Presence of a human chorionic gonadotropin-like substance in human sperm RICARDO
H.
EMIL10
0.
THERESA CARI.
M.D.
FERNANDEZ, M.
J.
ASCH.
M.D.
SILER-KHODR,
PAUERSTEIN,
PH.D. M.D.
Sn )I .-Intnnin, TPW/.S An hCG-like material has been extracted from human sperm. These experiments were designed to characterize this material. Sperms of 10 volunteers were separated from seminal fluid, washed in PBS three times, and resuspended in 0.5 ml of the same buffer. Samples were pooled; cells were disrupted by sqnication and extracted in alkaline buffer by constant agitation at 4” C. The extract was ultracentrifuged at 4” C. Supernate was lyophilized and reconstituted in 2 cc of distilled water. This material iresented a dose-response curve parallel to those of ISBhCG and CR1 19 in phCG RIA. When chromatographed in a Sephadex G-l 50 column the extract eluted within the hCG range and immunoreacted in the specific phCG RIA. When absorbed onto a concanavalin A-Sepharose column, all recovered immunoreactive material eluted after exposure to a-o-methylglucoside, indicating that it is a glycoprotein. The extract stimulated progesterone and testosterone secretion in porcine granulosa cells and decapsulated rat testis, respectively, indicating its biologic potency. (AM.J.OBSTET. GYNECOL. 135:1041,1979.)
FOR MANY YEA KS reproductive endocrinologists assumed that the only nonneoplastic extrapituitary sources of gonadotropins were the trophoblastic tissues of the placentas of primates and equines. Some gonadal neoplasms were also known to produce choFrom the Department of Obstetrics and Gwwlogy, Center for Research and Training in Reproduct&e Biology, The (fnkwr.: (I) Would the suspect material elutc af’ter gel filtration on a Sephadex column in the location appropriate to pure hCG? (2) Could we, utilizing specific radioimmunoassays, demonstrate immunoreactive parallelism with hCG? (3) Could we demonstrate the presence of a carbohydrate moiety by Concanavalin .4 column chromatography? (4) Would the substance demonstrate hCG-like biologic activity in bioassay systems? ‘I’his report documents physical, immunologic, and biologic data, all of which support the presence of an hCG-like substance in extracts from human sperm.
Materials and methods General procedure. Ejaculates from 10 volunteers of proved fertility, with known normal spermograms, were obtained by masturbation ,into glass jars (number of sperm per ejaculate: 241 2 39 millions, X t S.E.M.). Within I hour after collection, cells were separated from seminal plasma by centrifugation at 800 x g fox 30 minutes at 4” C. Sperm were washed three times, each time for 10 minutes in 2 ml of 0.01 M phosphatebuffered saline (PBS) pH 7.8, than resuspended in 0.5 ml bf the same buffer and stc?red at -20” C until processed. Samples were pooled and the cells were disrupted by sonification (Heat System Ultrasonics, Inc. W-37.5) at 30 watts for 1.5 minutes with 25 /J glass beads (‘/3 volume) in an ice bath. They were then extracted in the same buffer for 16 hours at 4” C under constant agitation, centrifuged at 800 x g for 30 minutes at 4” C, and the supernatant fluid was ultracentrifuged at 78,000 X g for 120 minutes at 4” C. The supernate was lyophilized and reconstituted in 2 ml of distilled water (8.2 mg/ml protein concentration by Lowry determination). Physical identification. Aliquots of the extract 0.5 ml were applied to a I .5 by 85 cm column of Sephadex G150 at 4” C, equilibrated with O.OlM PBS at pH 7.6; 1.3 ml fractions were collected. The column was calibrated on successive days with purified hLH (LER 960), hCG (CR1 19) and its subunits: ahCG (NIH) and PhCG (generously provided by Dr. Vernon Stevens). Void volume was determined by adding Blue Dextran
