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Regeneration of testis in the fowl and its bearing on germ-cell theory.

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Department of Xedicine, iSckool of Medicine, mid Department of Zoology,
Duke Cniversitg, D w h a m , N o r t h Carolirka
Among the arguments advanced to support his germ-plasm
theory Weismann has written (’04, vol. 1,p. 411) that ‘‘Even
the familiar fact that the excision of the reproductive organs
in all animals produoes sterility proves that no other cells
of the body are able to give rise t o germ cells; germ-plasm
cannot be produced de novo.” Although sterility following
gonadoctomy hardly proves that somatic cells cannot or indeed have not transformed into germ oells, the development
in the adult animal of a gonad de novo containing definitive
germ cells tvoiild provide unequivocal evidence of the capacity
of somatic cells to form germ cells.
I n the present paper are recounted observations which are
believed to establish regeneration of testis in the fowl following complete bilateral castration, and in certain cases evidence is presented f o r the formation of mature spermatozoa
from cells previously differentiated as somatic cells. A preliminary report of these findings was made before the
American Association of Anatomists in April, 1936 (Hooker,
Over a period of 5 years tve have had occasioii for various
reasons to castrate approximately 500 White Leghorn
cockerels. In performing these operations it occasionally
happened that a testis was inadvertently divided with the
C. TV. €IOOKEB A N D B. C U N N l X G l I A M
result that a fragment was left in the animal. In such cases
growth of the comb arid other head furnishings invariably
mas resumed within a month after operation, indicating that
the fragment had hypertrophied, attaining a functional les7el
comparable to that prior to operation. In a few cases, on the
contrary, comb growth mas not resumed until G months to
2 years after castrations which had heen recorded a s complete. The great delay in rcsumption of comb growth in these
cases, as contrasted with the much earlier growth in birds
known to have been incompletely gonadectomized, aroused
our suspicion that w e had encountered de iiovo developnient
of testis subsequent to total bilateral castration.
Upon the accumulation of five cases of the latter class a
study was made to determine the correctness of this suspicion. The study consisted in a n attempt to determine
whether the origiiial castration had bccn complete, and to
ascei*tainwhether the t e d e s in the two types of animals present
any distinguishable differences. While the circumstances
preclude any one test serriiig as an infallible index, several
points have been assembled which strongly suggest that
these five animals have shown de novo testis formation.
At each castratioii the operator recorded his impression of
the completeness of the removal of gonad tissue, and during
the last 2 years all testes taken at operation have been fixed
for histologieal study. From three of the five animals in
question the testes removed at the original operation have
been preserved. Upon examination with a dissecting microscope these testes present no breaks which would be present
if any fragment, however small, had bcen left in the animal.
It seems that no further information could be gained by
sectioning these testes.
Another evidence of the totality of thc original castration
may be had from a study of the relation of the testis to the
peritoneum. The various conditions which might obtain a r e
shown schematically in figure 1,in which each diagram reprcsents a transverse section of a lateral half of the animal at
the gonad levcl. I n a the relations in a normal bird are indicated; the testis is suspended by a mesorchiuni froni the
rcnal pcritoncuni. In total excisioii of the testis a cut is made
through the mesorchium at level To, whereas if a fragment
is left, the cut will bc made at a lc\.-el similar to that at S.
I n a suhtotal excision the fragment will be at the eiid of the
mesorchium ; and when hypertrophy occurs the testis will be
peiitlarit in the coclomc, suspended by a mesorcliiuiii as shown
i n b. If the removal has heen complete, the condition upon
healing will he that shown in c. Should it happen that the
peritoneum in the aclnlt retains gonad-forming capacity, any
Fig. 1 l h g r s m of a transverse section of a lateral half of the animal a t the
gonad leTel. Explanation in text. I<, kidncy; P, peritoncum; T, testis: M,
gonad which might be produced in the adult would presumably arise in a rnaiirier similar to that followed in the
embryo; namely, by proliferation of sex cords from the peritoneum, with thc cords formed in a retroperitoiieal position.
