THE PHYSIOLOGY
OF THE BROOD POUCH OF
THE MALE SEA HORSE HIPPOCAMPUS
ERECTUSI
J. R. LINTONZ AND B. L. SOLOFF3
Institute of Marine Science, University of Miami
ABSTRACT
In the spotted seahorse, Hippocampus erectus Perry, the male carries
the eggs and young in an abdominal brood pouch, which provides a
controlled environment for development. During the incubation period,
the sodium ion concentration of the fluid within the pouch increases
while the serum sodium remains relatively constant. The calcium concentration of the pouch fluid decreases as incubation progresses. Also,
radioactive calcium is taken up by the embryos in the pouch. Young
removed from the pouch show a greater mortality when placed into full
strength sea water than do those introduced into 0.4 sea water. The
pouch provides optimum ionic and osmotic conditions for development
and serves as an osmotic adaptation chamber for the young. The
anatomical relationships between embryos and paternal tissue, and the
histology of the pouch epithelium are described. Histochemical analysis of
glycogen, lipid, and phospholipid are given for animals in the various
stages of brOOding and non-brooding.
INTRODUCTION
The spotted seahorse, Hippocampus
erectus Perry, is a tropical and
subtropical marine teleost found in the coastal waters of the southeastern
United States, Mexico, and the West Indies. Its distribution is confined to
the shallow waters of these regions, where it usually inhabits beds of
turtle grass, Thalassia sp.
The seahorses as a group have attracted wide attention since ancient
times because of their unusual structure, their peculiar mode of life, and
their unique manner of providing parental care for the young. The male
seahorse possesses a large abdominal brood pouch, into which the female
deposits the eggs. Fertilization occurs at the time of deposition, and the
eggs are retained by the male until development is complete and the young
can swim and feed for themselves.
Since it was known that seahorse eggs and embryos would not develop
after being removed from the pouch and placed in sea water, many early
workers, reviewed by Huot (1902), thought that the young were dependent
upon the male for nutrition. Leiner (1934) later investigated the osmotic
IContribution No. 518 from The Marine Laboratory, Institute of Marine Science, University
of Miami. This research was conducted while the authors were Post-Doctoral Fellows
under a training grant from the Heart Institute of the National Institutes of Health.
2Present address: University of South Florida, Tampa, Florida.
3Present address: Department of Anatomy, University of Tennessee, Medical Units, 62 South
Dunlap Street, Memphis 3, Tennessee.
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Bulletin of Marine Science of the Gulf and Caribbean
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pressure of the fluid in the pouch of incubating males of H. brevirostris
and H. gluttulatus and found this pressure to be similar to that of the blood
during the early incubation period and to approach that of sea water in
the later stages. Leiner was also able to get a few embryos to develop
partially in dilute sea water. These findings imply that the male provides
an optimum osmotic environment
for the developing embryos.
It is the purpose of this investigation to determine some of the osmotic
and ionic changes which occur within the pouch during incubation, as
well as to describe some other physiological and anatomical relationships
between the embryos and the paternal tissue.
MATERIALS
AND METHODS
All fish used in this investigation were adult male H. erectus collected
by trawl from Biscayne Bay, Florida. The fish were allowed to equilibrate
in the laboratory in running sea water for a period of from one week to
ten days before treatment. Blood samples for freezing point depression
and ion analysis were taken by decapitating the animals and collecting the
blood directly from the heart in capillary tubes. Pouch fluid samples were
taken in capiIlary tubes from the cut posterior end of the pouch. Freezing
point depression
determinations
were made using a microcryoscope,
according to the method of Gross (Prosser, 1961).
Sodium and calcium determinations
were made with a Beckman DU
spectrophotometer
with a flame photometer attachment.
Sodium and calcium flux studies were made using an upright separation
chamber (Fig. 1) consisting of two tubes, one inside the other. The inside
tube was 20 cm long and 5 cm in diameter. The posterior part of the fish
was placed into this tube, a known volume of water added, and a watertight seal was made about the pectoral region using a proctologist's finger
cot as a diaphragm. This tube was then placed inside the larger tube and
an equal volume of sea water added. This effectively separated the water
surrounding the gills from the water surrounding the pouch openin<? and
urogenital orifice. Tracer doses of Na22 and Ca45 (2-4 JLc) were added
to one or the other of the chambers or injected into the fish. The direction
of movement of the ions was determined by samoling the water in each
of the chambers, the serum, the pouch fluid, ani the embryos. Na22 samples
were counted in an ECKO scintillation unit, and Ca45 was counted in a
gas flow unit. Embryos, serum and pouch fluid samples were digested with
concentrated
nitric acid and allowed to dry before counting.
