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Cytologic and hormonal activity of trophoblast in explant culture
FETUS AND
NEWBORN Cytologic and hormonal activity of trophoblast in explant culture ROLAND A. PATTILLO, M.D. THOMAS C. SMITH, PH.D., M.D. ELEANOR DELFS, M.D. RICHARD F. MATTINGLY, M.D. Milwaukee, Wisconsin
T H E T R o P H o B L A s T was shown to produce gonadotropin in tissue culture by Gey, Seegar, and Hellman 1 in 1938. Since that time, the development of a sensitive biologic assay 2 has made possible the more accurate determination of gonadotropic hormone in minute quantities. Concentration of the hormone by alcohol precipitation allows detection of small quantities present in pooled tissue fluid in which placental tissue is grown. Previous studies 3 have emphasized various cell types and growth patterns associated with short-term gonadotropin production. No unanimity exists at this time as to the cell type responsible for gonadotropin production.4 Stewart, Sano, and Montgomery 5 described three types of cells in explants of immature placentas: the oblong cell with an oval nucleus, taken to represent the syncytial celL the round cell with a round nucleus,
and a perinuclear halo, believed to represent the Langhans cell and the stromal cell with a round nucleus. Thiede 6 described the pleomorphic epithelioid or polygonal cell, the stromal cell, and a multinucleated giant cell observed in 16 hour cultures. Jones, Gey, and Gey 7 in their early report described broad and elongated cells, interconnected by multiple long narro\A/ cytoplasmic processes. When definite tissue culture colonies of this type were produced they could be maintained in continuous culture as a uniform strain. Repeated demonstrations of gonadotropic honnones were obtained for as long as 6 months. These assays, however, were achieved using the original Aschheim-Zondek technique and were not quantitated for chorionic gonadotropin content. The intent of our study is to correlate cytologic differentiation with quantitati\c gonadotropin production from trophoblastic tissue propagated in prolonged explant cultures.
From the Department of Gynecology and Obstetrics, Marquette University School of Medicine. Supported by Public Health Grant No. 43-64-52 and Arnerican Cancer Society, Milwaukee Division, Inc.
Materials and methods Sterile trophoblastic tissue is obtained for culture at the time of surgery for ectopic pregnancies. Hydatidiform mole tissue is obtained as available by hysterotomy or cervical
Presented at the Twenty-third International Congress of Physiological Sciences Tokyo, Japan, Sept. 9, 1965.
337
338
Pattillo et a/.
:\111 .
evacuation. The specimen is dissected in a ste rile Petri dish. Decidual elements, fibrin, and blood are meticulously removed. The villi arc washed in f[,·e changt'S of Hanks' bala nced salt solution and are mechanically minced. One drop of the suspension is mixed on a cover slip with similar amounts of chick embryo extract and chicken pl a sn1a and allowed to clot. The CO\Tr slip is placed in a culture tube containing meditim NCTC I 09 ; 20 p er u·nt calf sl'rurn with 1Oil units nf p enicillin, and 50 gamma streptomycin per milliliter. The cultme tube is transferred to the roller drum in an incubator a t 370 C. T-flasks are explanted at the sanw time. The m edium is changed every :"l to days and subcultures of flask cultures m a d e as indica ted. Gro\o\·th obsen at ions of the living cultures are made using the Plankton Jnicroscope . Cowr slips are periodically stained witl1 hematoxylin and eosin frolll roller tube cultures for growth observation and permanent mounts.
+
Results Morphologic observations. Several cell types were observt:>d in actively proliferating trophoblastic cultures. The broa d and elonga ted ce ll showing multiple cytoplasmic projec tions, often with interconnections between a djoining cells (Fig. 1) , w a s present in all cultures demonstrating gonadotropin produc-
Fig. l. Broad and elongated cell type showing cytoplasmic projections and interconnec tion of adjoining cells ( ectopic placenta in c ulture one month) . (x lOO.)
