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The transfer function of certain cells in the wall of the Graafian follicle as revealed by their reaction to previously stained fat in the cat.

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THE TRANSFER FUNCTION OF CERTAIIV CELLS
I N THE WALL OF' THE GRAAFIAN FOLLICLE
AS REVEALED BY T H E I R REACTION TO
PREVlOUSLY STAINED F A T I N THE CAT
R. M. WOTTON AND P. A. VILLAGE
Tlie Department of Btologiml Sczences, D?rqwsne Cniwrsity, Pittsburgh, Pa.
THREE FIGURBS
INTROD ZTCTION
Wilson ('47) and others have suggested that the nutrition
of the oocyte, during a major portion of its development,
is associated closely with the activity of certain accessory
niirse cells. I n the cat, and other mammals, these are separated from the egg cell by the barrier of the Bona pellucida.
This lnayer, however, j s penetrated by protoplasmic connections which maintain contact betwcen the enclosed ovum and
its accessory cells in the follicle wall and which may serve
as chaiinels for the transport of nutritive materials into the
oocgte. It would be desirable to test this predication experimentally by administering some food substance tagged in
such a way that its passage could be followed to the ovum.
Gage and Fish ('24), Wotton arid Zwcmer ('39), and
Zwemer and Wotton ( '44) hav shown that previously stained
fat can be fed, and subsequently traced through the body.
Due to the differential staining and other physical properties
of lipids, permanent microscopical preparations can he made
by the gelatin imbedding mid frozen section technique
(Zwemer, '33), showing the distribution of previously stained
fat in the tissues. Zwemer and Wottoii ('44) employed such
121
T H E BXATOMICkb RECORD. VOL. 110. KO. 2
JUNX
1981
122
R. &I. WOTTON AND P. A. VILLAGE
a procedure in their study of kidney function during fat
excretion, and the present experiments apply a similar approacli to a study of the function of the protoplasmic channels
through the zona pellucida.
XATERIALS AND METHODS
The ovaries from 24 young kittens and two mature cats
mere used in this study. These animals were fed cod liver
oil saturated with Sudan IV through a small rubber tube
attached to tlic tip of a glass hypodermic syringe. Gentle
pressure on the plunger of this syringe forced the test inen1
of about 5 em3 into the stomach.
Following an interval of one to two hours each animal, was
sacrificed and immediately dissected. The ovaries with the
adjacent parts of the fallopian tubes were fixed for 24 hours
in 10% pyridine formalin (Burke, '37).
Tissues were embedded in gelatin and sectioned on the
freezing microtonie. The mounted sections were immersed in
a solution of Sudan I V to intensify the color already present
in the previously stained fat in the tissues, and countcrstained in Harris ' hematoxylin for nuclear differentiation
F o r coniparison others were left completely unstained. All
slides were mounted in glychrogel (Wotton and Zwemer, ' 3 5 ) .
As a check on the formalin fixation, several ovaries \\-ere
preserved in Cliampy 's osmic-bichromate-chromic acid mixture, and sectioned by the paraffin technique. The blackening
of the f a t with osinic acid was sufficient to mark the oil, droplets (Glick, '49), while staining with eosin was helpful in
bringing out cytoplasmic structures.
In addition to the slides prepared from the tissues of the
animals employed in this investigation, sections of ovaries
from a series of cats used in a former study on fat absorption
in the intestine (Wotton and Zwenier, '39) were reviewed.
This brought t o 40 the number of kittens observed for a
reaction to f a t by the ova and their accessory cells.
FAT TRANSPORT BY FOLLICLE CELLS
123
OBSERVATIONS
T h e accessory n u r s e cells
The accessory cells in the follicle wall8surrounding tlie ova
appear to he of two morphologically different types. The
most commonly seen cells are oval in shape, with a n elongated process terminating in a slight swelling upon the outside
of the zona pellrucida. F r o m this enlargement a filose projection of cytoplasm could be observed passing through an
aperture in the zona into the oocyte (fig. 2). The other a r e
flat, closely adherent, and spread out upoii tlie surface of
the zona with several lobose, protoplasniic elongations tliroagli
this membrane into the egg cell beneath (fig. 3). Wliile it was
usual f o r one accessory cell to occupy with its cytoplasmic
extension a single opening through the zona pellncida, often
tu7o or more cells were seen to employ oiie aperture in common
f o r this purpose.
Fat droplets mere observed in tlie accessory nurse cclls,
and more frequently they w r e situated in their blunt terminations whicli projected into the oocyte. Fat drops with two
lobes, one lying just inside the cytosoine of tlie ovum and
still connected by a narrow strand to the other p a r t remaining
within .the projection from the follicle cell were seen (fig. 2 ) .
The ova
In each section studied, stained oil was observed in almost
all ova inclosed in vesicular o r Graafian follicles. These cells
were ovoid in section and contained a lolnulated nucleus with
a prominent, vesicular plasmosonie and soreral Iraryosornes.
Large clear vacuoles were seen wliich tended to arrange the
cytoplasm into a peripheral zone from which thick strands
passed inward to the nucleus. Granules arid spheroids of
lipid were observed i n this peripheral zone directly bencatli
the cell membrane ; and also densely packed in the pathways
approaching the nucleus (fig. 1). I n many ova certain oil
drops could be detected in very close contact with the nuclear
membrane.