A gonad arising in this fashion TI-ould occupy the position
shown in d. If this gonad were to undergo any corisidcrable
growth, the relations would be of the type shown in e, since
the peritoneum is more o r less taut while the kidney is
capable of dcformation.
C. 1
A large number of animals which have shown comb growth
subsequent t o castration have been examined. I n cvery case
in which it is known that the original castration ~"v'asincomplete tlic relations of the gonad have been precisely as postulated and diagrammed in b. On the other hand, i n each
of the five cases which showed delayed resumption of comb
growth the coridition has been either that shown in d or in e,
precisely as postulated for gonad formation following total
Comb growth in the fowl is a relatively sensitive indicator
of endocrine function of the testis. I n the absence of comb
growth there is no testis tissue present in the animal, or such
testis as is present is elaborating androgenic material at a
sub-threshold level. In this connection the condition in
animal 51 is interesting. At the time the regenerated testis
was removed the comb had attained a size of 7.0 x 4.4 em.,
and removal resulted in prompt regression of the comb. The
mass excised weighed but 10 mg. When this minute mass
of testis tissue produced comb growth of such magnitude it
is by no means unreasoiiable t o consider that no testis tissue
is present in an animal which shows n o comb growth over
an extended period.
Upon histological examination testes known to have grown
from retained fra,ments (fig. 2) are morphologically almost
indistinguishable from normal bird testis o r from autoplastic
testis grafts (fig. 3). The tubules of these testes arc uniformly large and present active spermatogenesis; very f ~ w
or no immature tubules are seen. The testes from the five
animals under consideration, on the other hand, differ strikingly from this pattern (figs. 4, 5, 6). There is an increased
internal lobulation of these gonads with.the lobules of rarying
size and irregular arrangement in contrast to the radial
disposition and fairly uniform size of the lobules of the
normal testis or hypertrophied fragment. The tubules are
markedly smaller in diameter and are, with few exceptions,
immature, with the degree of maturity ranging from a
typical medullary cord through all the stages to a n infrequently encountered completely mature tubule. Of these
gonads those of one animal contain only sterile cords, those of
two animals contain cords in which spermatogenesis has gone
no further than syermatocytes ; and those of two animals
contain tubules in which spcrrnatogenesis is in places complete.
If these testes do originate in the manner postulated, it
does not necessarily follow that sex cord proliferation would
be seen in sections. If, however, formation of sex cords from
the peritoneum were seen in but one case, it would confirm
the hypothesis regarding these gonads. I n three of the five
animals cords are quite obviously forming at the periphery
and migrating into the substance of the gonad (fig. 7). Occasionally transitional stages between rnesothelial cells of
the serosa and larger, rounded cells under tlie serosa may
be seen. I n rarer instances i t appears not impossible that
fibroblasts of the tuiiica allnuginea may be the cells of origin.
I n both circumstances the enlarged cells aggregate into cords
and migrate inward, with tlie picture strikingly resembling
that sometimes seen in the formation of cords iii the ovarian
cortex. As these cords grow deeper into the substance of the
testis they increase in size and degree of development until
ultimately mature spermatozoa may be found in certain of
Goitad regeneration
Regeneration of gonad follo~ringcomplete gonadectomy
lias been claimed by Davenport ( '25), Parkcs, Fielding and
Krambell ('27), and Pallot ('28). Davenport reports that
a very large number of his ovariectomized mice (64%) regenerated ovaries within a few weeks o r months after their
removal. His argunieiits for de novo formation of ovary in
certain of his cases are the care with which the operation
was done, many regenerated ovaries were not enclosed in
a capsule, two ovaries regenerated in the place of one, and
one case in w l i i ~ hthe regenerated ovary was found at a point
5 mm. distant from the site of tlie prex-ious ovary. The
originally excised ovaries were apparently not examined t o
ascertain their integrity, and the time of appearance of the
regenerated ovary is iincertain.