( kt obt'r I ! ~ lt lL & ( ;y uq· .
J. Oh ~ t.
tion. Fine vacuolation of tlH· cvtoplasm '' a~ regularly observed. Its gro\o\·th pattern \1 a~ one of migration from the t·xplant a~ 'inglt· cells (Fig. 2 1 . This cell was usu;dh- ohsnwcl after .'i to 7 clays of cultme. Tht· nucle i m-rt · nmnd tu m·a l, light staining, and conta im:cl several nucleoli ( Fig. :{ · . :\1 ul tinucl<'a tinu was obscn·ed ( Fig. In so11w cases, ln11~ continuous sheets of n·lls g re11 out in a syncytium. [n other instanu·s. rnultinuckatl'tl trophoblas ti c buds could lw sct·n tc' ptt ,;lt out from the expla nt. The pleomorphic epithelioid cdl or ectopic placentas and hyda tidiforlll ntolt •s . Cttlturcs of hydatidiforn1 Jllnles dell1onstratec1 a strong tendency to isolat<'d liqllefa rtion and circular growth formation '. Fi!.!. (), Rounded cells with perinuclear halos. spindk. oval, oblong. and bilobecl cells IHTC obse rved in a more d ense area of tfw explant (Fig. 7 of both hyda tidiform mole and nonua l tr<>phoblast cultures. At the peripherv. tht' broad and elongated cells with cl"topbsrnic pw.icc1ions \H' n' obsen·ed (Fig. B ' . Endocrine assays. T!w tissue culture fluid was a ssa\T d for chorionic gonadotropin u ' in.t; the inunatttrc rat uterirH· 1\t"ight m e thod. " Test 111atnial is gi1·en in -;j:-; in jl' ctit>n' '" t'l 2 days 11itlt sa nifice at 72 l)(ltJrs . Till' ttkrirw
+·,.
Fig. 2. Photograph from living trophobl as t utilure taken in T-Aask. In culturP 9 wPeks. R ('frac til c granuks in cytoplasm of cell in upper l't· ntn
Volume 96 :"Jumht'J" 3
Fig. 3. Ectopic placenta in culture one month. Light stain in g, round and O\'al nuclei with sevC'ra l nucleoli. ( · 100.)
Fig. 4. Multinucleated cells having migrated from thC' clot and growing on glass. (><100.)
Fig. 5. Pleomorphic, multiangular cell with fine brush border on inferior surface of cell on left. (x400.)
Trophoblast activity
1n
explant culture
339
response end point is secondary to ovarian stimulation and is sensitive to 0.5 I. U. Cellfree supernatant fluid is either assayed directly or the gonadotropin is concentrated by extraction and precipitation to a dry powder and reconstituted in a known volume of saline prior to biologic assay. Ether extraction removes steroids which would interfere with the assay. All tissue culture medium, calf serum additives, embryo extract, and plasma clot are assayed for controls to rule out an exogenous source of gonadotropin and estrogens. Dose levels comparable to the maximum quantity received by the animal during any of the assays are tested . Calf serum in volumes of 3 to 15 mi. and chicken plasma and chick embryo extract at 5 m i. levels were negative for gonadotropin. Tissue fluid media from the first week in culture is discarded. This eliminates false positive results from gonadotropin present in the original tissue. Assays on tissue fluid after the first week were usually negative in our laboratory if trophoblastic growth had not occurred. The total number of days each specimen was maintained in culture is shown in Table I to range from 21 to 94 days. The initial endocrine assays were conducted at 14 days (first week media changes discarded) . Gonadotropin content is reported per total pooled tissue fluid to reflect total cellular output. This is converted to units per 1nilliliter
Fig. 6. Liquefaction centers with circular growth formation. Hydatidiform mole in culture 3 weeks. (xlOO.)
340
t ktobt·l I . I ~U .b
Pattillo et al.