2000, at a concentration of‘ 0.5 mgiml. 11) the samples. Aliquots of the fractions i0. I to 0.3 ml) \verc assa! ed with specific r-adioimmunoassa),s (RIA) ti)r phC:(; and hCG-hLH. Eluates that immunoreacted in thr /3hCC; KI:\ (Ii-a,tions 10 to 24 postvoid volume). as well as those obtained prior to the void volume (fractions I to 13) and those from fractions 45 to 55 postvoid volume, \vere processed as follows: all tubes from each group wc’re pooled, lyophilized. and reconstituted in 2 ml of distilled water. These specimens, as well as the original “crude material” (prechromatograph\-) were rlrcn tcstcd in various assays as follows: Immunologic and partial chemical characterization. Dose-response curves, performed with serial dilutions of. the crude material and various eluates from the column, were compared for parallelism lo reference standard preparations (CR1 19. lS2. LER 960) in a PhCG RIA.“, I2 In control experiments. the additon of commercial proteases (Protease Crude P4630, Pepsin P7012, Tryspin T8003, Sigma Chemical Co.) did not alter the binding of hCG in the /3hCG RIA. The crude extracts and the eluates were sent to Dr. G. D. Braunstein for concanavalin A column chromatograph?- according to the method of Braunstein. Basor, and Wade.l The eluted fractions were analyzed in a PhCG RI,4. Recover) of radioiodinated glycoproteins of the immunoreactive hCG in pregnancy serum was 94.7% ? 3.5 (S.E.M.). Biologic activity. Testosterone production by drcapsulated rut testis. Samples were sent to Dr. Brij B. Saxena for determination of their abilitv to induce testosterone production in the rat testis. Adult Sprague-Dawley rat testis was cultured according to the method of Catt, Dufau, and Tsuruhara.’ Triplicate vials were inoculated with IO0 ~1 of PBS containing 1 mg/ml of bovine gamma globulin; 0.33, 0.5, 0.75, 1.25. 2.5, or 5 ng/lOO ~1 of hCG iBahl, 1971, 12000 IUimg), or unknown sample. After incubation at 37” C in a metabolic shaker under 95% 02-5%, COZ for 4 hours, the media from triplicate incubation vials were centrifuged at 1,500 ng for 10 minutes and the supernates were stored at - ljo C until rapid chromatography radioimmunoassay was utilized for the measurement of testosterone, as follows: Assaysha@: PBS with I gm/L of sodium azide and 1 gm/L of’ gelatin. Solvents and reagents: analytical grade anhydrous ether, isooctane, ethylacetate (Mallinckrodt Chemicals), ethylene glycol (Chromatoquality), and toluene (Seintillar). Norit A charcoal (Matheson, Coleman and Bell), dextran T-70, (Mann kesearch Lab.), and sterile irrigation water were used throughout the assay. Omnifluor was
Volume Number
Presence
135 8
N
ISZ-hCG
of hCG-like
substance
in sperm
1043
mIU
t-20
r-40
Fig. 1. Dose-response curves of human chorionic gonadotropin reference standard preparations (IS2 and CR1 I’& and the crude human sperm extract in a specific PhCG RIA. obtained from New England Nuclear. Nonradioactive steroids were obtained from Sigma. Celite, analytical filter aid (Johns Manville Co.) supplied by Fisher Scientific Company. Procr~rt~: To each 0.1 ml of sample was added 0.1 ml of assay buffer containing 2,000 dpm of H:‘T. These tracers served as internal standards for recovery estimations. After mixing and equilibration of the tracers with the samples at room temperature for 30 minutes, extraction was carried out twice with 5 ml of ethyl ether by vortexing. After clear separation of the two phases. The lower phase was quickly frozen in ethanol containing Dry Ice. The upper phase was then decanted for evaporation to dryness under filtered air. The dried residue was transferred to celite microcolumns for chromatography. The celite was combusted to 1,000” F overnight, then mixed with ethylene glycol (2 gm/tnl) for packing the columns according to a modification of Abraham’s system #2.’ Isooctane saturated with ethylene glycol (1 ml) was used to transfer the dried samples to celite columns. The column was washed with 1 ml of isooctane to eliminate lipids, cholesterol, and nonpolar compounds. Elutions were carried out with 3.5 ml of 15% ethyl acetate in isooctane (testosterone fraction). All eluates were dried immediately after each collection. Assay buffer (1 ml) was added to the dried eluates. They were mixed and incubated for 1 hour and 0.1 ml aliquots were taken for RIA. One aliquot of each eluate was pipetted into counting vials for recovery estimation. Standard curves ranging from 0 to 800 pg were set up in triplicate. Total incubation volume of the samples, antibodies, and tracers was 0.6 ml at 4” C for 16 hours. Separation of bound and unbound tracers was achieved by adding 0.5 ml of 0.2 gm/lOO ml of charcoal with 0.02%’ dextran in assay buffer. After centrifugation, supernates
were decanted into glass counting vials containing 10 ml of counting solution in a Packard Scintillation Counter Model 3330 with 50% efficiency in H” channel. Samples were counted long enough to reduce counting error to less than 2%. Computer program was used for construction of log-it/log transformation of the standard curves. Blank values were not subtracted as they were always below the sensitivity of standard curve (logit above 2.2). Values were corrected for recoveries. Pqyst~um podu&)~ 1)~ pan-iur gmn~~1o.w calls. The ability of the extract and the eluates to stimulate progesterone production by granulosa cells was ascertained by Dr. Cornelia P. Channing. utilizing methodology developed in her laboratorv.” Porcine ovaries were collected and the 3 to 5 mm follicles were aspirated with a 20 gauge needle and 10 cc syringe. The aspirate was centrifuged at 500 x g for 5 minutes to separate granulosa cells from follicular fluid. The granulosa cells were washed twice in medium 199 and counted in a hemocytometer in 0.06% trypan blue. The cells were diluted in culture medium 199. containing 25 mM Hepes buffer, 10% pig serum, and 50 wgiml of fungizine to yield 0.1 x IO6 live cells (cells which exluded the trypan blue dye) in 0.2 nl. The cell suspension was mixed well and inoculated in 0.1 ml of medium onto Falcon microtest plates at a concentration of 0.05 0.1 x lo6 cells/well. In each experiment, 60 replicate wells were inoculated with cells. The test solutions and standard hCG (Canfield CR 121; 12-14,000 IUimg) Lvere diluted in culture medium (100 ~1 of test solution in 1.0 ml of medium) and filtered through a 0.22 p Millipore filter. The unknown was added to four replicate culture wells in a volume of 0.1 ml of culture medium. This made the total volume of culture tnedium 0.2 ml. Each unknown was examined in two
1044
Asch
et al.
hs
Column Chrcmoi ophy sephodex GE0 %( xl5cm) Flow. IO-12ml/hr 001 M Pf3s pH76 4°C ,dhCG RIP.
(CR 119)
9
Fraction hJJ(LER
hlumbw
960)
6C
5c
40 e 3 -3c E
VO I
zc IC
0
5
*
I5
30
F~IK&I
Fig. 2. Sephadex (CR119), purified volume determined @hCG RIA.
33
40
45
f’&%w
Cl50 gel chromatographic patterns human luteinizing hormone (LER by blue dextran elution. Elutions
of purified human chorionic gonadotropin 960) and human sperm extract. Vo is void from the column were assayed in a specific
Volume Number
Presence
135 8
of hCG-like
substance
in sperm
1045
ml Fig. 3. Sephadex Cl50 gel chromatographic pattern of human sperm extract. c’o is void volume determined by blue dextran elution. Elutions from the column were assayed in a hCG-LH RIA.