124
B. M, WOTTON A N D P. A. VILLAGE
None of the fol~liclesobserved in this study showed signs
of atresia, such as the resorption of the liquor folliculi and
epithelial destruction accompanied by karyolysis.
1)~scussIoN
I n the early development of a new individual, the ovum
has a dual function, for, as well as furnishing a genetic component, it snpplies some food substances for the cells of the
future embryo. The nutritive materials are absorbed and
retained in the cytosome by the oocyte during its growth
within the follicle. This storage of food chiefly accounts for
the relatively large size of the mature egg cell. The acquisition of these materials seems to be carried out through the
intermediary activity of the accessory follicle cells, acting
as transfer agents across the barrier of the zona pellucida.
These cells, situated in the wall of the Graafian follicle,
pick up fat as droplets released from the capillary circulation
and transport them by way of their protoplasmic projections
through the apertures in the zona pellncida into the ovum.
Once within the oocyte the oil droplets appear to pass along
the cytoplasmic pathways from the peripheral zone toward
the circumnuclear region (fig. 1). The close association between certain fatty globules and the nuclear membrane seemed
to suggest some reciprocal activity (Boyd, '35). Histologically no change could be detected in the previously stained
cod liver oil during its passage through the accessory nurse
cells ; and presumably the physiological activity of such cells,
a s far as lipids are concerned, is confined mainly to the
function of its physical transfer from the tissue spaces into
the oocyte.
Our observations on the appearance of coiistricted oil
droplets seen at the tips of the projections from thc follicle
cells, where they come in contact with the periphery of the
cytosome of the ovum, clearly demonstrate the existence of
an interface or membrane between these cells. Singer and
Zwemer ( '34), and Zwemer, Wotton and Norkns ('38), Wotton and Zwemer ('39), and Zwemer and TTTotton ('44) have
FAT TRANSPOBT BY FOLLICLE CELLS
125
reported similar pictures for the transfer of lipid in such
hourglass shape in and out of cells. The constriction is
caused by the compression of the plastic oil as it squeezes
through a tiny pore in the cell membrane. Thus it would
seein that the question of cytoplasniic coiitiiiuity (Wilson,
’47) between these nurse cells and their oocyte in the cat can
be answered in favor of a close contact between the two cell
surfaces across w2iicl.i fats, and presumably other food substances, are transferred.
STTNMARY
In a series of 40 kittens the cellnlar channels through the
zoiia pellucida by which the ooc2yte may receive food substances were studied. They were found to he projections
belonging t o two types of accessory nurse cells situated in
the follicle wall.
By following the path of previously stained cod liver oil
from the hlood capillaries into the ovum the transport function of these accessory cells for this substance, and prcsumably for other materials needed by the ovum, was established.
LITERSTURE CITED
BOYD,E. M.
1935 The relation of lipid romposition to physiological activity
in the ovaries of pregnant r a b b i t s J. Biol. Chem., l o g : GOT.
BURKE,F. V. 1933 The pH of formalin. A factor in fixation. Am. J . Path.,
9: 915-920.
GAGF, S., BND Y. A. FISH 19?1 F a t digestion, absorption and assimilation in
man and animals as determined by the dark-field microscope, and a
f a t soluble dye. Am. J. Anat., 8 4 : 1-77.
OrmK, D. 1949 Techniques of Histo- and Cytoeliemistry. Interscience Pnblishers, Inc., X e w York. 68.
SINGER,
E., AND R. L. ZWE%EER 1934 ~icroscopieobservat~onsof the structural
c l l a n g e ~in the adrenal gland of the living frog under experimental
conditons. Anmat. Rec., 60: 183-187.
WILSON,E. B. 1947 The Cell in Development and Heredity. The Sfaemillan
Co., Kew York. 3 ed.: 331-338.
WOTTO;N,R. &
AND
I.,
R. L. ZWEMER 1935 A note on “Glgchrogel” mounting
solution. Stain Tech., 10: 21-22.
1939 Studies on direct and visible ingestion of f a t by differentiated
body cells of the eat. Anat. Ree., 75; 493-507.
~-
126
E. M. WOTTON AKD P. -4. VILLAGE
ZWEYER, R. L. 1933 A nietbod for studying adrenal and other Iipoids by a
modified embedding and mounting technique. Anat. Rec., 57 : 41-11.
ZWEMER, R. L., AND R. M. WOTTON 1944 Fat excretion in the guinea pig
kidney. Anat. Ree., 90 : 107-114.
ZWFXER, R. L., R. M. W o n o ~AND M. G. NORKUS 1938 B study of cortieoadrenal cells. Anat. Rec., 73: 249-263.
FAT TRASSPOBT BY FOLLICLE CETALS
PTJATE 1
R . X.W O T T O N A 6 D P . A . VILLAGE
1 Graafian follicle shon-itig fat inclusions. x 200.
Camera lucidtt drawing of iiume cell typc found in the wall of Graafian follicle. X 3000.
Caulera lucicla clraming of second iiurae re11 type. Compare with figure 2. X 3000.
2
3
127
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staines, reaction, cells, certain, wall, cat, transfer, fat, revealed, function, previously, follicle, graafian
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