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Experimentally produced sterile gonads and the problem of the origin of germ cells in the chick embryo.

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'I'he present study is an outgrowth of experiments primarily designed to test the validity of Swift's theory of the
extra-gonadal origin of the germ cells in the chick. I n the
original experiments whole hlustoderrns, mostly of the headprocess stage (about 19 liours incubation), wcre grown in
chorio-allantoic grafts to ascertain thcir capacity l o differentiate a gonad following the excision of the crescentic area of
the so-called primorclial germ cells (JTillier, '26). It was
fourid that gonad and other trunk organs failecl to dc~velop,
anterior organs only being formed. Since in these experinients t h e w appeal-ed to bc a causal comectioii between the
position of the primitive knot arid capacity to form emhryonic parts, experiments were designed to analyze such a relutions11ip (Hunt, '31; Willier and Rawles, '31) and as a conwquencc methods of obtaining gonads free of germ wlls
were discovered. By transplanting entire blastoderms of
stages later than the liead process, i.e., of 3 a i d 9 somites, a
gonad free of germ cells was obtained for the first time
(JYilIier, '32, p. 131). A similar result was obtained by transplanting tlic region containing the primitive knot of a hlastoderm of thc head-process stage. Subsequently a rather
thorough slndy has heeii made using thme two basi, mclthods
of analysis or modifications thereof.
' It
is a pleasiire to ackiioi\lcdgr the excelleiit ns\istxnvc of L h . Mary I:. Ranlpc
this investigation.
Tn all cases the gonatl mhich differentiates iii such grafts is
sterile and consists of typical male-like sex cords, ovarian
cortex iiever having formed. Tlii s singular resilllt not on1.cprcseiits a problem of considerable sigiiificancc in itself but
iiidicates that gonad origin and t o a certain extent its differentiation, is indepenclen t of the so-called primordial germ
cells. These prohlems together with a consideration of their
bearing cm the theory of the extra-eiiihryonic origin of germ
cells in the cliiclr furnish the chief topics f o r analyis in this
A preliniiiary report of thc priiicipal geneidizatioiis of the
studies made on experimcntallp prothiced sterile goiiads has
been rnadc briefly elsewliere (Willier, ’32 ; IViIlier and Rawles,
Ry mcaiia of the clioi-io-allantoic graft chick blastodcrms
at stages ranging from the head process (ca. 18 hours incuhation) i o those having 25 somites mere analj-zed f o r their
capacity to foriri a gonad. Such a range includes a number
of stagtls in the clevelopmciital history of the primordial
germ cells. Large amoeboid cells, which were first descrihetl
by Dnntschakoff ( ’08) a s ‘ entodermallen TTanclerzellen’ and
subsequeritly identified by Swift ( ’14) as ‘primordial germ
cells,’ arise from the gerrn-~i-allcntoclerm just a t the anterolateral iliargiI1 of the pellucid area, the so-called ‘germ cell
crescmit’ (figs. 1, 2 and 3 ) . They first make tlicir appearance
at the primitive streak stage aiitl continue to f o r m until the
embryo has several somites. Thus they accumulatc at the
site of origin in groups in the apace betmccii the riitoclerm
a i d ecloclerm (fig. 3). With the ingrowth of mesoderm into
this region the germ cells enter it, a few being found therein
in 6- and %somite embrvos. As blood vessels clcrelop within
the mesoderm, they a r e caarriecl ( or move by their own
activity) to e w r y p a r t of the vascular area and embryo.
This is the sitnation begiiining with the 10- and 12-somite
stages. They reinain thus clisti-ibntecl until about t h e 22somite stage, at which time the)- liavc entirely clisappearecl
(Dantschakoff). According t o Swift all tlie geriri cells are
to be found in the blood vessels until the 21-somite stage,
after which they gradually dwi*easc in numbers until finally
after the 25-somile stage nolie js found. Duriiig this period
they are entering and aecumulatiiig in the tissues of the
embryo, especially in the splancliiiie rncsoderm of the future
Fig. 1 Map showiiig distribution of the primordial gerrri cells in a head-process
blastoderm. X 29.
Fig. 2 Nap showing distrihution of the priiiiordial g e r m cclls i n a 3 soinite
blnstoderm. >: 27.
Summarily stated, we may recognize f o r purposes of the
present experimental analysis three critical stages in the developmental history of tlie germ cells : blastoderms in which
the germ cells 1) are at the sitc of origin 2 ) are in the blood
vessels and 3) have entered the splanchnie mesoderm of the
future gonad.
