-GelA - 0 5 m K cl” Y/ELD:

0 19-o

*

/ON-EXCHANGE CHROMATOGRAPHY

38 fraction*

DEAE B,o-Gel 0 15-O 20 M NaCi

A fraction

*

13 mg

YIfLD:

20 mg

FIG. 1. Extraction of newborn calf testis and further fractionation of extracts and fractions with Mullerian inhibiting substance activity by density gradient sedimentation, gel chromatography, and ion-exchange chromatography. The protein yields of the freeze-dried samples obtained at each step are included.

ated at a flow rate of 20 ml/hr, and lo-ml fractions were obtained. The column eluate was monitored for the presence of peptide constituents by measuring the absorbance at 280 nm. After applying the sample, the column was washed with buffer until a steady baseline was obtained. The column was then eluted with a linear sodium chloride gradient which was monitored by conductivity measurement. Chemical Analysis

of Tissue Fractions

The protein content of fractions

was de-

termined by amino acid analysis (Moore et al., 1958). Glucosamine and galactosamine were also determined using the amino acid analyzer, following hydrolysis of samples with 6 N HCl for 3 hr at 100°C (Swann et al., 1977). Other carbohydrate constituents were determined by gas-liquid chromatography after hydrolysis of samples with methanolic HCl and formation of the trimethylsilyl derivatives (Rheinhold, 1972). The distribution of peptide constituents was determined by SDS gel electrophoresis (Furthmayr et al., 1971).

76

DEVELOPMENTAL BIOLOGY VOLUME69, 1979

Preparation of Antiserum dine Extract Fraction

to the Guani-

An antiserum (ASoucl) was prepared in rabbits against a guanidine extract supernatant fraction (Grade III regression; Fig. 5E) after dialysis of the extract against distilled water, 1 A4 NaCl, and 0.15 M NaCl. The supernatant obtained by centrifugation was lyophilized and mixed with complete Freund’s adjuvant. New Zealand white rabbits were injected in the foot pads, and then intramuscular booster injections of incomplete Freund’s adjuvant were given at 14,23,36, and 42 days. The animals were bled on Days 23, 57, and 65. Antisera from the fourth and fifth bleedings on Days 57 and 65 were used in these experiments. Antisera were absorbed repeatedly (9x) by the addition of lyophilized calf serum, followed by incubation at 4°C for 16 hr and centrifugation, until no serum components were detected by double-diffusion analysis. Pretreatment of Fractions prior to Testing for MIS Activity Aliquots of the guanidine extract supernatant or the gradient and column fractions to be tested in the in vitro organ culture system were dialyzed overnight against distilled water, for 8 hr against 1 M sodium chloride, and for 48 hr against five changes of 0.15 M NaCl, pH 7.2. The fractions were then concentrated on an Amicon ultrafilter fitted with a PM10 membrane to yield solutions of approximately 5 ml. Aliquots of the retained solution were mixed 1:l with the culture medium used in the assay system, and sterilized by filtration under pressure through a 0.22~pm filter (Milhpore). The concentration of samples in the test procedures corresponded to 0.3-1.5 g of testis/organ culture dish. Organ Culture Assay System The assay system used was described by Donahoe et al. (1977b). The urogenital ridge was dissected from the 14-day female rat embryo and transferred to an organ culture dish (Falcon, 3010). Specimens were

placed on stainless-steel grids coated with a thin layer of 2% agar and incubated for 72 hr at 37°C in 5% CO* and 95% air over 2 ml of culture medium [CMRL 1066 containing 10% fetal calf serum, 1% penicillin (10,000 units/ml), and 1% streptomycin (10,000 pg/ml)] or a 1:l mixture of culture medium and the supernatant or gradient fraction to be tested. The incubated tissue was then coated with a mixture of 2% agar and albumin at 44”C, fixed in buffered formaldehyde, dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Eight-micrometer serial sections were stained with hematoxylin and eosin for viewing by light microscopy. Sections from the cephalic end of the Mullerian duct were assigned a coded number and graded for regression (Donahoe, 1977a) on a scale of 0 to V. Five slides with six to eight sections per slide were read for each assay. A grade of activity was listed as the nearest whole number to the mean. A test group for the fractionation procedures represents at least 10 assays. If the mean fell midway between two numbers, then both numbers were listed. Grade 0 refers to no regression. The Mullerian duct, which is lined with columnar epithelial cells whose nuclei have a basilar orientation, has a widely patent lumen (Figs. 5B and G). Grade I is minimal regression. The duct is slightly smaller, and either the surrounding mesenchyme is condensed around the duct as seen in plastic sections or there is a clear area around the duct as seen in paraffin sections (Fig. 5D). Grade II refers to mild regression. The duct is smaller, and the mesenchymal condensation or the clear area around the duct is more pronounced. The nucleii of the shorter epithelial cells loose their basilar orientation (Figs. 5H and I). Grade III is moderate regression. The duct is very small and disorganized. The tip of the urogenital ridge develops poorly distal to the Wolffian duct (Fig. 5E). Grade IV is severe regression. The duct is replaced by a whorl of cells (Fig. 5A). Grade V refers to complete regression. No remnant of the duct can be detected. Positive tissue con-

SWANN

ET AL.