The brooding stages were determined by measuring the length of the
embryo from the tangent of the tail to the crown, and from the crown
to the tip of the tail. Eyeless embryos up to 2.5 mm were considered as the
early brooding stage; middle-stage embryos were those 2.5 bv 5 mm to
4 by 6 mm; late-brood embryos were those 4 by 6 mm to 8 by 10 mm in
length.
1964]
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47
I.
FIGURE
1. Separation
c!1amber
HISTOLOGICAL
used in the ion-ft·JX studies.
METHODS
Pouch material was dissected from animals in the various stages of
incubation and placed in several fixatives. The choice of fixative and
subsequent treatment of the tissue was dictated by the histological method
to be used. The number of tissues used for each method is outlined in
Table 1.
Lipid was detected in two ways. Some pouch tissue was fixed in
10 per cent buffered formalin at 7°C, embedded in gelatin or polyvinyl
alcohol and cut at 10-15 microns on a freezing microtome. The sections
were colored with Sudan black B in ethylene glycol (Chifelle and Putt,
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TABLE 1
NUMBEROF POUCHTISSUESUSED AT EACHBROODINGSTAGE
FOREACHMETHOD
METHOD
Lipid
Osmium fixed
Frozen
Elftman's
PAAS
PAS
Toluidine Blue
Alcian Blue
NonBrooding
EarlyBrooding
MidBrooding
5
1
3
2
2
5
5
3
5
9
3
4
9
9
4
Late
Brooding
2
PostBrooding
1
2
]
2
2
2
2
1
1
2
1
1951) or in 60 per cent triethyl phosphate
(Gomori,
1952). Control
sections were extracted with a 1: 1 mixture of methanol and chloroform for
one half hour at 37°C before emersion into the Sudan black. Other pieces
of pouch epithelium were dissected free of the pouch envelope and fixed
in weak or strong Flemming's solution. These tissues were embedded in
paraffin and sectioned at 4 or 5 microns. The paraffin was removed with
chloroform and the sections were mounted in neutral Canada Balsam. The
sections were examined with phase contrast as well as normal optics.
Phospholipid was identified by using Elftman's "controlled chromation"
technique (Elftman, 1958). The tissues were fixed in dichromate-sublimate
for 3-4 days, dehydrated
rapidly, embedded in paraffin and sectioned
at 6-8 microns. The sections were colored by Sudan black in ethylene
glycol. Mallory's modification of Kaiser's glycerol gelatin (Lillie, 1954)
was used as the mounting medium. Some sections were stained with the
peracetic acid-Schiff (PAAS)
reaction (Lillie, 1954) and mounted in
glycerol gelatin.
Glycogen was identified by using the periodic acid-Schiff (PAS) reaction. The tissues were fixed in cold Rossman's alcoholic picro-formol fluid,
embedded in paraffin, and sectioned at 8 microns. Control sections were
incubated for 45 minutes at room temperature in a 1 per cent aqueous
solution of malt diastase. Some sections were counterstained with Celestin
blue and Lillie's modification of Mayer's acid hemalum.
Metachromasia
was investigated by using the method of Kramer and
Windrum (1955). However, we found that staining had to be prolonged
to overnight instead of the 20-30 minutes suggested. Sections were
observed in water mounts as well as dehydrated and mounted in balsam.
The tissues used were those that had been fixed in either Rossman's fluid,
dichromate-sublimate,
or 1'0 per cent buffered formalin.
Acid mucopolysaccharides
were sought in tissues fixed in Rossman's
fluid or in dichromate-sublimate.
The method of Steedman (Pearse, 1960)
1964]
Linton & Soloff: Physiology
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49
was used, which employs alcian blue. Neutral red was used as the
counterstam.
The general oversight methods of hematoxylin (or hemalum) and eosin
were performed on some paraffin sections from tissues which had been
fixed in 10 per cent buffered formalin or dichromate-sublimate.
However,
the bulk of the general observational
data was taken from the counterstained PAS preparations and from phase contrast observation of osmium
tetroxide fixed tissues.