Tlie manner of isolation of tlie portion of the blastoclerm to
be grafted is somewhat variable. In the majority of cases
examined the trarisplaiit consisted of the entire pellucid area
or the greater portion of it isolated in various ways. First,
the transplant included all of the pellucid area except a
narrow marginal zone, the line of excision falling just inside
the junction of the opaque aiicl pellucid areas. I t was originally thought that snch an isolation w ~ d dfree the traiisplant
of all primordial germ cells. It is now apparent after a careful study of their distribution that while tlie locus of origiri
as well as the greater portion of them are discarded with the
opaque area some undoubtedly remain in the pellucid area
(fig. 1). This is generally true for the head-process blastoderm, the stage used in this type of isolation. In some cases
particularly at later stages the germ cells are distribiited in
such a manner, I%., (as in fig. 2 ) that tlie majority woulcl
be located in the isolated pellucid area rather than in the
cliscardetl portion of the blastoderni. A second type of isolation was made so that all of the germ cells 11-ould be included
in the pellucid area. In this case the line of excision falls external to the junction of the pellwid and opaque areas. The
stages used varied from head process to 11-somites. A third
type of isolation inclucted all of the pellucid area caudal to
the level just anterior to the heart primorclium. As in the
first case the line of excision of the portion grafted lap just
inside the junction of the pellucid aiid opaque areas. The
donor blastoderms ranged from 3- t o 13-somite stages. A
fourth type of implant was isolated so as to includc all of the
pellucid area loetmeen the level of either the twelfth o r fourteenth pair of somites arid the node level (but not including
it). The donor embryos ranged from 1 2 to 25 soinite stages,
during which period the germ cells are supposedly in the
blood circulation or are in the process of entering tlie tissues
of the embryo.
In a smaller group of experiments the transplaiit included a
small piece of blastoderm of the head-process stagc containiiip
the primiti1-e knot of Hensen. Sucli pieces ils tested nieasiired
approximately 0.3 mm. square.
I n still anothor set of experiments the uriiio-genital i-iclges
wcrc isolated from donors having 29 l o 41 sornites. At these
stages ilie germ cclls a r e visible in the gonad-forming areas.
Ririce ihcsc grafts were considered iii a former report (Willier,
’33, fig. 14, p. 641) they are included here for comparative
~ ) u r l ) o s only.
Fig. 3 Photograph of primordial germ crlls situat(d betwccii the crtoderm
entodcrm of :t 3-somite hlastoderm. X 1500.
~ I E S C R I P T I O N o i c TTIE R I ~ X T J L T S
1. Enfire pdliccid area with niost of t h e germ cell cresceizt
czciscd. Thirtccn grafts, all from blastodcrms of the head-
process stage, were examined. They were large grafts coiltaining usually all parts of the embryo back to and in two
cases including the mcsonephros. Neither gonad, adrenal
nor metanephros differentiated. Germ cells were not observed ( V17illier and Rawles, ’31).
2. Entire pellucid area inclicdiiay t h e entire germ cell
crescerzt. From this type of transplant fourteen grafts were
examined, two from blastoderms of the head-process stage
a i d twelve of the somite stages (one each from 2-, 3-, 4- and
5-somite donors; two froin 7-somite, three from 9-, one each
from 9-, 10- and 11-somite donoi-s). These grafts were similar
to those described above in size and in parts of the embryo
formed. Mesonephros but not metanephros has developed in
six grafts, all from blastoderms of somite stages. Both adrenal and gonad are associated with the mesonephros in two
cases (3- and 9-somite donors) and adrenal only in another
case (7-somite donor).
Structure of gonad from 3-sornite donor. This gonad is a
tiny body lying next t o the middle portion of a somewhat
attenuated adrenal body. Both gonad and adrenal protrude
into a coelom-like space in the mcsenchyme of the graft.
When the adrenal is traced through the sections it becomes
more or less continuous with a much drawn-out body of mesonephric tissue. Several germ cells occur in the mesenchyme
of this graft.
Structurally thc gonad is composed of a very few male-like
sexual cords each circumscribed by a thin connective tissue
envelope. The cords are short and unbranched and consist
of non-germinal cells somewhat irrcgularly arranged as a
rule, but in three cords their nuclei occupy a more regular
position next to llie wall of the sex cord, resembling somewhat
closely a normal testicular cord of a corresponding age except for the absence of germ cells. The amount of intercordal
connective tissue is small.