MIS Extraction

trols, using fetal testis, and negative tissue controls, where the Mullerian ducts were incubated alone or with muscle (Fig. 5B), were included in each experiment. Mullerian ducts exposed to extracts from nontestitular tissue, to inactive testicular fractions (Figs. 5G and 6A and D), or to saline served as biochemical controls. Aliquots of all fractions were dialyzed against distilled water and freeze-dried, and protein content was measured. MIS activity measurements were also performed using a guanidine extract supernatant fraction before and after absorption with normal rabbit serum or with an antibody to guanidine extract. The absorption was carried out three times by the addition of undiluted antiserum to the guanidine extract fraction, followed by incubation at 4°C for 16 hr and centrifugation to remove the precipitate formed. RESULTS

Preliminary experiments showed that extraction of calf testis with saline or urea solutions did not yield active extracts. Further experiments using 1 M guanidine chloride solutions produced active extracts, but there was considerable variation in the degree of activity obtained and some were negative. Benzamidine (0.005 M), a protease inhibitor, was then added to the guanidine solution used for the tissue extraction and consistent results were obtained. Aliquots in the range of 1 testis/plate or 0.5 testis/ml were added 1:l with culture media. Attempts to concentrate further or to lyophilize resulted in a loss of biologic activity. The MIS activity in the extracts concentrated within this range varied from I to IV in different preparations. Photomicrographs of the urogenital ridge of the 14.5-day female fetal rat after incubation with calf testis (Grade IV, positive), calf muscle (Grade 0, negative), and the guanidine chloride extract of newborn calf testis (Grade III) are shown in Figs. 5A, B, and E. Parallel experiments carried out with adult bull testis and calf heart, two tissues that do not possess MIS activity in

77

from Whole Testes

the organ culture test system, showed that the guanidine chloride extracts obtained from these tissues were also inactive (Fig. 5G). Following extraction with guanidine chloride, the solutions obtained were always extremely viscous. These solutions had a high absorbance at 260 nm and presumably contained DNA. Dialysis of the tissue supernatant prior to testing in the organ culture assay system produced an insoluble precipitate. This precipitate was removed by centrifugation (120,OOOg at 4”C), and after extensive washing and resuspension in culture medium, portions of the resuspended precipitate were tested for MIS activity. Negative results were always obtained and the residue was therefore discarded. Fractionation of whole tissue guanidine chloride extract by sedimentation in a cesium chloride density gradient yielded 11 fractions (Fig. 2). When these were tested Test

Group

MIS ACTIVITY

,

I

1

0

0

II

2

0

0

I-II

3

0

0

II

4

0

0

n-m

Fraction:

A f---+-lI$

-\

1.8

; ? L

1.6

-$ \

b 1.4

; IL 2;

1.2 1

3

5

7

9

z Q

11

fRAC T/ON NUMBER FIG. 2. Cesium chloride density gradient fractionation of guanidine chloride extract supernatant. Activity was always located in the protein fraction (C). The MIS activity of each test group represents the nearest number to the mean of more than 10 assays.

78

DEVELOPMENTAL BIOLOGY

for MIS activity (the result for each test group represents the nearest number to the mean of more t.han 10 assays), fraction 10 had minimal or no activity (Fig. 6A) and fraction 11 possessed high activity (Fig. 6B). All other fractions were biologically

-0

10

20

VOLUME

inactive in the organ culture assay. Ionexchange chromatography of protein fraction 11 obtained by density gradient sedimentation produced a series of fractions. In the example shown in Fig. 3b, seven fractions were obtained by pooling the column

30 FUA c NOM

69, 1979

40

50

60

MumE~

b Test Group 1

MIS

ACTIVITY

I

I 0

0

0

1-m

0

0

0

2

0

0

0

n-m

I

0

0

3

0

0

0

II

I

0

0

4

0

0

I

II

0

0

0

Fraction :