RESULTS
Sodium levels of serum and of pouch fluid were determined for nonbrooding males and males in various stages of brooding. The results
represented in Figure 2 show clearly that pouch fluid sodium levels increase
during incubation while the serum levels remain relatively constant. It
is thought that the rather wide variations within the groups is due to the
method of staging the embryos.
TABLE 2
CALCIUM CONTENT OF SERUM AND POUCH FLUID
Serum
Pouch Fluid
NonBrooding
Early
Brooding
Middle
Brooding
Late
Brooding
(8)
7.8
(8)
(10)
(9)
7.1
6.3
7.2
6.1
7.5
6.8
5.6
Non-brooding males are divided into two groups, those with Na values
exceeding 250 MEqll are considered post-brooding; those with Na levels
less than 250 MEq/l
are presumed to be prepared to receive eggs. Males
which were observed giving birth to young in the laboratory were shown
to return to the low Na state within three weeks. This low Na state is,
then, considered to be the normal resting condition of the male.
Table 2 represents the Ca levels in serum and pouch fluid during incubation. A slight drop in pouch fluid Ca is noted as incubation proceeds.
Freezing point depressions of serum and pouch fluid of early and nonbrooding males are shown in Table 3. Note that in these stages the osmotic
concentration of the pouch fluid is slightly below that of the serum.
Early and middle-stage embryos were removed from the pouch and
placed in petri dishes containing sterile sea water and sterile 0.4 sea
water. The differential mortality rates of these two groups of embryos are
shown in Table 4. A small percentage of the embryos in 0.4 sea water
survived to the stage of complete yolk absorption in about two weeks. At
this time they were placed in full-strength sea water where they survived
for another ten days.
The directions of Na movement were determined
using separation
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Bulletin of Marine Science of the Gulf and Caribbean
400
440
Pouch Fluid ---Serum
-----
420
. 400
.•.•.
I!!
-J
z
w
380
I
....• 360
~
::J
~ 340
-J
-J
:Ii
~
•...
z
w
•...
z
320
300
280
0
U
II:
260
.!!
0
z
240
220
T
I
T
I
I
._.
---f----i---
jc;:'"'-
200
180
160
I
1
tl/21
Eorl,
Mlddl.
BROODING
FIGURE
Lol.
Poll. PorI.
-
f20)
NoIbroocIlllt IIoIb,.l••
(high)
(low)
STAGE
2. Na-ion concentration of pouch fluid and serum of males in the
various brooding stages.
cham bers (Fig. 1) and tracer doses of N a 22. Males in various stages of
brooding, and non-brooding males were placed in the separation chambers
and Na22 was introduced into the posterior chamber which enclosed the
pouch. Twelve hours later the pouch fluid, the serum, and the water in the
anterior chamber were sampled and found to contain no Na22• In the next
experiment, Na22 was introduced into the anterior chamber surrounding
the gills. In three hours, the pouch fluid and the serum were radioactive,
and the water in the posterior chamber contained only a trace of Na22•
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Linton & Soloff: Physiology
51
of the Seahorse
TABLE 3
FREEZING POINT CEPRESSION
(PER CENT)
OF SERUM AND POUCH FLUID
Pou~h Fluid
32.2%
29.2%
27.0%
38.0%
28.2%
25.0%
Serum
31.7%
26.1 %
43.6%
31.7%
31.2%
55.0%
X 29.9%
X 36.5%
TABLE 4
PER CENT MORTALITY OF SEAHORSE EMBRYOS REMOVED FROM
POUCH AND PLACED IN SEA WATER AND 0.4 SEA WATER
0.4
Sea Water
(66)
Sea Water
(71 )
6 hr
18 hr
24 hr
40 hr
48 hr
10%
39%
48%
63%
70%
23%
38%
84%
93%
99%
When Na22 was injected into the pouch of early brooding males, the serum
and water in the anterior chamber became radioactive within two hours.
These experiments indicate that there is no direct exchange between
the pouch fluid and the environment, but that there is Na ion exchange
between the serum and the pouch fluid.
Calcium45 was introduced into the anterior chambers containing brooding males and the embryos were removed in twelve hours, washed carefully,
ashed with nitric acid, and counted. The embryos were found to contain
traces of Ca 45.
HISTOLOGICAL
RESULTS
The pouch of the seahorse is lined by an envelope of epidermis-covered,
dense, white connective tissue that protects the delicate epithelium which
lines the lumen. The epithelium is supported on a layer of loose connective
tissue which contains many capillaries. The presence or absence of a
basement membrane is problematical and will be discussed below.