Gonad from 9-somite donor. ‘I’his is the first sterilc gonad
ever obtained in our study. T t is a cylindrical body, measuring 386 .u i n length and 290 p in diameter. It is surrounded
with a coclom-like cavity, except on one side where it is contiiiuous with the graft mesenchyme, and at one end where it
joins the mesonephros. Lying in the mesenchyme near its
nriion wiih the mesonephros is a mass of suprarenal tissue.
l~iliethe gonad described above, it consists of male-like
sexual cords, some intercordal connectivc tissue and tl tuniclike envelope of mesenchyme. I n the intercordal tissue there
is a considerable number of blood spaces containing red blood
corpuscles. T h e sexual cords range in form from irregular
collections of non-germinal cells t o definitely circumscribed
cords which a r c somewhat elongated and even branched
(Willier, '32, p. 131, fig. 16). I n some of Ihe highly differentiated scx cords tlic nuclei of the non-germinal cells show a
tendeiicy to bc arranged at thc periphery of the cord as in
the normal. Morc atypically, however, they are irregularly
arranged, a condition which is characterislic of young scx
cords. After a careful search no gcrm cclls werc recognized
either in Ihc sex cords, or i n a n y other p a r t of the gonad,
or even clscwheiv in the graft.
3 . Entire pcllucid arca cxccpt portion aiztciior t o hcart
l e u d Twciity grafts of this category were examined from
donors liaving From 3 to 13 somitcs (one each from 3- and
4-soinitc, two from 5-somite, thrcc from 6-somite, two from
7-somite, onc from 8-somitc, five from 9-somitc, two from
10-soniite, and one cach from 11-, 12- and 13-somite embryos).
I n -this ippe of graft wl1er~tho head rcgion as wcll as thc
germ cell crescent were discarded, it was thought that if the
size of tlie implant or tlic dominant head region wcrc reduced,
growth and differentiation of trunk parts might he favored,
rcsulting in ail increased frequency of gonad formation.
Evidcncc indicates that this is thc casc, since mesonepliros ant3
adrcnal haw appcarcd with far greater f requeney, the former
in sixtecn and tlic latter in thirteen grafts. Furthermore, a
more posterior organ, the mctancphros, has appearcd definitely in two grafts and probably is present in two others.
This organ occurs only in grafts containing both mcsonepliros
and adrenal. With tlie few exceptions noted these grafts are
similar in thc kinds of p a r t s differentiated to those dcscribed
above. A small gonad associated with tlic mesoiicphros has
developed in two grafts (6- and 10-somite donors). Metanephros and adrcnal a r e also present in each of these grafts.
Gonad from 6-somite donor. I n this graft, grown on a
female host, two separate adreno-gonad-mesoncphros complexes have developed very much as in the normal embryo.
T o one side (apparently dorsal) of them is a rather typical
spinal cord surrounded by cartilaginous vertebrae. On the
K. H. WILT,IF,ll
opposite side (ventral) there a r e two coelom-like cavities in
the mesenchyme each partially surrounding a mesonephros.
Into these cavities two small gonads a r e suspended from each
mesonephros. Adjacent to each mesonephros on the side
toward the spinal cord is a mass of adrenal tissue. Each
gonad is a definitely circumscribed cylindrical body of small
dimensions (the one shown in fig. 4 measures 156 p in length
by 145 in diameter ; the other three have similar dimensions).
The two gonads of each mesonephric complex a r e separated
by a gonad-free zone which apparently marks the line of constriction of aii originally single gonad-forming area.
Histologically each gonad consists of three kinds of tissues,
viz., male-like sexual cords, intercordal tissue, and next to the
coelom-like space a tunic of connective tissue. The intercordal tissue is a stroma of connective tissue containing blood
capillaries and occasionally a rounded cell. The sexual cords
although relatively few in number a r e well-formed structures,
only occasionally showing any indication of convolutions, or
branches. They arc made up of non-germinal cells only. As
seen in figure 4 the sexual cords show a high degree of cliffei-entiation. The riuclei of the non-germinal cells a r e situated
in a row at the periphery of the cord and from them cytoplasmic strands extend inward. This histological picture is
similar to that seen in the sex cords of a normal testis of a n
embryo of the corresponding ngc (i.e., 1 2 days). I n some of
ihe cords the nuclei a r c less definitely arranged.
Gonad from 10-soniite donor. The gonad in this case is a
small cylindrical body about 198 p in length and 146 p in
diameter, resembling in form and size the gonad just described. It, together with a mass of adrenal tissue i n juxtaposition, is situated at one end of a much elongated mesonephros. The mesonepliros and adrenal lie embedded in mesenchyme, while the gonad is surrounded, except where it unites
with the niesonephros, by a coclom-like cavity. A t the opposite end of the mesonephros lies a metaiiephric body.
With respect to the kinds of tissues, this gonad is very
similar to the one just described. Thc tunica albuginca is
thinncr and tlic sexual cords fewer and of smaller diameter.
Some arc dcfiriitely branched and the nuclei of the iiongerminal cell a r e only occasionally typically situated at the
periphery oP certain cords.