A

S

C

D

E

F

G

FRACTION

NUYBER

3. Column chromatography of the protein fraction obtained by cesium chloride. (a) A column (200 X 2 cm) packed with Bio-Gel A-0.5M was eluted with 1 M guanidinium chloride, 0.05 M Tris-HCl, pH 7.2, at a flow rate of 20 ml/hr, and 11-ml fractions were collected. V, indicates the elution position of blue dextran, and K the elution position of 3Hz0. The protein fraction was prepared for chromatography by dialysis against the column eluents. Active fractions are shaded. (b) A column (20 x 2 cm) containing DEAE Bio-Gel equilibrated with 0.05 M Tris-HCl, pH 7.2, was used. The gradient protein fraction was dialyzed against this buffer and then applied to the column, which was washed with the buffer until a stable baseline had been reestablished. The column was then eluted with a linear NaCl gradient, monitored by conductivity, at a flow rate of 16 ml/hr, and 8-ml fractions were obtained. The column effluents were monitored for protein constituents by measuring the absorbance at 280 nm. Fractions for testing in the organ culture assay system were obtained by pooling the column fractions as indicated. The results obtained for a series of preparations are shown. Each test group represents the nearest number to the mean of more than 10 assays. Active fractions are shaded. FIG.

SWANN ET

AL.

MIS Extraction

from Whole Testes

79

a

1

2

FIG. 4. Electrophoresis of active column fractions (Fig. 3) on 5% polyacrylamide gels in the presence of sodium dodecyl sulfate. Gel 1 is Bio-Gel fraction B (Fig. 3a), and gel 2 is DEAE fraction D (Fig. 3b). Approximately 150 pg of sample was applied to each gel, and peptides were detected by staining with Coomassie blue. The arrows indicate the mobilities of (a) calf skin collagen a chains and (b) ovalbumin, run on duplicate gels.

effluent. Although essentially the same profile was obtained with different preparations, variations occurred in the resolution obtained between the constituents eluted by the gradient. Testing the pooled column effluent fractions for MIS activity, however, indicated that the constituents eluted between 0.15 and 0.20 N NaCl possessed positive activity, while the other fractions were negative. Gel filtration chromatography of the pro-

tein fraction obtained by the cesium chloride density gradient procedure (fraction 11, Fig. 2) on a Bio-Gel A-0.5M column (Fig. 3a) produced three major fractions: a void volume, a large heterogeneous peak eluted in the included volume, and a third peak eluted close to the total volume of the column. Based on the elution profile obtained, the column effluent fractions were pooled as indicated in Fig. 3a. Testing these fractions for MIS (Fig. 6C) showed that

80

DEVELOPMENTAL BIOLOGY

constituents eluted with K,, values between 0.19 and 0.38 (fraction B) were active. The other fractions were inactive (Fig. 6D). The overall fractionation scheme and the yield of the freeze-dried samples obtained are given in Fig. 1. Analysis of the active column fractions by disc gel electrophoresis (Fig. 4) showed the presence of several peptide constituents. The major constituent, with a mobility intermediate between those of collagen (x chains and ovalbumin, stained with periodic acid-Schiff reagent, and is therefore a glycoprotein. Amino acid and carbohydrate analyses showed that active fractions obtained by gel chromatography contained between 85 and 89% (w/w) amino acids and 7% (w/w) carbohydrates (mannose, 1.5%; galactose, 4.5%; glucosamine, 1.0%; and Nacetylneuraminic acid, 2.0%). The amino acid analysis indicated that the major components in active fractions did not have an unusual amino acid composition. Using undiluted antiserum (A&X-& prepared in rabbits against a guanidine extract supernatant fraction, which had been absorbed nine times with fetal calf serum, up to four precipitate bands were detected in the active guanidine extract and column fractions. However, precipitate lines could not be detected against calf serum, indicating that the observed antigenic components

VOLUME 69, 1979

present in the active fractions were derived from the testis. Precipitate bands were detected against an active fraction when the antiserum was serially diluted to 1:300. As shown in Fig. 5H, the guanidine extract supernatant fraction exhibited Grade II regression of the Mullerian duct. Similar regression was also observed when extract fractions were absorbed with freeze-dried normal rabbit serum (Fig. 51). When the samples were absorbed with the antiserum raised to a guanidine extract with Mullerian inhibiting substance activity (AScnc~),however, the MIS activity of the guanidine extract fraction was virtually eliminated (Fig. 55). DISCUSSION

These experiments were carried out over an extended period of time, and many tissue sampling, handling, and extraction procedures were employed. The most reproducible series of results was obtained with the methods described. The cesium chloride density gradient fractionation procedure proved to be the best method to separate an active protein fraction from solubilized nucleic acids. Further fractionation of the density gradient protein fraction by gel filtration consistently yielded on active tissue fraction eluted at K,, values between 0.19 and 0.38.