\JJ I.
Fig. 7. De nsely pop ula t< ·cl an ·a of t'x plan t show ing seve ral cell types: ob long ce lls, ro und ce lls w ith perinuclear halos. bilolw d n ·ll, and mitotic cell ( ldt cen ter 1. H ydatid ifo r m mole culture, :l weeks. (
Fig. 8. Per iph e ra l an·a fro 111 s:llllt · ;· ul tun·
& f;y 1w1
:JS
Fi ~.
I. In lt•ss d rnsc area of cu lt un·. tb !· r,-- lls d isplaY thf' long- thin cy toplasm ic proj cn ioth .
1
Table I. H CG assays* of tissue fluid from ectopic trophoblast in explant culture
I Specime n
( '1'-fiask )
-
- ----
I. U ./total 1 Total dav.< fwoled ti.u ue ! I . U. / ml. of in cult u re 1 fluid ! tiH u e fluid 2+ ~+
39 39 :' I 9+
79+.00 IH15 9. 1H 13.75 +~.6 6
5.60
150
INT.
H CGauayafta l4day.<
~ - - ---- - --
K.M . V. M . S. E. C.L. M. W. S. E.
.J . ()h., t .
UNITS HCG
100
10.7 00 0. 190 0. 12-J. 0.1 70 0.576
o.:wo
•·M ethod of De ifs."
7
21
35
49
63
77
91
da y s in cu lt ure
by dividing by the total volume of culture medium used. The most ac tive culture p roduced 794.0 I. U. ; the least active p roduced 5.60 I. U . In order to further elimina te any possibil ity of gonadotropin from the original fluid or tissue, Table II records endocrine assays only afte r a minim um of 3 weeks in cu lture (first 2 weeks media cha nges excluded ). A decrease to 392.70 I. U . is seen in the most active culture. Of 8 additional ectopic trophoblast cultures not shown in these tables, 4 demonstrated similar cytologic pa tterns, a nd growth was observed from 13 to 20 days. Positive assays for H C G were found in 3 cases ; one was not assayed. Decidual growth' occurred
Fig. 9. Chori on ic gona dotropin response in t iss Ut· culture. Th,· po ints at 1+ and :?! days re presen t pai red assays on 15 and 5 speci m en . res pectively. The points at 37, 6 3, and 9+ days re present ertupi c placental spec imen S. E .. m ain ta ined in flask culture for 94 days.
m one specimen, no growth occ urred in the remaining 3. Assays on 2 hydatidiform m oles m aintained in culture for 21 and 24 days were also perform ed (T able III ) . The dedine in gonadotropin production previous ly observed with increasing age of the cultures cannot be evaluated in one of the specimens dtt c to loss of a p ortion of the tissue flu id. The average gonadotropin response derived in this study from p rolonged trophobl astic cultures is graphically illustrated in
Volume 96
Trophoblast activity rn explant culture 341
.'lumber 3
Table II. HCG assays after 21 days in culture
Specimen
I. U./total pooled tissue fluid
K.M. V. M. S. E. C. L. M.W. \ S. E. ) (T-Rask)
:l92.70 1.70 18.20 12.05 19.47 8.49 2.19
I. U./ml. of tissue fluid
3.850 0.015 0.076 0.110 0.190 0.152 ().(155
Table III. HCG assays of hydatidiform mole in explant culture
Days S pecimen
culture
B.A. H. L.J.