The conclusion of Parkes, Fielding arid Erambell is based
on the following observations: of a large number. of ovariectomized mice eleven showed resumption of oestrous cycles
subsequent to an initial cessation of 1 2 to 113 days duration ;
serial section of the extirpated ovaries of six of these animals
revealed that in five cases they had been removed intact ; arid
ovarian tissue could be identified in these animals subsequent
t o operation.
The observations of Pallot are essentially the same as those
of Parkes and his co-workers. He observed a return of
ocstrus in three of a series of rats sliomn by section of ovaries
to have been totally ovariectomized. Tlie lion-oestrous phase
was approximately 6 months in duration. In two of these
animals ovary was found a t autopsy.
These coiiclusions have not remained unchallcngetl. €Innson
and Hcys ('27) and Heys ('29, '31) have ovariectomized a
large number of rats varying in age from 10 to 200 days at
the time of operation. None of their animals under 40 claps
of age at operation showed any ovarian tissue subsequent
to operation, a fact they attribute to the greater ease of complete extirpation in the young animal. I n eight instances
among the adults ovarian tissue was identified at autopsy,
six of which had been completely ovariectomized as shown by
serial section of the original ovaries. These authors entertain
the opinion that ovarian regcncration occurs only following incomplete ovariectomy.
Haterius ('28) also denies de novo formation of the ovary
following complete ovarieetomy. His opinion is based
largely, it seems, on the rarity of such apparent cases in his
experiment (five out of ninety-six), although histological
study revealed the original ovaries to be entire.
Peiicharz ('29) insists that in neither rats nor mice does the
ovary arise de iiovo, providing all ovarian substance has been
removed. His conclusions are based on a study of the vaginal
smears of completely and sub-totally ovariectomize'i animals.
He observed an initial cessation of oestrous cycles, of variable
duration, in partially ovariectomized animals.
The most serious flaw in Davenport's work is his f ' '1urc
to determine whether the ovaries hacl been removed intact.
Another criticism lies in the fact that his only criterion of
the presence of ovarian tissue is its detection at autopsy,
which gives no indication of the time intervening between
operation and regeneration or hypertrophy. The evidence
of Parkes, Fielding, and Brambcll and of Pallot is much more
convincing, and Hate riu s ' a r guriient that histologic t o t a lit y
of the extirpated ovaries is not proof of completeiicss of removal is difficult to accept. Moreover, infrequency of regeneration seems to us by no means to constitute a n argument
for incompleteness of the original operation. The same remarks may be made concerning the views of Heys and
Hanson. It would seem to niakc no difference how difficult the
operation may be, the age of the animal, o r the rarity of
regeneration de novo ; if the ovaries are removed entire, the
animal is totally ovariectomized, and any ovarian tissue which
may be observed subsequently cannot represent a hyyertrophied fragment. I n regard to the absence of oestrous
phenomena, the commonly observcd irregularities, and even
absence of cycles, in unopcrated animals constitute a m o i ~
serious indictment of this criterion of absence of ovary than
does the observation by Pencharz that the cessation and ultimate resumption of cycles in subtotally ovariectoniized rats
and mice is similar t o that reported by Parkes and his collaborators in their anirnals. It seenis reasonable that tlie attainment of threshold activity would present the same general
picture, whether the minute ovary is growing from a fragment or from ovary formed de novo. There seems t o be a
widespread conviction that de novo formation of gonad is
an impossibility. F o r example, Domm ( '27) concludes that
his birds were regenerating gonad from a fragment, despite
his definite impression that all gonad tissue had been removed.
The relative freedom from cyclic activity on the part of the
testis should make the male f a r more farorable than the
female for a study of this nature; and the possession of an
external character which responds quantitatively to testis
hormone, and which reacts sharply to castration, should serve
to render the male fowl a n ideal test animal. The period
of time during which no comb growth was exhibited by the
animals in this report points rather definitely t o absence of
testis as its explanation. It is reasonably well established
that a pale, capon comb is never seen in a fowl containing
even a minute quantity of testis; it is reported by Benoit
(’27) that 0.3 gm. of testis i s sufficient to maintain the noncastrate condition, and 1 mg. of testis will produce growth
of the comb. Greenwood and Crew ( ’ 2 7 ) in a study of
‘developmental capons’ found that the presencc of 0.016 gm.