FIG. 5. (A) Newborn calf testicular fragment incubated with the urogenital ridge of the l4-day female embryonic rat causes almost complete regression (Grade IV) of the Mullerian duct (M). The Wolffian duct (W), by comparison, is stimulated. (B) Muscle fragment causes no regression (Grade 0) of the Mullerian duct (M). (C-F) Supernatant after guanidine extraction of newborn calf testis is added to the organ culture media in increasing doses, and the urogenital ridge of the ll-day female embryo is examined after 3 days of incubation. (C) Two and five-tenths milligrams of protein per milliliter, the equivalent of 0.1 testis starting material/organ culture plate, causes no regression; (D) 6.25 mg of protein/ml, the equivalent of 0.25 testis/plate, causes Grade I regression; (E) 12.5 mg of protein/ml, or 0.5 testis/plate, causes Grade III-IV regression. (F) More than 25 mg of protein/ml, or 1 testis/plate, is toxic to the urogenital ridge. Differentiation does not occur. Therefore, the protein concentration for assay of the guanidine extract was kept between 7 and 15 mg/ml. (G) Urogenital ridge incubated with guanidine extract of adult bull testis. Thirty grams of whole tissue was extracted as described, and after dialysis, the extract was added to the incubation medium at concentrations of 7-15 mg of protein/ml. No regression of the Mullerian duct (M) could be detected at these concentrations by this assay. Higher protein concentrations were toxic, as in F. (H-J) Detection of MIS activity in a guanidine extract supernatant fraction from newborn calf testis (H), after absorption with normal rabbit serum (I) and after absorption with antiserum to guanidine extract (J). Regression of the Mullerian (M) duct (Grade II) occurred in H and I, whereas activity was lost (Grade 0) after absorption with antiserum to an active guanidine extract. N=8 assays per group. (A-G) x 238; (H-J) x 190.

82

DEVELOPMENTAL BIOLOGY

VOLUME 69, 1979

FIG. 6. (A,B) Density gradient sedimentation fractions of guanidine extract supernatant were added to the organ culture media, and the urogenital ridge of l4-day female embryos was examined after 3 days of incubation. In this experiment, only fraction 11 (B) demonstrated regression (Grade III) of the MuIIerian duct (M). Fraction 10 (A) showed no regression. (C,D) Density gradient fraction 11 was further fractionated on a Bio-Gel A-0.5M column, and ahquots were added to the organ culture media for assay. Only fraction B, with a K., between 0.19 and 0.38 (CT),had MuIIerian inhibiting substance activity. Fraction C, by comparison (D), was inactive.

DEAE-cellulose chromatography gave more variable results. When Bio-Gel fractions were refractionated on the DEAE column to achieve a higher degree of purification of the MIS active fraction, the DEAE fractions were sometimes negative or exhibited low levels of activity. MIS stability experiments have indicated that the loss of activity when the DEAE column procedure is used is probably related to the length of time that fractions are exposed to conditions of low ionic strength. The BioGel column was calibrated, using standard protein solutions, but since elution on gel columns is related to molecular shape and diffusion and these properties are not known for the substance(s) possessing MIS activity, the elution position of the MIS

active fraction was therefore calculated as a K,, value. The initial studies carried out to detect the presence of MIS in the tissue extract fractions employed a wide range of concentrations. These experiments showed that a concentration-dependent graded response was obtained within a restricted range when each fraction was added to the organ culture dish at concentrations corresponding to 0.3-1.5 g wet weight of the original testis starting material. Below 0.3 g, diminished activity was observed, whereas at concentrations greater than 1.5 g per dish, toxic reactions were frequently detected. For this reason, the quantities of sample used in the assay were standardized to correspond to approximately 0.9 testis/dish

SWANN ET AL.