4. Portion of the pellucid area from a p p r o z i m a t e l y t h e
15-somite lcvrl to node l c v d (donors 12 to 25 somitcs).
Twenty-four gi.afls have bcen exarriiiied from the level of tlic
Fig. 4 A sterile gonad differentiated on a female host froin n. (i-somite blastoderin following the removal of the head and the entire germ cell cresccnt. X 650.
R. 11. V I ~ I L L I E ~
pellucid area destined t o form the mesonephros (from fifteenth to thirtieth somite in the normal embryo). The
anterior margin of the transplant lay hetween the fourteenth
and fifteenth somite level except in the 12- and 13-somite
donors where it was between the last two somitcs formed. In
all cases the posterior margin lay just in front of the node.
Of the grafts examined, one came from a 12-somite, one from
a 13-somitc, two from lLsomitc, t h i w from lG-somitc, two
from l'I-somite, 1wo from B s o m i t e , two from 20-somite, two
from 2l-somite, one from 22-somitc, two from 23-somite, four
from 24-somiter and two from 25-somite donors. The typical
structures found in thcsc grafts a r c spinal cord, ganglia, nototissues a s
cliord, mesonephros and such gcncr~~lly-occurring
gut, cartilage, bone, miisclc, skin and feather germs.
Siricc we arc dcaliiig her-e with thc level of blic pellucid area
normally concerned with the formation of the adreiial-gonadniesoncphros complcx these tissues would be expected to occur
with a high freqimicy. The results, howevcr, turned out to
be rather disappointing in this respect, since only fourteen
of the twciity-four grafts showed any developmciit of the
mesoncphros, and only about half of thcsc showed adrenal.
(lollad, appearing rriiich less frequently than either mesonephros or adreiial, has differentiated in juxtaposition to these
tissues in two cases as a small body devoid of gcrm cells.
These two gonads, obtained from 15- and 18-somite donors
respectively will now be described.
Gonad from 15-somite donor. 'I'hc gonad in this case is an
exceptionally small body (120 11 X 160 1 1 ) . One side of it is
continuous with a small mesonephros, while Ihe other protrudes into a coelom-like cavity. Nearby in the mcscnchyme
is a small mass of adrenal cords. The free surface of the
gonad is covered with a thin mesenchynial wall.
The sexual cords a r e quite few in nixmlner, short and slender,
and in some cases not very distinctly outlined. Tn several
cords the nuclei of the non-germilia1 cells are arranged at the
surface of the cord, thus presenting a somewhat typical
picture of a inale sex cord.
Gonad from 18-somite donor. This gonad is a sphcrical
body (ZOO p ill diameter) situated at one end of a somewhat
clongated mcsoncphros, wherc it protrudcs into a large
coelom-like cavity. Adrenal tissuc is not associated with the
gonad bnt with one or two othcr masses of mesoncphric tissuc
found elsewliere in the samc graft.
r i
I h e gonad is made up of sterile sex cords ranging from a
few which are typically male-ljke in structure (peripheral
position of nuclei of lion-germinal cells) to many which appear in section as ncsts of two o r more non-gcrminal cells.
Between the scx cords is an abundance of loosc connective
tissue containing iiumerous blood channels. Next to the
‘coelome’ the gonad is covcred with a single layer of flattened
cells. Betwcen this and the sex cords is a comparatively thick
layer of loosc mesenchyme.
5. Piece of blastoderm coiataiiuhg the primitive knot.
Fifteen grafts of the node region of thc blastoderm in the
head-process stage have been examined histologically.
Mesonephros has differcntiated in eight of these. Associatcd
with it in two grafts arc adrenal, gonad and metanephric
tissues and in two others adrcnal tissue only.
Gonad 223-5-1. I n juxtaposition t o onc end of an elongated
mass of mcsonephric tissue lies a cylindrical gonad body of
small dimensions (175 p X 117 p ) . Adrcnal docs not appear
in this particular complex but is connected with a second mass
of mcsoiiephric tissue found clsewhcre in the graft. The
gonad, although a definitely circumscribcd body, is neither
surrounded by a coelom nor covcred with a tunica albuginea.
Its surface is continuous with the surrounding loose mcsenchyme cxccpt where it comes in contact with mesonephric
tubulcs. It is made up of sex cords containing only iiongerminal cells a s may be seen in figure 5. The cords exhibit
sonic variation in structure. A fcw are wcll defined with the
nuclei of non-germinal cells peripherally situated and
oriented. Many slender cords are found in which the nongerminal cells and their nuclei are irregularly arranged. Both
types of cords may be branched or slightly convoluted.
Masses of lion-germinal cells not formed into definite cords
also occur.