The pouch in the non-brooding cnimal.- The epithelium of the non-brooding pouch, which is composed of 2-3 cell layers (Fi<;. 3), is arranged in
folds which limit the area of the pouch lumen. The cells of the proximal
layer are small and cuboidal to oval i:1 shape; those of the distal layer vary
in shape, sometimes appearing columnar, club-shaped,
or oval and, at
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times, prismatic. Under the phase microscope at 1250 a series of short
spines can be seen projecting from the apical surface of these cells. A
tangential section of these cells reveals a girdle of processes which resemble
those of the "prickle cells" of the mammalian epidermis.
The osmium preparations revealed little or no lipid, and the preparations colored for phospholipid showed only a few scattered granules. However, several of the frozen sections colored for lipid showed scattered fine
and coarse droplets which were absent in the methanol/chloroform
controls.
Three of the PAS preparations showed positive, diastase-removable
particles in the basal portion of the proximal cells. Only one of these
preparations showed a great abundance of this material. PAS-positive
material that was not removed by diastase was observed in four locations:
in the area at the base of the cells, the gland cells, the apical cytoplasm,
and the apical surface of the distal layer of cells. The PAS reaction at
the apical surface of the distal cells was confined to small granules which,
under phase contrast, appeared to coincide with the spines seen in the
osmium preparations.
The PAS reaction of the apical cytoplasm of the distal layer of cells
was a rather constant observation, although the degree of reaction was variable. The reaction was more intense in the folded areas of the epithelium,
and PAS positive globules were present in some of the tissues in the areas
between the epithelial folds. In one of the tissues, an intensely positive,
broad, cuticle-like layer was observed in limited areas of the epithelium.
A few scattered pycnotic nuclei and some entire cells were observed on
and within this layer, which in some areas was stripped off the epithelium.
Most epithelia examined had varying numbers of rather large, oval or
rounded profiles which were presumably gland cells (Fig. 4). In only a
few instances could these be observed to open onto the surface of the
epithelium. Many of these cells appeared empty, although in others, PASpositive clumps of material were present. This material was not removable
by diastase treatment. At times, a few granules of pigment were associated
with this material, and when the plane of the section was favorable a
nucleus could be seen compressed against the cell membrane. In one
specimen a few of the gland cells had enlarged into cysts.
The presence or absence of a definitive basement membrane was not
established with certainty. The presence of numerous capillaries pressing
against the base of the epithelium obscured observation in many instances.
At times, a very thin PAS-positive line was observed and interpreted to be
a basement membrane. However, in all cases the loose connective tissue
immediately beneath the epithelium was at last faintly PAS-positive and
compression of the epithelium against this may have given the appearance
of a basement membrane. In one preparation the supporting connective
1964J
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53
FIGURES3-4. 3, Section through a villus of a non-brooding animal. Note compound epithelium and connective tissue core. The large black masses beneath
the epithelium are composed of pigment (Flemming's) .--4. Cross section
through villus of a non-brooding animal. Note gland cells. One of these can
be seen penetrating the epithelium. (Frozen section, Sudan black B.)
tissue was extremely PAS-positive, and there was, immediately beneath the
epithelium,
a thick, amorphous
PAS-positive
layer. This layer, which
contained fibroblasts, appeared to surround the bases of the proximal
cells of the epithelium.
The toluidine blue preparations
showed the same variability exhibited
by the previous methods. Basophilia ranged from extreme to slight and
was located usually in the infranuclear
region of the distal cells. The
epithelia of five of the nine preparations showed a noticeable violet staining when observed in water mounts and which was retained after dehydration. This staining appeared restricted to the distal cell layer of the epithelium. The connective tissue adjacent to the epithelium in three of the preparations showed a slight degree of metachromasia when observed in water
mounts. However, dehydration destroyed this reaction. The aldan blue
preparations showed very slight staining at the apical border of the distal
cells, and in some places seemed separated from the epithelium by a nonstaining layer.
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The pouch of the brooding animal.-In the pouch of the brooding animal,
the broad folds present in the non-brooding animal have been compressed
to a honeycomb system in which the eggs lie (Fig. 5). The eggs at the
periphery of the pouch were more completely surrounded by the septa of
this honeycomb than were the eggs situated more deeply in the pouch. The
eggs in the center of the pouch appeared to float freely in the pouch fluid.