MIS Extraction

from Whole Testes

83

(testis weight=15g). In the case of the minor bands that might not be seen otherguanidine extract fraction, this corre- wise (Fig. 4). Using the guanidine extract which had been absponded to a freeze-dried sample weight of antiserum (A&& sorbed with calf serum, several components 12.5 mg/ml (Fig. 5E). For the cesium chloride protein fraction and the Bio-Gel B were also detected in active fractions obThe fraction, the corresponding weights of sam- tained by column chromatography. ple used for each assay were 3 and 0.4 fact that this antiserum does not form premg/ml, respectively. In terms of the quan- cipitate lines with calf serum indicates that tity of constituents present in active Bio- lines observed with the testis fractions are Gel B fractions (0.4 mg/ml), compared to due to the presence of testis-derived antithose present in active guanidine extracts genie components. These data, together (12.5 mg/ml), the fractionation procedure with the methods used to purify the testis employed results in a 30-fold purification of extracts and the amino acid analysis data, a fraction with MIS activity. indicate that the testis-derived components The results obtained show that MIS ac- are proteins or glycoproteins. tivity can be extracted from the testis and These results are in agreement with methat this activity was present in the protein dia incubation studies and provide addifraction following sedimentation in a ce- tional evidence that regression of the Mulsium chloride density gradient. Since low lerian duct is mediated by a testis protein molecular weight constituents tend to as- or glycoprotein. The active partially purisume a diffuse distribution in these gra- fied fractions, however, contained several dients, the presence of activity predomitestis-derived constituents, and thus it renantly in fraction 11 (Fig. 2) suggests that mains a possibility that one, or more than the activity is associated with a macromoone, testis component causes duct regreslecular constituent. This was confirmed by sion. This problem cannot be so,lved until the finding that activity was retained upon more is known about the chemical nature dialysis and after ultrafiltration on an Amiof MIS. The ability to extract and purify con PM10 filter, and agrees with earlier MIS active substances, however, should famedia incubation studies (Picard and Josso, cilitate the isolation and identification of 1976). Because of the highly dissociative this embryonic hormone. conditions employed in the gradient and The helpful suggestions of Doctors J. Gross, R. L. extraction procedures, it appears unlikely Trelstad, T. F. Linsenmayer, J. W. McArthur, and I. that the distribution of activity obtained in Z. Beitins and the technical assistance of Myong Suk the density gradient procedure was due to Lee, Carmello Bondi, Thomas Manganaro, Marcia an interaction between an active low mo- Sullivan, and Stuart Sotman are greatly appreciated. This work was supported by funds from the Shriner lecular weight component and an inactive Burns Institute, Boston Unit; the Pediatric Surgical macromolecule acting as a carrier. Further Research Fund of the Massachusetts General Hospifractionation on the Bio-Gel column (Fig. tal; and NIH Grant No. CA 17393-2. 3a) using a relatively high ionic strength dissociative solvent system (1 M guanidine REFERENCES hydrochloride) also indicated that the acDONAHOE, P. K., ITO, Y., MARFATIA, S., and HENtive component behaves like a macromoleDREN, W. H. (1976). The production of Mullerian cule. Analysis of active Bio-Gel B and inhibiting substance by the fetal, neonatal, and adult DEAE D fractions by disc gel electrophorat. Biol. Reprod. 15, 329-334. DONAHOE, P. K., ITO, Y., PRICE, J. M., and HENDREN, resis showed that each fraction contained W. H. (1977a). Mullerian inhibiting substance activa mixture of protein constituents, several of ity in bovine fetal, newborn, and prepubertal testes. which had the same electrophoretic mobilBiol. Reprod 16, 238-243. ities. Relatively high sample concentrations DONAHOE, P. K., ITO, Y., and HENDREP;, W. H. (197713). A graded organ culture assay for the detecwere added to the gels in order to detect

84

DEVELOPMENTAL BIOLOGY

tion of Mullerian inhibiting substance. J. Surg. Res. 23, 141-148. DONAHOE, P. K., ITO, Y., MORIKAWA, Y., and HENDREN, W. H. (1977c). Mullerian inhibiting substance in human testis after birth. J. Pediatr. Surg. 12, 323-330. FAHEY, J. L., MCCOY, P. F., and GOULIAN, M. (1958). Chromatography of serum proteins in normal and pathologic sera: The distribution of protein-bound carbohydrate and cholesterol, siderophiiin, thyroxin-binding protein, Blz-binding protein, alkaline and acid phosphatases, radioiodinated albumin and myeloma proteins. J. Clin. Invest. 37,272-284. FURTHMAYR,H., and TIMPL, R. (1971). Characterization of collagen peptides by sodium dodecyl sulfate-polyacrylamide electrophoresis. Anal. Biothem. 41,510-516. Josso, N. (1971). Interspecific character of the Mullerian inhibiting substance: Action of human fetal testis, ovary, and adrenal on the fetal rat mullerian duct in organ culture. J. Clin. Endacrinol. Metab. 32,404-409. Josso, N. (1972). Permeability of membranes to the Mullerian inhibiting substance synthesized by the human fetal testis in vitro: A clue to its biochemical nature. J. Clin. Endocrinol. 34, 265-270. Josso, N., FOREST,M. D., and PICARD, J. Y. (1975). Mullerian inhibiting activity of calf fetal testis: Re-

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