24 21
zn
Assay after 14 days
Assay after 21 days
I. U./ I pooled I. U./ ml. fluid
I. U./ I pooled I. U./ ml. fluid
3.09 34.65
0.08 0.47
6.01 3.64
0.790 0.035
Fig. 9. The average gonadotropin content of all cultures assayed is plotted against days in culture. A low but stable level is observed through 94 days. The sharp drop in gonadotropin content may reflect lack of some specific substance needed in the culture environment or an accumulation of inhibitory products by the natural degenerative changes in some of the explants. Further studies toward elucidating these factors and improving growth and survival are being pursued. Comment
Persistent gonadotropin production in prolonged trophoblast tissue cultures was associ-
ated with the presence of a broad and elongated epithelial cell displaying multiple cytoplasmic processes or pseudopodia. This type of cell was predominant in cultures which were maintained for 94 davs with continued gonadotropin production. Increased knowledge of normal trophoblastic cell function is a prerequisite to the study of its abnormal forms. Accurate measurement of gonadotropic hormone production provides a sensitive indicator of cellular activity. The tissue culture system offers a convenient means by which pathologic and neoplastic states of the placenta may be studied. Further knowledge of cellular function of the trophoblast at a biochemical and metabolic level should contribute to an understanding of the clinical problems of ITproductive failures, abortions, and neoplasms of the placenta. Summary
Trophoblastic tissue from 13 ectopic pregnancies and 2 hydatidiform moles have been grown in prolonged explant cultures. Quantitative gonadotropin hormone production and characteristics of cell growth are described. From these studies, it is observed that a characteristic cell, which migrates from tlw explant and displays a broad and elongated form with multiple cytoplasmic projections as described originally by Jones, Gey. and Gey,' is consistently associated with persistent gonadotropin production and prolonged survival of the trophoblast in culture. Gonadotropic hormone output was shown to decline sharply after explantation and to maintain a low level for a prolonged period thereafter.
REFERENCES
1. Gey, G. 0., Seegar, G. E., and Hellman, L. M.: Science 88: 306, 1938. 2. Delfs, E.: Endocrinology 28: 196, 1941. 3. Soma, H., Ehrmann, R. E., and Hertig, A. T.: Obst. & Gynec. 18: 704, 1961. 4. Waltz, H. K., Tullner, W. W., Evans, V. J., Hertz, R., and Earle, W. R.: J. Nat. Cancer Inst. 14: 1173, 1954.
5. Stewart, H. L., Jr., Sana, M. E., and Montgomery, T. L.: J. Endocrinol. 8: 175, 1948. 6. Thied~, H. A.: AM. J. 0BsT. & Gv:-mc. 79: 635, 1960. 7. Jones, G. E. S., Gey, G. 0., and Gey, M. K.: Bull. Johns Hopkins Hosp. 72: 26, 1943. 8. Hellweg, G., and Shaka, J. A.: Obst. & Gyncc. 13: 519, 1959.
NEWBORN Cytologic and hormonal activity of trophoblast in explant culture ROLAND A. PATTILLO, M.D. THOMAS C. SMITH, PH.D., M.D. ELEANOR DELFS, M.D. RICHARD F. MATTINGLY, M.D. Milwaukee, Wisconsin
T H E T R o P H o B L A s T was shown to produce gonadotropin in tissue culture by Gey, Seegar, and Hellman 1 in 1938. Since that time, the development of a sensitive biologic assay 2 has made possible the more accurate determination of gonadotropic hormone in minute quantities. Concentration of the hormone by alcohol precipitation allows detection of small quantities present in pooled tissue fluid in which placental tissue is grown. Previous studies 3 have emphasized various cell types and growth patterns associated with short-term gonadotropin production. No unanimity exists at this time as to the cell type responsible for gonadotropin production.4 Stewart, Sano, and Montgomery 5 described three types of cells in explants of immature placentas: the oblong cell with an oval nucleus, taken to represent the syncytial celL the round cell with a round nucleus,
and a perinuclear halo, believed to represent the Langhans cell and the stromal cell with a round nucleus. Thiede 6 described the pleomorphic epithelioid or polygonal cell, the stromal cell, and a multinucleated giant cell observed in 16 hour cultures. Jones, Gey, and Gey 7 in their early report described broad and elongated cells, interconnected by multiple long narro\A/ cytoplasmic processes. When definite tissue culture colonies of this type were produced they could be maintained in continuous culture as a uniform strain. Repeated demonstrations of gonadotropic honnones were obtained for as long as 6 months. These assays, however, were achieved using the original Aschheim-Zondek technique and were not quantitated for chorionic gonadotropin content. The intent of our study is to correlate cytologic differentiation with quantitati\c gonadotropin production from trophoblastic tissue propagated in prolonged explant cultures.