Gonad 226-4-1. About mid position of a n elongated mass
of mesoiicphric tissue a i d in contact with it is found a gonad
Fig. 5 A sterile gonad differciitiatcd on n male host froiii a small portion coiltniniiig Nensen 's node of n head-process blastoderm. X 500.
of small dimensions (168 p x 117 p ) . At the surface of the
mesonephros, about 90" away appears a small adrcnal of
poorly diffcrentiatcd cords. This gonad is a definitcly circumscribed cylindrical body lying in loose mesenchyme. 111 juxtaposi tiori are two slender extensions of coclom-like spaces, one
of which actually comes in contact with the sex cords of the
gonad. Struoturally the sex cords arc very similar to those
just described; in number, however, they are fewer.
Estra-golzadal origin of the g e r m cells. The development
of a gonad Pree of germ cells as found and described in the
present series of grafting experiments furnishes strong evidence that ‘the germ cell crescent’ of Swift is the source of
the germ cells of the gonad rudiment. This result is in
agreement with the findings of other investigators who used
different methods of freeing blastoderms of primordial germ
cells in early stages of development (just prior to formation
of first somite). The ‘germ cell crescent’ of the blastoderm
has been excised (Reagan, ’16), irradiated with ultraviolet
(Benoit, ’30) and destroyed by means of electric cautery
(Dantschakoff et al., ’31). A histological examination of
blastoderms thus treated and incubated for 2 or more days reveals the entire absence of germ cells from the gonad-forming
areas. It is thus seen that regardless of the method used for
removing germ cells from the blastoderm the gonad-forming
area which subsequently develops is sterile. If such a gonadforming area is permitted to develop as is done in the present
grafting experiments, it forms a definite sex gland which is
also sterile.
Can these results be accepted as proof of the extra-gonadal
theory of origin of the germ cells in the chick? Certain evidence obtained from our graft studies indicates the necessity
of exercising caution in making such an interpretation. I n
the first place such a conclusion is rather difficult to reconcile
with the finding that a sterile gonad forms in those implants
of the pellucid area which included the germ cell crescent.
I n such grafts the germ cells may be found scattered in the
mesenchyme or missing altogether. Whether they are present
in the graft makes no difference in the result. This is expected since the pellucid area in the graft develops no blood
vascular system of its own, coiisequently the germ cells of the
crescent have little o r no chance of getting t o the developing
gonad. The complete disappearance of the germ cells from
the implant may be explained as the result of their entrance
into the blood circulation of the host embryo or of phagocytic
A second disturbing point in this connection is the discovery
that a sterile gonad may form in a graft of the gonad-forming
area at a stage when germ cells are already present in
it (Willier, '33, p. 641). This has occurred in two cases out
of seventy-five examined. Also in a few grafts a part of the
same body of gonad tissue may be sterile. Sirice germ cells
are found in the mesenchyme of these grafts, it is likely that
they have migrated from the differentiating gonad-forming
area, or a part thereof, leaving it sterile.
A third significant point is the occurrence of germ cells in
a graft of a tiny median piece containing the anterior half of
the node of a blastoderm of a head-process stage (Rawles,
'36, p. 291). The germ cells found in such a graft, are identical
as regards structure to those found in lateral pieces which
included some of the germinal crescent and are apparently
similar to those occurring in grafts of the gonad-forming0 area
The significance of this result is somewhat puzzling. It is
conceivable in the first place (especially since these so-called
germ cells appear in grafts from practically all but the most
posterior regions of the pellucid area) that they represent a
special type of cell characteristically developed in the grafted
pieces. A second interpretation is that the node area, which
seems to have considerable powers of reconstitution, forms
germ cells de novo. It is suggested, in the third place, that
the site of origin of germ cells is much more extensive than
has been described by Swift and others. A preliminary study
of the distribution of the primordial germ cells in blastoderms
of pre-somite and early somite stages reveals that they are
not confined to a narrow crescentic zone at the anterolateral
boundary of the pellucid area but occur in small numbers
medially and posteriorly from it (figs. 1 and 2). I n view of
such a scattered distribution the results with the median piece
can be fitted into the view that germ cells are extra-gonadal
in origin.
A fourth consideration bearing on the question of germ
cell origin is that conditions peculiar to the graft may inhilit
the differentiation of germ cells similar to what appears to
happen in the case of the gonad. The very small size of the
gonad formed and low frequency of formation in the grafts
of the present experiments, suggest that conditions are not
ideal for its origination. This is apparently not due to the
absence of the primordial germ cells since a similar low frequency was found t o be characteristic of grafts of the gonadforming area at a stage when germ cells are present in it
(Willier, '33). There is evidently some condition lacking in
grafted pieces from blastoclerms prior to 72 hours incubation,
that accounts for the failure of gonad differentiation. This
is somewhat peculiar to gonad for under the same conditions
mesonephros and suprarenal differentiate well and with a
somewhat higher frequency. It sccrns reasonable t o expect,
therefore, that under such unfavorable circumstances germ
cells might fail t o differentiate.