of testis sufficed to produce and maintain vascularity of the
comb. Our animal 51 exhibited a not inconsiderable growth
of the comb associated with the presence of but 0.010 gm. of
testis, the removal of which promptly produced regression
and paling of the comb. A period of 6 months or more during
which there is no comb growth can be satisfactorily explained
only on the basis of total testis deficiency, especially since
partial castration is followed, as is well known, by prompt
and marked ‘compensatory hypertrophy. ’
It is unfortunate that not all the original testes removed
from these animals were saved. However, those icstes which
were saved are intact and it is thus clear that a t least three
of the animals were totally castrated.
A striking evideiice of dc novo testis formation in these
fowls is provided by the peritoneal relations. It is remarkable that the conditions postulated should have been met so
precisely following the two types of operation. It is just
possible, of course, that following incomplete removal of a
testis the mesorchium might in some maiiner contract resulting in the establishment of an apparent retroperitoneal
location of the fragment. Such an occurrence appears improbable, and the invariably pendant character of the numerous testes knomm to have developed from fragments decreases
the likelihood of mesorchial retraction. On the contrary, it
appears more likely that a de novo gonad could simulate
superficially the relations of a fragment.
If it be accepted that these animals were totally gonadectomized, there are at least two possible sources of the new
testis. The possibility of a minute, accessory gonad cannot
be overlooked, although such a structure has not been reported in the fowl, and we have never seen accessory gonads
in chick embryos despite exmiination of large numbers. The
other, aiid postulated, possibility is proliferation of cords
from the peritoneum. The finding of this particular occurrence in certain of our cases is a demonstration of de novo
testis formation, even if thc possibility of incompleteiiess of
castration be admitted.
Germ-cell history
While earlier writers considered the germinal epithelium
the site of origin of the germ cells in the chick, the history
given by Swift ( '14, '15, '16) is the currently prevalent view.
A4ccording to this account the primordial germ cells arise in
the endoderm of the area pellucida during the primitive
streak stage. Upon the extension of the mesoderm into this
region the germ cells enter the mesoderm and migrate into
the vascular channels. With the onset of circulation these
cells a r e transported over the body in the blood stream, some
of them arriving ultimately a t the gonad-forming area where
they leave the blood stream arid enter llic germinal cpithclium.
The cells so derived become the spermatogonia and presumably the definitive germ cells. This account has received
confirmation from Woodger ('25) and Goldsmith ('as), with
Goldsmith empliasizirig this failure t o observe any extensive
degeneration of primordial germ cells and his belief that
these germ cells a r e retained in an unbroken line to definitive
spermatozoa aiid ova.
I n addition to the foregoing purely descriptive studies the
experimental approach has been applied to the germ cell
problem in the chick. Heagan ('16) operated on chick
embryos in pre-somite stages and succeeded in removing the
crescent shaped area of the proamnion i n which the germ
cells a r e thought to arise. The gonads which formed in his
operated embryos were completely sterile, showing o d y the
presence of stroma with its peritoneal covering. Benoit ('30)
subjected the germ cell area to the ultraviolet radiation and
observed almost total destruction of the germ cells and only
a n infrequent germ cell in the gonads which formed.
Dantsclzalroff, Dantschakoff and Bereskina ( '31) have destroyed the germ cell crescent in part or entirely by cautery.
Their results point to the cells of the crescent a s the ancestors
of the germ cells of the early gonad. Willier ('37) has removed the germ cell crescent of Swift and transplanted the
remainder of the blastoderm to the chorio-allantoic memhrane. Such gonads as developed were devoid of germ cells.
The results of these experiments are in accord with the concept of a n extra-gonadal origin of the primitive germ cells;
but they obviously have no bearing on the equally fundamental question of the origin of definitive spermatozoa arid
ova. It is i n connection with the latter problem that the
results of Domm ('29) a r e of interest. This author found
that if the left ovary is removed during the period when the
primordial germ cells a r e still present i n the rudimentary
right ovary (as late a s 3 meelis post-hatching, according to
Rrode, 'as),the right ovary may transform into a testis containing male germ cells ; if the same operation is performed
upon chicks subsequent to the disappearance of the primordial germ cells i n the right ovary, the latter becomes a
sterile testis.