From the Department of Gynecology and Obstetrics, Marquette University School of Medicine. Supported by Public Health Grant No. 43-64-52 and Arnerican Cancer Society, Milwaukee Division, Inc.
Materials and methods Sterile trophoblastic tissue is obtained for culture at the time of surgery for ectopic pregnancies. Hydatidiform mole tissue is obtained as available by hysterotomy or cervical
Presented at the Twenty-third International Congress of Physiological Sciences Tokyo, Japan, Sept. 9, 1965.
337
338
Pattillo et a/.
:\111 .
evacuation. The specimen is dissected in a ste rile Petri dish. Decidual elements, fibrin, and blood are meticulously removed. The villi arc washed in f[,·e changt'S of Hanks' bala nced salt solution and are mechanically minced. One drop of the suspension is mixed on a cover slip with similar amounts of chick embryo extract and chicken pl a sn1a and allowed to clot. The CO\Tr slip is placed in a culture tube containing meditim NCTC I 09 ; 20 p er u·nt calf sl'rurn with 1Oil units nf p enicillin, and 50 gamma streptomycin per milliliter. The cultme tube is transferred to the roller drum in an incubator a t 370 C. T-flasks are explanted at the sanw time. The m edium is changed every :"l to days and subcultures of flask cultures m a d e as indica ted. Gro\o\·th obsen at ions of the living cultures are made using the Plankton Jnicroscope . Cowr slips are periodically stained witl1 hematoxylin and eosin frolll roller tube cultures for growth observation and permanent mounts.
+
Results Morphologic observations. Several cell types were observt:>d in actively proliferating trophoblastic cultures. The broa d and elonga ted ce ll showing multiple cytoplasmic projec tions, often with interconnections between a djoining cells (Fig. 1) , w a s present in all cultures demonstrating gonadotropin produc-
Fig. l. Broad and elongated cell type showing cytoplasmic projections and interconnec tion of adjoining cells ( ectopic placenta in c ulture one month) . (x lOO.)
( kt obt'r I ! ~ lt lL & ( ;y uq· .
J. Oh ~ t.
tion. Fine vacuolation of tlH· cvtoplasm '' a~ regularly observed. Its gro\o\·th pattern \1 a~ one of migration from the t·xplant a~ 'inglt· cells (Fig. 2 1 . This cell was usu;dh- ohsnwcl after .'i to 7 clays of cultme. Tht· nucle i m-rt · nmnd tu m·a l, light staining, and conta im:cl several nucleoli ( Fig. :{ · . :\1 ul tinucl<'a tinu was obscn·ed ( Fig. In so11w cases, ln11~ continuous sheets of n·lls g re11 out in a syncytium. [n other instanu·s. rnultinuckatl'tl trophoblas ti c buds could lw sct·n tc' ptt ,;lt out from the expla nt. The pleomorphic epithelioid cdl
+·,.
Fig. 2. Photograph from living trophobl as t utilure taken in T-Aask. In culturP 9 wPeks. R ('frac til c granuks in cytoplasm of cell in upper l't· ntn
Volume 96 :"Jumht'J" 3
Fig. 3. Ectopic placenta in culture one month. Light stain in g, round and O\'al nuclei with sevC'ra l nucleoli. ( · 100.)
Fig. 4. Multinucleated cells having migrated from thC' clot and growing on glass. (><100.)
Fig. 5. Pleomorphic, multiangular cell with fine brush border on inferior surface of cell on left. (x400.)