A significant problem for discussion at this time is the
nature of the so-called primordial germ cells in the chick. On
this subject considerable controversp has arisen among investigators (for a recent review of the literature on this subject see Blocker, ' 3 3 ) . The chief point at issue is whether
these cells are germinal or non-germinal in nature. That
these cells are germinal in nature from the time of their early
segregation is the more commonly, but not too critically, accepted view. Swift ( '15 and '16), Goldsmith ('28) and others
claim not only to have traced their migration into the developing gonad rudiment but to have followed the cytological
changes as they transform into definitive sex cells. An argument of more recent origin, based on the experiments of
Benoit, Reagan and Dantschakoff, is that since injury o r
destruction of the germinal crescent results in the appearance
of a gonad area free from germ cells, they must be the
forerunners of definitive sex cells. These descriptive and
experimental studies seem to indicate that the primordial
germ cells are the sole source of all definitive sex cells. That
the experimentally produced sterile gonad rudiment may have
the ability to form sex cells at some later stages is not ruled
out. Indeed Fell ('23) and Gatenhy ('24) have brought forward evidence that germ cells may arise from the peritoneum
of the adult fowl. Others, such a s Firket ('14 and '20) and
de Winiwarter and Sainmont ('09) have arrived at an interpretation, not too convincing, for the existence of two sets
of germ cells. The first or primary ones degenerate and are
replaced by germ cells which arise from the epithelial components of the gonad. A related interpretation is that the
definitive sex cells come from these two sources.
The strongest evidence for the interpretation that the
primordial germ cells are the sole source of the definitive sex
cells comes irom an experiment,al analysis of the sexual nature
of the right ovary of the domestic fowl. It is found that the
primordial germ cells (assuming their extra-gonadal origin)
persist in the riglit ovary until the third week after which
they gradually disappear (Brode, '28). I f the left ovary is
excised before the time of their disappearance, the right ovary
transforms into a testis containing male sex cells which may
even reach the stage of mature spermatozoa; on the other
hand, if removed after the time of their disappearance no sex
cells whatever differentiate in the testis (Benoit, '23; Domm,
The alternative view for examination is that primordial
germ cells are non-gcrminal in nature. Berenberg-Gossler
('14),although able t o confirm in all essentials Swift's account of their origin and migration to the genital ridges, regarded them not as germ cells at all but as entodermal
wandering cells which transform late into mesodermal cellsand being like any other mesodermal cell may give rise to
sex cells. I n studying the development of the germ cells in
the human testis Stieve ('27) reaches a similar conclusion.
I n seems highly probable that the primordial germ cells are
not germ cells in the strict sense but are relatively undifferentiated cells which remain in this condition until gonad
differentiation sets in. This is beautifully brought out in ovotestes produced in chick embryos by the injection of sex
hormones (Willier et al., ’3i). I n such gonads, irrespective
of their zygotic sex constitution, the germ cells differentiate
in the ovarian cortex into oogonia and ih the testicular cords
into spermatogonia. If by chance a germ cell comes to lie
outside of either cortical or testicular tissiie, i.e., in the mesenchyme, it remains undifferentiated. A similar lack of differentiation of germ cells is seen in grafts of the germinal
crescent itself (Willier, ’ 3 3 ) . It is thus evident that the
primitive germ cells not only have the capacity to differentiate
in either the male o r female direction but are dependent upon
a specific sex-cord environment for their differentiation into
epecific sex cells. Such a labile state of the primordial germ
cell is indicative of its generalized nature.
Granting the germinal nature of the primordial germ cells
an interesting problem is presented as to the conditions of
their origin at a particular time and place in the blastoderm.
That they are conditioned in the direction of germ cells by
correlative processes seems probable. Certainly such an
interpretation is more in accord with the principle of correlative development, shown t o hold in the determination of other
parts of the embryo, than with the view that they represent
a ‘segregation of germ plasm’ in the Weismannian sense.
The nature of the processes involved in their origin remains
for future elucidation.
Gonad diff eremtiation iwlependeiat of primordial germ cells.