doses in two separate culture experiments. The standards were diluted to yield 0.01, 0.10, 0, 0.5, 1.0, and 10 ng hCG per culture and each dose was added to four replicate cultures. The cultures were incubated for 2 days in humidified 5% CO2 in air-gas phase. The culture medium was collected by aspiration from the control, standard, and experimental cultures and replaced with fresh medium. Culture was continued for an additional 3 days followed by a second aspiration of the media, and fixation and staining of the cultures with Oil Red 0 for lipid and hematoxalin. The progesterone content of the spent media in each sample after 2 and 5 days of culture was measured by radioimmunoassay (RIA). Niswander’s anti- 11 OH progesterone-BSA antiserum was used in the progesterone RIA and the assay was carried out on unextracted diluted culture medium as detailed previously.” The coefficient of variation in progesterone secretion between replicate cultures of the same experiment was 11% in 15 groups of cultures. Progesterone secretion between days 3 and 5 was used for the final calculation. Determination qf odenylyl cyclase-stlmulatillg activity. The crude (prechromatography) extraction was sent to Dr. Mary Hunzicker-Dunn for determination of adenyl cyclase-stimulating activity. To this end, serial dilutions of the crude extract were added in 10 ~1 aliquots to triplicate adenylyl cyclase assays carried out for 10 minutes at 37” C in medium containing 3.0 mM ((~-32~) adenosine triphosphate (ATP) (10 to 12 x lo6 cpm; New England Nuclear), 5.0 mM MgCL*, 10 mM EDTA, an ATP-regenerating system consisting of 20
mM creatine phosphate (Calbiochem/Behring, Dallas, Texas) and 0.02 mgiml myokinase (Sigma Chemical Co., St. Louis, Missouri), and between 8 and 15 pg of protein of homogenates containing adenylyl cyclase prepared from Graafian follicles (1.0 to 2.0 mm in diameter) dissected from estrous rabbits.” Stimulating activity was calculated by interpolation ofthe activity found in the presence of diluted “unknown” samples into concentration-effect curves obtained concurrently with NIH-LH-B9 (0.001 to 10 pg/ml) standard. RIA procedures. All protein RIA were performed by means of a second antibody precipitation method. LH-hCG was determined as described previously.” The standard was the Second International Standard for hCG (IS2-hCG). In this system, 1 mIU of ISS-hCG was equivalent to 0.5 mIU of Second IRP-HMG, 0.09 ng of purified hLH (LER 960), or 0.18 ng of hCG (CR1 19). Assay sensitivity- with this procedure is 0.380 mIU/tube and the intra- and interassay coefficients of variation are 3.1% and 8.4%. respectively, at 70% of the maximum binding. Cross-reactivity with either the beta (Stevens) or alpha (NIAMDD) subunits of hCG was 12% (at 50% of the B,): however, the alpha subunit had a flatter nonparallel slope. Quantitation of a ahCG was done with a specific antiserum for this subunit (SAG from the NIAMDD), at a final dilution of l/100,000. Purified hCG (from the NIAMDD) was radioiodinated and 0.100 ng added to every tube. Standard is the ISS-hCG; 1 mlC’ (ISS-hCG) was equivalent to 0.006 ng of purified ahCG
B-KG s9 r8
RIA
1
MEG
10
20 FRACTION
FRACTIONS I-20 ELUTED WITH p1El.S. DM m. 7.8ph. FRACTIONS 21-u) ELUTED WITH 0.2 tn d-D-METHYLOLUCOSIDE
30 NUMBER
40
50
Fig. 4. Concanavalin A-Sepharose column chromatography of the PhCC immunoreactive material from the human sperm extract after gel filtration in a Sephadex Cl50 column.
(NIAMDD). Incubation of antibody. label, and standard or sample was for 16 hours at 4” C; then ARGG was added. The assay sensitivity was 0.4 mIU/tube and the intra- and interassay coefficients of variation were 3.0% and 9.4%. respectively, at 70% of the maximum binding. Intact hCG (CR1 19) or purified hLH (LER 960) was indistinguishable from ahCG in this assa) with a potency of 18% on a weight-to-weight basis. Cross-reactivity with /3hCG according to this system was 2.0% and
H.