The mucosa of the brooding pouch was compressed into a series of
septa which formed the walls of the honeycomb. When observed in cross
section, a septum is composed of a loose connective tissue core on either
side of which is arranged the epithelium (Fig. 6). The epithelium at the
bottom of the pocket in which the egg lies is flattened and compressed
against its supporting loose connective tissue. The epithelium varies in
thickness from one to two cell layers, seemingly dependent upon the
pressure exerted in each particular area. This is illustrated by observations
on tissues which had been dissected off the pouch envelope and freed
of adhering young. These tissues usually showed a compound epithelium.
Two of the frozen tissues colored with sudan black showed scattered
droplets of lipid situated in the basal region of the epithelium. The third
preparation showed some fine, scattered lipid droplets which were most
apparent in the areas of the epithelium that exhibited the least compression. The preparations fixed in Flemming's solution showed little or no
lipid in the tissues taken from the early brooding animals. However, lipid
was seen scattered through both layers of the epithelium in those tissues
taken from middle-brooding
and late-brooding animals. The lipid droplets
were found in greater quantity in some areas of the epithelium than in
others. The phospholipid and PAAS preparations
were essentially ne,<;ative.
A PAS-positive reaction was found in both the loose connective tissue
and the epithelium of all the specimens examined. The diastase treatment
was ineffective in localizing removable material due to the extent of the
PAS reaction and the compression of the epithelium. The loose connective
tissue in many areas of the epithelium reacted strongly, and the most dense
reaction appeared in the tissues taken from the late-brooding animals. In
the tissues from all the brooding stages, the reaction was most marked
in the connective tissue composing the core of each septum. Three of the
tissues from the early brooding animals and one of the tissues from the
middle brooding animals had lightly reactive areas in which a possible
basement membrane could be seen.
The PAS-positive reaction in the epithelium appeared greatest in the
apical cytoplasm of the surface cells. In many instances, a heavily PAS
reactive, granular surface layer of variable thickness was present on
these cells. The "spikes" described previously were also seen in this layer.
When present, the chorion of the eggs overlaid this layer and appeared
1964]
Linton & Soloff: Physiology
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55
I
1
•••
5. Cross section through a seahorse at the level of the brood-pouch.
Note thickness of the wall of pouch, and arrangemnet of embryos. (HematoxyJ:n and eosin,)
FIGURE
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c
FIGURES6-7. 6, Cross section of a septum in the pouch of an early brooding
animal. Note extreme flattening of epithelium and vascularity of connectivetissue core. The flanking membranes lined with globules are part of the yolk
sacs of two embryos (Hematoxylin and eosin) .-7. A section through several
villi of the pouch of a post-brooding fish. Note compound epithelium and
presence of large amounts of lipid. (Flemming's.)
torn and incomplete. Precipitated pouch fluid material and perivitelline
material gave a variable reaction in the PAS preparations, ranging from
negative to intensely positive.
The more highly compressed portions of the epithelium appeared damaged. Some cells seemed to have stripped off the epithelium, and small
excavations in the epithelium were seen, in which the lumen of the pouch
was separated from the capillaries of the paternal connective tissue by
only a thin rim of cytoplasm. In two of the PAS preparations, one from
an early-brooding animal and the other from a middle-brooding animal,
PAS-positive clumps of material were found in several of the capillaries.
One preparation from an early-brooding animal contained what were
presumably leucocytes. These cells were rounded and contained a small
nucleus and PAS-positive granules. They could be seen in the blood vessels adjacent to the epithelium, in the connective tissue adjacent to the
epithelium, and in the epithelium itself.
1964]
Linton & Solof}: Physiology of the Seahorse
57
The precipitated pouch fluid material and perivitelline material stained
red with toluidine blue whether observed in water or in balsam. This
material stained positively with the alcian blue. The material between the
chorion and the epithelium, and the chorion itself, stained with neither
toluidine blue nor alcian blue. The cytoplasm of the surface layer of cells
stained a light violet with the toluidine blue even after dehydration.
When a second layer of cells could be detected, their cytoplasm appeared
non-stained. Cytoplasmic basophilia was found in an infranuclear position,
and was more marked in the distal layer of cells than in any underlying cells.
The pouch of the brooding animal.- The epithelium of the pouch of the
post-brooding animal appeared stratified. The proximal layer of cells was
cuboidal to oval in shape. The cells composing the distal layer appeared
columnar, club-shaped, or oval. Separations between cells were noticeable,
and many capillaries were seen abutting against the epithelium. One
toluidine blue preparation gave the impression that the epithelium was
pseudo-stratified in some areas.