Although Reagan, Benoit and Dantschakoff have shown that
the gonad-forming area of an embryo may be freed of germ
cells by removing o r destroying the germinal crescent, its
power to differentiate into a gonad recognizable as to sex has
hitherto not been ascertained. The failure of the embryos to
live long following the destruction of the germinal crescent
largely explains the lack of data on this point. On this
‘1’111: A N A T 0 3 1 I C A I , RECORD,
VOL. 70, NO. 1 AND S U P P L E X E N T NO. 1
problem Dantschakoff ( ’31, ’32) has argued that since no
gonad rudiment forms when the entire crescent is cauterized
and one with a reduced number of germ cells forms when the
germinal crescent is only two-thirds o r three-fourths destroyed, the entodermal wandering cells (germ cells) act as an
‘organizer’ of the gonad. I n other words the germinal epithelium alone cannot ‘organize’ a gonad. I n most of the
cases of complete cauterization of the crescent the embryos
are, it seems probable, not old enough a t the time of their
examinatioii-in most cases less than 80 hours old-to have
formed a distinct gonad rudiment. The injuries brought
about by the drastic treatment necessarily given such embryos
has certainly retarded their de\Telopment as compared with
the normal. According to our studies the germinal epithelium
malies its first appearance at approximately the 35-somite
stage as a definite thickening of the coelomic epithelium. This
stage may be reached in embryos incubated from 60 hours
(Willier, ’33) to 84 hours (Swift, ’15) depending upon the
temperature of incubation, season of the year, and other
The abnormal conditions, especially the changes in vascular
circulation, may inhibit the development of the gonad rudiment in such embryos. That the origination of the gonad
rudiment is easily inhibited is indicated by certain studies on
its frequency of formation in chorio-allantoic grafts. I n
grafts containing the portion of the embryo normally forming
the adi*enal-gonad-mesonephros complex, i.e., the level between the fifteenth sornite and the node of embryos of 15- to
17-somite stages, mesonephros differentiates in the majority
of cases, adrenal in half of the cases and gonad somewhat
rarely. It is evident therefore that these results of Dantschakoff cannot be accepted as proving that the gonad rudiment is
unable to develop in the absence of primordial germ cells.
On the contrary the gonad rudiment not only has the power
to arise but it may differentiate into a sterile gonad independently of primordial germ cells. This has been shown by
testing in chorio-allantoic grafts the power of the whole
pellucid area or a certain small portion of it at selected stages
in its development to differentiate gonad. The stages chosen
were either early somite or head process, all prior t o any
possible migration of the primordial germ cells from their
seat of origin and prior to the formation of the gonad-forming
areas. Whether the germinal crescent is removed from the
implants of the whole pellucid area seems t o make no difference in the result, since if it is included there is no
mechanism for transporting the germ cells to the developing
gonad areas. The miniature sterile body wliieh has formed in
a total of ten cases, is without doubt a gonad as its structural
organization and topographical position, i.e., in juxtaposition
to mesonephros and adrenal, conclusively demonstrate. The
small size of the gonad is not to be correlated with the absence
of germ cells, since it has been shown that a transplanted
gonad-forming area in which they are present likewise forms
a small gonad. As previously pointed out (Willier, '33,
p. 636) differentiation without growth in chorio-allantoic
grafts is the rule not only for gonad but for other organforming systems.
Comparative studies on other animals also show that a
gonad may arise independently of the primordial germ cells.
In the amphibians both Kuschakewitsch ( 'lo) and Humphrey
( '27, '28) have reported the differentiation of a sterile gonad
or its rudiment under certain experimental conditions in which
the germ cells either failed to separate from the entoderm or
their exact history is unknown. A more convincing demonstration is furnished by the experiments of Gcigy ( '31) on the
eggs of Drosophila melanogaster. By exposure to ultraviolet radiation the primitive sex cells situated at the germinal
pole of the egg may be destroyed. If all of these cells are
destroyed the eggs develop normally except that both lobes
of the gonad of both sexes are sterile. If the sex cells of one
side only are destroyed the lobe of the gonad of the corresponding side only is sterile. Although retarded to some
extent in development such sterile ovaries and testes are
normal in form and histology. Similarly in the frog Rournoure ( '37) finds that the somatic elements may form a gonad
'pratiquement privi.cs de tissu fertile ' following ultraviolet
irradiation of the inferior pole of the unse,mented egg.
A question related to the one just discussed is the dependence of the gonad rudiment upon the mesonephros for its
origin and differentiation. Dantschakoff ('32) in her theor?
that all of the component parts (coelomic epithelium, entodermal wandering cells and mesonephros) are essential for
gonad development would support this view. So far as our
evidence goes it is clear that a gonad rudiment may differentiate somewhat independently of the mesonephros, since in
no case are urinogenital connections developed and in a
number of instances the gonad is somewhat distantly removed
from the mesonephros. That a gonad rudiment map arise
entirely independently of the mesonephros is difficult to conceive. Indeed Dantschakoff ('32) has found that the gonad
anlage separated from the mesonephros from embryos of
34 days, fails in chorio-allantoic grafts to form a gonad
with sex cords. Whether the failure to form a gonad is
due to a lack of correlative influences or t o injury effects to
the gonad-f orming areas during their isolation cannot of
course be decided.