EMIL10
0.
THERESA CARI.
M.D.
FERNANDEZ, M.
J.
ASCH.
M.D.
SILER-KHODR,
PAUERSTEIN,
PH.D. M.D.
Sn )I .-Intnnin, TPW/.S An hCG-like material has been extracted from human sperm. These experiments were designed to characterize this material. Sperms of 10 volunteers were separated from seminal fluid, washed in PBS three times, and resuspended in 0.5 ml of the same buffer. Samples were pooled; cells were disrupted by sqnication and extracted in alkaline buffer by constant agitation at 4” C. The extract was ultracentrifuged at 4” C. Supernate was lyophilized and reconstituted in 2 cc of distilled water. This material iresented a dose-response curve parallel to those of ISBhCG and CR1 19 in phCG RIA. When chromatographed in a Sephadex G-l 50 column the extract eluted within the hCG range and immunoreacted in the specific phCG RIA. When absorbed onto a concanavalin A-Sepharose column, all recovered immunoreactive material eluted after exposure to a-o-methylglucoside, indicating that it is a glycoprotein. The extract stimulated progesterone and testosterone secretion in porcine granulosa cells and decapsulated rat testis, respectively, indicating its biologic potency. (AM.J.OBSTET. GYNECOL. 135:1041,1979.)
FOR MANY YEA KS reproductive endocrinologists assumed that the only nonneoplastic extrapituitary sources of gonadotropins were the trophoblastic tissues of the placentas of primates and equines. Some gonadal neoplasms were also known to produce choFrom the Department of Obstetrics and Gwwlogy, Center for Research and Training in Reproduct&e Biology, The (fnkwr.: (I) Would the suspect material elutc af’ter gel filtration on a Sephadex column in the location appropriate to pure hCG? (2) Could we, utilizing specific radioimmunoassays, demonstrate immunoreactive parallelism with hCG? (3) Could we demonstrate the presence of a carbohydrate moiety by Concanavalin .4 column chromatography? (4) Would the substance demonstrate hCG-like biologic activity in bioassay systems? ‘I’his report documents physical, immunologic, and biologic data, all of which support the presence of an hCG-like substance in extracts from human sperm.
Materials and methods General procedure. Ejaculates from 10 volunteers of proved fertility, with known normal spermograms, were obtained by masturbation ,into glass jars (number of sperm per ejaculate: 241 2 39 millions, X t S.E.M.). Within I hour after collection, cells were separated from seminal plasma by centrifugation at 800 x g fox 30 minutes at 4” C. Sperm were washed three times, each time for 10 minutes in 2 ml of 0.01 M phosphatebuffered saline (PBS) pH 7.8, than resuspended in 0.5 ml bf the same buffer and stc?red at -20” C until processed. Samples were pooled and the cells were disrupted by sonification (Heat System Ultrasonics, Inc. W-37.5) at 30 watts for 1.5 minutes with 25 /J glass beads (‘/3 volume) in an ice bath. They were then extracted in the same buffer for 16 hours at 4” C under constant agitation, centrifuged at 800 x g for 30 minutes at 4” C, and the supernatant fluid was ultracentrifuged at 78,000 X g for 120 minutes at 4” C. The supernate was lyophilized and reconstituted in 2 ml of distilled water (8.2 mg/ml protein concentration by Lowry determination). Physical identification. Aliquots of the extract 0.5 ml were applied to a I .5 by 85 cm column of Sephadex G150 at 4” C, equilibrated with O.OlM PBS at pH 7.6; 1.3 ml fractions were collected. The column was calibrated on successive days with purified hLH (LER 960), hCG (CR1 19) and its subunits: ahCG (NIH) and PhCG (generously provided by Dr. Vernon Stevens). Void volume was determined by adding Blue Dextran