Trophoblast activity
1n
explant culture
339
response end point is secondary to ovarian stimulation and is sensitive to 0.5 I. U. Cellfree supernatant fluid is either assayed directly or the gonadotropin is concentrated by extraction and precipitation to a dry powder and reconstituted in a known volume of saline prior to biologic assay. Ether extraction removes steroids which would interfere with the assay. All tissue culture medium, calf serum additives, embryo extract, and plasma clot are assayed for controls to rule out an exogenous source of gonadotropin and estrogens. Dose levels comparable to the maximum quantity received by the animal during any of the assays are tested . Calf serum in volumes of 3 to 15 mi. and chicken plasma and chick embryo extract at 5 m i. levels were negative for gonadotropin. Tissue fluid media from the first week in culture is discarded. This eliminates false positive results from gonadotropin present in the original tissue. Assays on tissue fluid after the first week were usually negative in our laboratory if trophoblastic growth had not occurred. The total number of days each specimen was maintained in culture is shown in Table I to range from 21 to 94 days. The initial endocrine assays were conducted at 14 days (first week media changes discarded) . Gonadotropin content is reported per total pooled tissue fluid to reflect total cellular output. This is converted to units per 1nilliliter
Fig. 6. Liquefaction centers with circular growth formation. Hydatidiform mole in culture 3 weeks. (xlOO.)
340
t ktobt·l I . I ~U .b
Pattillo et al.
\JJ I.
Fig. 7. De nsely pop ula t< ·cl an ·a of t'x plan t show ing seve ral cell types: ob long ce lls, ro und ce lls w ith perinuclear halos. bilolw d n ·ll, and mitotic cell ( ldt cen ter 1. H ydatid ifo r m mole culture, :l weeks. (
Fig. 8. Per iph e ra l an·a fro 111 s:llllt · ;· ul tun·
& f;y 1w1
:JS
Fi ~.
I. In lt•ss d rnsc area of cu lt un·. tb !· r,-- lls d isplaY thf' long- thin cy toplasm ic proj cn ioth .
1
Table I. H CG assays* of tissue fluid from ectopic trophoblast in explant culture
I Specime n
( '1'-fiask )
-
- ----
I. U ./total 1 Total dav.< fwoled ti.u ue ! I . U. / ml. of in cult u re 1 fluid ! tiH u e fluid 2+ ~+
39 39 :' I 9+
79+.00 IH15 9. 1H 13.75 +~.6 6
5.60
150
INT.
H CGauayafta l4day.<
~ - - ---- - --
K.M . V. M . S. E. C.L. M. W. S. E.
.J . ()h., t .
UNITS HCG
100
10.7 00 0. 190 0. 12-J. 0.1 70 0.576
o.:wo
•·M ethod of De ifs."
7
21
35
49
63
77
91
da y s in cu lt ure
by dividing by the total volume of culture medium used. The most ac tive culture p roduced 794.0 I. U. ; the least active p roduced 5.60 I. U . In order to further elimina te any possibil ity of gonadotropin from the original fluid or tissue, Table II records endocrine assays only afte r a minim um of 3 weeks in cu lture (first 2 weeks media cha nges excluded ). A decrease to 392.70 I. U . is seen in the most active culture. Of 8 additional ectopic trophoblast cultures not shown in these tables, 4 demonstrated similar cytologic pa tterns, a nd growth was observed from 13 to 20 days. Positive assays for H C G were found in 3 cases ; one was not assayed. Decidual growth' occurred
Fig. 9. Chori on ic gona dotropin response in t iss Ut· culture. Th,· po ints at 1+ and :?! days re presen t pai red assays on 15 and 5 speci m en . res pectively. The points at 37, 6 3, and 9+ days re present ertupi c placental spec imen S. E .. m ain ta ined in flask culture for 94 days.