A final problem of considerable interest for discussion is
whether the germ cells have a stimulative action upon nongerminal cells and tissues such as the coelomie epithelium or
mesenchyme. This has been discussed somewhat fully in a
previous paper ('CVillier, '33, p. 639 ff.) and the conclusion
reached that the evidence for any such activation is inadequate.
The siizgular differentiation of gonads structurally male.
One of the most interesting results for a n a l y i s is the invariable development of a testis-like gonad, ovarian cortex having
failed to differentiate in each of the ten cases examined.
This is the case regardless of the method used for obtaining
them. Two possible interpretations suggest themselves.
First, these testis-like gonads are to be regarded as having a
male chromosome composition, a differential survival of the
sexes in favor of the males having occurred. Second, on the
basis of the expected sex ratio, approximately half of these
should possess the female chromosome composition. On
statistical grounds it seems somewhat improbable, although
theoretically possible, that all of these gonads should have a
male-determining constitution. Repeated attempts have been
made to ascertain the nature of the chromosome composition
of the gonads and although mitotic figures are moderately
abundant, none is sufficiently ideal to enable one to distinguish
with certainty the sex chromosomes. Should it be established
that some of the gonads have a female chromosome composition, a very significant problem in gonad differentiation
presents itself for analysis, namely: the inability to form
ovarian cortex in the absence of the primordial germ cells.
It may well be that ovarian cortex which in the normal contains by far a greater number of germ cells than the medullary
cords, is unable to form in their absence. Lastly, it should be
pointed out that upon the failure of ovarian cortex to differentiate, the medullary cords would be expected to assume tlic
male structural form of the primary sex cord since such a
transformation is known to occur in the right ovary following
the removal of the left ovary in female chicks after hatching.
I n this manner the invariable development of a male structural composition of the sterile gonads may be explained.
1. Isolated portions of chick blastoderms at certain critical
stages in the developmeiital history of the primordial germ
cells were tested in the chorio-allantoic graft for their power to
form a gonad.
The stages selected include blastoderms in which the primordial germ cells are 1) still at or near the seat of their
origin, the germinal crescent (head process to 10 somites),
2) in the blood vessels (10 somites to 25 somites) and 3 ) in
the gonad-forming areas.
A variety of isolated portions were analyzed : 1) The entire
pellucid area including a small part or all of the germinal
crescent, 2 ) the entire pellucid area except for the head
region and entire germinal crescent, 3 ) the pellucid area between fifteenth somite level and node (12 to 25 somite donors),
B. €
4) a tiny piecc containing the primitive knot of the headprocess blastodcrm and 5 ) gonad-forming areas of from 29to 41-somite donors. The last mentioned are introduced for
a comparative study (Willier, '33).
2. Rcgardless of the nature of the isolatcd piece from the
pellucid area the gonad which differentiates is invariably
sterile and consists of male-like sex cords, ovarian cortex
never having formed. This has occurred in ten cases out of
a total of eighty-six grafts, showing a low frequency of origin.
Typically the gonad lies adjacent to mesonepliros and suprarenal gland. The host may be either male or female. Whether
the germinal crescent is included in the implant or whether
the germ cells persist semis to make no difference in the result obtained, since in all cases the gonad is sterile. This is
accounted f o r in part by the lack of development in the graft
of a mechanism for transporting the primordial germ cells
to the developing gonad areas.
3. The formation of a sterile sex gland from the gonad area
in the absence of primordial germ cells supports Swift's
theory of their extra-gonadal origin. This is not regarded,
however, as a crucial experiment, since gonad development
is apparently easily and singularly inhibited by conditions
peculiar t o the graft, which likewise may inhibit the differentiation of germ cells.
The nature of the primordial germ cell is discussed and the
suggestion made that it is a generalized cell which is dependent upon correlative processes for its origin and for its
subsequent differentiation into a specific sex cell.
4. The result indicates that a gonad rudiment not only has
the pov7er to arise but may differentiate independently of the
primordial germ cells. The relationship of gonad origin
and differentiation t o the mesonephros is discussed and the
conclusion reached that although dependent at first, subsequent differentiation is probably independent of this body.
5. The invariable diff erentiation of gonads structurally
male is an unexpected result. Granting the probability that
some of them ham a female-dcterrnining constitution, a significant problem in gonad origin presents itself for analysis,
vie., the inability -lo form ovarian cortex in the absence of
germ cells, and the assumption by the primary sex cords
of a male rather than a female structure.
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V O ~ . 49,
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