2000, at a concentration of‘ 0.5 mgiml. 11) the samples. Aliquots of the fractions i0. I to 0.3 ml) \verc assa! ed with specific r-adioimmunoassa),s (RIA) ti)r phC:(; and hCG-hLH. Eluates that immunoreacted in thr /3hCC; KI:\ (Ii-a,tions 10 to 24 postvoid volume). as well as those obtained prior to the void volume (fractions I to 13) and those from fractions 45 to 55 postvoid volume, \vere processed as follows: all tubes from each group wc’re pooled, lyophilized. and reconstituted in 2 ml of distilled water. These specimens, as well as the original “crude material” (prechromatograph\-) were rlrcn tcstcd in various assays as follows: Immunologic and partial chemical characterization. Dose-response curves, performed with serial dilutions of. the crude material and various eluates from the column, were compared for parallelism lo reference standard preparations (CR1 19. lS2. LER 960) in a PhCG RIA.“, I2 In control experiments. the additon of commercial proteases (Protease Crude P4630, Pepsin P7012, Tryspin T8003, Sigma Chemical Co.) did not alter the binding of hCG in the /3hCG RIA. The crude extracts and the eluates were sent to Dr. G. D. Braunstein for concanavalin A column chromatograph?- according to the method of Braunstein. Basor, and Wade.l The eluted fractions were analyzed in a PhCG RI,4. Recover) of radioiodinated glycoproteins of the immunoreactive hCG in pregnancy serum was 94.7% ? 3.5 (S.E.M.). Biologic activity. Testosterone production by drcapsulated rut testis. Samples were sent to Dr. Brij B. Saxena for determination of their abilitv to induce testosterone production in the rat testis. Adult Sprague-Dawley rat testis was cultured according to the method of Catt, Dufau, and Tsuruhara.’ Triplicate vials were inoculated with IO0 ~1 of PBS containing 1 mg/ml of bovine gamma globulin; 0.33, 0.5, 0.75, 1.25. 2.5, or 5 ng/lOO ~1 of hCG iBahl, 1971, 12000 IUimg), or unknown sample. After incubation at 37” C in a metabolic shaker under 95% 02-5%, COZ for 4 hours, the media from triplicate incubation vials were centrifuged at 1,500 ng for 10 minutes and the supernates were stored at - ljo C until rapid chromatography radioimmunoassay was utilized for the measurement of testosterone, as follows: Assaysha@: PBS with I gm/L of sodium azide and 1 gm/L of’ gelatin. Solvents and reagents: analytical grade anhydrous ether, isooctane, ethylacetate (Mallinckrodt Chemicals), ethylene glycol (Chromatoquality), and toluene (Seintillar). Norit A charcoal (Matheson, Coleman and Bell), dextran T-70, (Mann kesearch Lab.), and sterile irrigation water were used throughout the assay. Omnifluor was
Volume Number
Presence
135 8
N
ISZ-hCG
of hCG-like
substance
in sperm
1043
mIU
t-20
r-40
Fig. 1. Dose-response curves of human chorionic gonadotropin reference standard preparations (IS2 and CR1 I’& and the crude human sperm extract in a specific PhCG RIA. obtained from New England Nuclear. Nonradioactive steroids were obtained from Sigma. Celite, analytical filter aid (Johns Manville Co.) supplied by Fisher Scientific Company. Procr~rt~: To each 0.1 ml of sample was added 0.1 ml of assay buffer containing 2,000 dpm of H:‘T. These tracers served as internal standards for recovery estimations. After mixing and equilibration of the tracers with the samples at room temperature for 30 minutes, extraction was carried out twice with 5 ml of ethyl ether by vortexing. After clear separation of the two phases. The lower phase was quickly frozen in ethanol containing Dry Ice. The upper phase was then decanted for evaporation to dryness under filtered air. The dried residue was transferred to celite microcolumns for chromatography. The celite was combusted to 1,000” F overnight, then mixed with ethylene glycol (2 gm/tnl) for packing the columns according to a modification of Abraham’s system #2.’ Isooctane saturated with ethylene glycol (1 ml) was used to transfer the dried samples to celite columns. The column was washed with 1 ml of isooctane to eliminate lipids, cholesterol, and nonpolar compounds. Elutions were carried out with 3.5 ml of 15% ethyl acetate in isooctane (testosterone fraction). All eluates were dried immediately after each collection. Assay buffer (1 ml) was added to the dried eluates. They were mixed and incubated for 1 hour and 0.1 ml aliquots were taken for RIA. One aliquot of each eluate was pipetted into counting vials for recovery estimation. Standard curves ranging from 0 to 800 pg were set up in triplicate. Total incubation volume of the samples, antibodies, and tracers was 0.6 ml at 4” C for 16 hours. Separation of bound and unbound tracers was achieved by adding 0.5 ml of 0.2 gm/lOO ml of charcoal with 0.02%’ dextran in assay buffer. After centrifugation, supernates
were decanted into glass counting vials containing 10 ml of counting solution in a Packard Scintillation Counter Model 3330 with 50% efficiency in H” channel. Samples were counted long enough to reduce counting error to less than 2%. Computer program was used for construction of log-it/log transformation of the standard curves. Blank values were not subtracted as they were always below the sensitivity of standard curve (logit above 2.2). Values were corrected for recoveries. Pqyst~um podu&)~ 1)~ pan-iur gmn~~1o.w calls. The ability of the extract and the eluates to stimulate progesterone production by granulosa cells was ascertained by Dr. Cornelia P. Channing. utilizing methodology developed in her laboratorv.” Porcine ovaries were collected and the 3 to 5 mm follicles were aspirated with a 20 gauge needle and 10 cc syringe. The aspirate was centrifuged at 500 x g for 5 minutes to separate granulosa cells from follicular fluid. The granulosa cells were washed twice in medium 199 and counted in a hemocytometer in 0.06% trypan blue. The cells were diluted in culture medium 199. containing 25 mM Hepes buffer, 10% pig serum, and 50 wgiml of fungizine to yield 0.1 x IO6 live cells (cells which exluded the trypan blue dye) in 0.2 nl. The cell suspension was mixed well and inoculated in 0.1 ml of medium onto Falcon microtest plates at a concentration of 0.05 0.1 x lo6 cells/well. In each experiment, 60 replicate wells were inoculated with cells. The test solutions and standard hCG (Canfield CR 121; 12-14,000 IUimg) Lvere diluted in culture medium (100 ~1 of test solution in 1.0 ml of medium) and filtered through a 0.22 p Millipore filter. The unknown was added to four replicate culture wells in a volume of 0.1 ml of culture medium. This made the total volume of culture tnedium 0.2 ml. Each unknown was examined in two
1044
Asch
et al.
hs
Column Chrcmoi ophy sephodex GE0 %( xl5cm) Flow. IO-12ml/hr 001 M Pf3s pH76 4°C ,dhCG RIP.
(CR 119)
9
Fraction hJJ(LER
hlumbw
960)
6C
5c
40 e 3 -3c E
VO I
zc IC
0
5
*
I5
30
F~IK&I
Fig. 2. Sephadex (CR119), purified volume determined @hCG RIA.
33
40
45
f’&%w
Cl50 gel chromatographic patterns human luteinizing hormone (LER by blue dextran elution. Elutions
of purified human chorionic gonadotropin 960) and human sperm extract. Vo is void from the column were assayed in a specific
Volume Number
Presence
135 8
of hCG-like
substance
in sperm
1045
ml Fig. 3. Sephadex Cl50 gel chromatographic pattern of human sperm extract. c’o is void volume determined by blue dextran elution. Elutions from the column were assayed in a hCG-LH RIA.