m one specimen, no growth occ urred in the remaining 3. Assays on 2 hydatidiform m oles m aintained in culture for 21 and 24 days were also perform ed (T able III ) . The dedine in gonadotropin production previous ly observed with increasing age of the cultures cannot be evaluated in one of the specimens dtt c to loss of a p ortion of the tissue flu id. The average gonadotropin response derived in this study from p rolonged trophobl astic cultures is graphically illustrated in
Volume 96
Trophoblast activity rn explant culture 341
.'lumber 3
Table II. HCG assays after 21 days in culture
Specimen
I. U./total pooled tissue fluid
K.M. V. M. S. E. C. L. M.W. \ S. E. ) (T-Rask)
:l92.70 1.70 18.20 12.05 19.47 8.49 2.19
I. U./ml. of tissue fluid
3.850 0.015 0.076 0.110 0.190 0.152 ().(155
Table III. HCG assays of hydatidiform mole in explant culture
Days S pecimen
culture
B.A. H. L.J.
24 21
zn
Assay after 14 days
Assay after 21 days
I. U./ I pooled I. U./ ml. fluid
I. U./ I pooled I. U./ ml. fluid
3.09 34.65
0.08 0.47
6.01 3.64
0.790 0.035
Fig. 9. The average gonadotropin content of all cultures assayed is plotted against days in culture. A low but stable level is observed through 94 days. The sharp drop in gonadotropin content may reflect lack of some specific substance needed in the culture environment or an accumulation of inhibitory products by the natural degenerative changes in some of the explants. Further studies toward elucidating these factors and improving growth and survival are being pursued. Comment
Persistent gonadotropin production in prolonged trophoblast tissue cultures was associ-
ated with the presence of a broad and elongated epithelial cell displaying multiple cytoplasmic processes or pseudopodia. This type of cell was predominant in cultures which were maintained for 94 davs with continued gonadotropin production. Increased knowledge of normal trophoblastic cell function is a prerequisite to the study of its abnormal forms. Accurate measurement of gonadotropic hormone production provides a sensitive indicator of cellular activity. The tissue culture system offers a convenient means by which pathologic and neoplastic states of the placenta may be studied. Further knowledge of cellular function of the trophoblast at a biochemical and metabolic level should contribute to an understanding of the clinical problems of ITproductive failures, abortions, and neoplasms of the placenta. Summary
Trophoblastic tissue from 13 ectopic pregnancies and 2 hydatidiform moles have been grown in prolonged explant cultures. Quantitative gonadotropin hormone production and characteristics of cell growth are described. From these studies, it is observed that a characteristic cell, which migrates from tlw explant and displays a broad and elongated form with multiple cytoplasmic projections as described originally by Jones, Gey. and Gey,' is consistently associated with persistent gonadotropin production and prolonged survival of the trophoblast in culture. Gonadotropic hormone output was shown to decline sharply after explantation and to maintain a low level for a prolonged period thereafter.
REFERENCES
1. Gey, G. 0., Seegar, G. E., and Hellman, L. M.: Science 88: 306, 1938. 2. Delfs, E.: Endocrinology 28: 196, 1941. 3. Soma, H., Ehrmann, R. E., and Hertig, A. T.: Obst. & Gynec. 18: 704, 1961. 4. Waltz, H. K., Tullner, W. W., Evans, V. J., Hertz, R., and Earle, W. R.: J. Nat. Cancer Inst. 14: 1173, 1954.
5. Stewart, H. L., Jr., Sana, M. E., and Montgomery, T. L.: J. Endocrinol. 8: 175, 1948. 6. Thied~, H. A.: AM. J. 0BsT. & Gv:-mc. 79: 635, 1960. 7. Jones, G. E. S., Gey, G. 0., and Gey, M. K.: Bull. Johns Hopkins Hosp. 72: 26, 1943. 8. Hellweg, G., and Shaka, J. A.: Obst. & Gyncc. 13: 519, 1959.