Numerous studies thus support the views of Swift on germ
cell history i n the fowl. Firket ('go), however, favors two
generations of germ cells in the chick, the primordial germ
cells which degenerate and a generation arising from the
germinal epithelium which gives rise to the defiiiitive germ
cells. Moreover, in the adult fowl it has heen reported by
Fell ('23) and Gatenby ('24) that peritoneal cells may transform into germ cells. Gatenby's report deals with a single
hen possessing a n adenomatous and atrophic left ovary, anterior to which were found two small testicular masses. I n
these masses trailsformation of p e r i t o n ~ a l cells into germ
cells is described ; however, none of the germ cells is adranced
beyond the stage of the spermatogonium. Fell describes the
gonads of eight liens in different stages of sex reversal which
had been preceded by disease arid atropliy of the ovary. The
formation of testicular tissue was accomplished by a Iliicliening of the germinal epithelium and a proliferation of sex
cords which enlargcd to form tubules. Three of the gonads
contained mature spermatozoa while the reniaiiiing five were
less mature.
The significance of our observations lies i n thcir providing
further instances of the origin of germ cells in the adult fowl
from somatic cells, with the added feature that i n our birds
the process was not initiated hy any demonstrable disease.
Moreover, if we interpret our observations correctly, the
germ cells in our specimens could have arisen old37 from
somatic cells, and in so doing refute one of Weismann’s
original arguments.
These instances of the origin in the adult of germ cells
from cells of the peritoneum are not construed as conflicting
necessarily with an extra-gonadal origin of germ cells in this
species; but the57 do demonstrate that there may he two
seats of origin of germ cells, arid that a transition of somatic
cells into germ cells is possible i n a species usually regarded
as presenting a n unbroken germ line.
Of 500 V h i t e Leghorn fowls castrated, five are considered
from the following evidence to have regeiierated testis subsequent to total bilateral gonadectomy :
1. From three of these five birds the original gonads were
preserved and proved to be intact.
2. Comb growth was arrested in these fis-e animals for
a period varying from 6 months to 36 months following castration before being resumed, whereas following subtotal
castrations comb growth was resumecl within 1month.
3. I n one instance extensive comb growth after a prolonged
arrest was produced by a regeiierated tegtis weighing but
10 mg., an observation indicating that absence of comb growth
is possible only when there is total absence of testis.
4. All of the regenerated testes were retroperitoneal in position, while hypertrophied fragments were always pendant in
the body cavity.
5. The regenerated testes are readily distinguishable
histologically from hypertrophied fragments which, in turn,
resemble normal testis and autoplastic testis grafts.
6. I n the regenerated testes cords may occasionally be seen
forming from the peritoneum in a manner similar to that
followed in the embryo.
Since the intermediate stages between mesotlielial cells and
mature spermatozoa have been seen in these regenerated
testes it is concluded that somatic cells can transform into
germ cells, this in a species whose definitive germ cells are
generally regarded as arising only from primordial germ
cells of endodermal origin.
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Testis growing from a fragmcnt known to have been left a t operation. X $0.
Autoplastic testis g r a f t in juvenile bird. Residence 2 weeks. X 70.
4 Testis in bird 60 which showed no comb growth f o r 6 months following a
castration recordcd as complete. This gonad was retroperitoneal. Note immatnrity of cords and increase of strorna. X 70.
.5 Retroperitoneal gonad recovered from bird 38 which showed no growth of
the comb until 29 months after a castration believed to have been complete.
The tubules arc immature. X 70.
6 Retropcritoneal gonad from bird 56 which showed no comb growth for
10 months following castration which was recorded a s complete. The tubules
are immature and t h e stroma is inercased. x 70.
7 Area of gonad of bird 38 showing formation of (aords from the peritoneum.
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