doses in two separate culture experiments. The standards were diluted to yield 0.01, 0.10, 0, 0.5, 1.0, and 10 ng hCG per culture and each dose was added to four replicate cultures. The cultures were incubated for 2 days in humidified 5% CO2 in air-gas phase. The culture medium was collected by aspiration from the control, standard, and experimental cultures and replaced with fresh medium. Culture was continued for an additional 3 days followed by a second aspiration of the media, and fixation and staining of the cultures with Oil Red 0 for lipid and hematoxalin. The progesterone content of the spent media in each sample after 2 and 5 days of culture was measured by radioimmunoassay (RIA). Niswander’s anti- 11 OH progesterone-BSA antiserum was used in the progesterone RIA and the assay was carried out on unextracted diluted culture medium as detailed previously.” The coefficient of variation in progesterone secretion between replicate cultures of the same experiment was 11% in 15 groups of cultures. Progesterone secretion between days 3 and 5 was used for the final calculation. Determination qf odenylyl cyclase-stlmulatillg activity. The crude (prechromatography) extraction was sent to Dr. Mary Hunzicker-Dunn for determination of adenyl cyclase-stimulating activity. To this end, serial dilutions of the crude extract were added in 10 ~1 aliquots to triplicate adenylyl cyclase assays carried out for 10 minutes at 37” C in medium containing 3.0 mM ((~-32~) adenosine triphosphate (ATP) (10 to 12 x lo6 cpm; New England Nuclear), 5.0 mM MgCL*, 10 mM EDTA, an ATP-regenerating system consisting of 20
mM creatine phosphate (Calbiochem/Behring, Dallas, Texas) and 0.02 mgiml myokinase (Sigma Chemical Co., St. Louis, Missouri), and between 8 and 15 pg of protein of homogenates containing adenylyl cyclase prepared from Graafian follicles (1.0 to 2.0 mm in diameter) dissected from estrous rabbits.” Stimulating activity was calculated by interpolation ofthe activity found in the presence of diluted “unknown” samples into concentration-effect curves obtained concurrently with NIH-LH-B9 (0.001 to 10 pg/ml) standard. RIA procedures. All protein RIA were performed by means of a second antibody precipitation method. LH-hCG was determined as described previously.” The standard was the Second International Standard for hCG (IS2-hCG). In this system, 1 mIU of ISS-hCG was equivalent to 0.5 mIU of Second IRP-HMG, 0.09 ng of purified hLH (LER 960), or 0.18 ng of hCG (CR1 19). Assay sensitivity- with this procedure is 0.380 mIU/tube and the intra- and interassay coefficients of variation are 3.1% and 8.4%. respectively, at 70% of the maximum binding. Cross-reactivity with either the beta (Stevens) or alpha (NIAMDD) subunits of hCG was 12% (at 50% of the B,): however, the alpha subunit had a flatter nonparallel slope. Quantitation of a ahCG was done with a specific antiserum for this subunit (SAG from the NIAMDD), at a final dilution of l/100,000. Purified hCG (from the NIAMDD) was radioiodinated and 0.100 ng added to every tube. Standard is the ISS-hCG; 1 mlC’ (ISS-hCG) was equivalent to 0.006 ng of purified ahCG
B-KG s9 r8
RIA
1
MEG
10
20 FRACTION
FRACTIONS I-20 ELUTED WITH p1El.S. DM m. 7.8ph. FRACTIONS 21-u) ELUTED WITH 0.2 tn d-D-METHYLOLUCOSIDE
30 NUMBER
40
50
Fig. 4. Concanavalin A-Sepharose column chromatography of the PhCC immunoreactive material from the human sperm extract after gel filtration in a Sephadex Cl50 column.
(NIAMDD). Incubation of antibody. label, and standard or sample was for 16 hours at 4” C; then ARGG was added. The assay sensitivity was 0.4 mIU/tube and the intra- and interassay coefficients of variation were 3.0% and 9.4%. respectively, at 70% of the maximum binding. Intact hCG (CR1 19) or purified hLH (LER 960) was indistinguishable from ahCG in this assa) with a potency of 18% on a weight-to-weight basis. Cross-reactivity with /3hCG according to this system was 2.0% and