Figure 7 illustrates the large amount of lipid which can be seen in the
cells.
A broad PAS-positive zone was present at the base of the epithelium
and appeared to invade the interstices at the bases of the proximal cells.
Some cells, identical to the leucocytes mentioned previously, could be seen
in the loose connective tissue, the capillaries, and in the epithelium itself.
The cytoplasm of the distal layer of cells was PAS-positive at the apical
pole. These cells were basophilic in the infra-nuclear region. A relatively
thick, PAS-positive layer overlaid the epithelium in many areas. Some
dense nuclei were apparent within and on this structure. The cytoplasm
of the proximal cells was PAS-positive. Some of these cells contained
PAS-positive granules which were removed by diastase treatment. Gland
cells, some empty and some containing PAS-positive material, were seen.
The apical layer of the epithelium stained intensely with toluidine blue.
Dehydration
showed that this was caused by the heavy basophilia
of the
distal cell layer. The basophilia was located in the infranuclear and basal
region of the cells. After dehydration, it could be seen, also, that the
non-basophilic cytoplasm of the distal cells stained a faint violet. The
proximal layer of cells was virtually unstained, as was the PAS-positive
surface layer and the connective tissue. The results of the alcian blue
technique were negative.
DISCUSSION
As Leiner (1934) found for H. brevirostris and H. guttulatus, the
osmotic concentration of the pouch fluid of H. ereetus is nearly the same as
that of the blood in the early stages of incubation. It has also been demonstrated that the embryos of H. ereetus, when removed from the brood
pouch, show a markedly higher mortality rate in sea water than in 0.4 sea
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Bulletin of Marine Science of the Gulf and Caribbean
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water. The fact that in these experiments, a small percentage of the embryos
in 0.4 sea water survived to the stage of complete yolk absorption seems to
preclude any dependence of the young upon the male for nutrition.
Furthermore, the sodium ion concentration of the fluid within the pouch
increases during incubation and drops back to the level of the blood sodium
after parturition. Fish that were observed delivering young in the laboratory
returned to the low sodium state within two weeks. Apparently this low
sodium state is the normal resting condition and these fish are prepared
to receive eggs.
The serum is the source of the sodium in the pouch and this sodium
must be actively transported across the pouch epithelium. As incubation
progresses, the sodium gradient increases. These observations clearly indicate that the pouch epithelium is a highly efficient osmoregulatory organ
operating under a sensitive control mechanism. The nature of this control
mechanism is the subject of current studies.
The calcium
concentration
of the pouch
fluid decreases
as incubation
progresses. That calcium45 is taken up by the embryos in the pouch ~uggests that this calcium may be incorporated into the embryonic skeleton.
The bony body rings of the seahorse undoubtedly create a high calcium
requirement in the developing embryos.
Although it has not yet been demonstrated, the impermeable nature
of the pouch makes it almost certain that the exchange of gases and nitrogenous waste products occurs across the pouch epithelium. The results
of the histological examinations clearly show that this tissue is particularly
well suited for such exchange. The flattening of the epithelium during
brooding, combined with the richness of the sub-epithelial capillary bed,
would appear to facilitate diffusion and transport. Furthermore, there
may be no basement membrane to impede exchange. The histological
results are not at all clear-cut, and indicate that the situation may resemble
that in the human skin where the "epidermis is separated from the dermis
by a matting of argyrophilic and PAS reactive reticular fibers, the meshes
of which contain the cytoplasmic processes of the basal epidermal cells,
when these are present. There is no homogenous membrane that corresponds to a true basement membrane" (Montagna, 1961). The increased
PAS reactivity of the loose, sub-epithelial connective tissue during brooding and after release of the young is probably caused by concentration of
the PAS-positive reticular fibers due to compression and the presence of
increased amounts of glycoprotein (Montagna, 1961). However, the results of our histological examination do not shed much light on the mechanisms of active transport in this tissue. Although one source of energy
for the cells is glycogen, only limited quantities of this PAS-positive,
diastase-removable material were observed. This probably indicates that
the turnover of glycogen is very rapid, presumably due to a high meta-
1964]
Linton & Soloff: Physiology
of the Seahorse
59
bolic activity of the epithelium. However, such speculation must be leavened by the mention of two alternative possibilities for the relative lack
of glycogen. These animals fed very little during captivity. This undoubtedly would cause a rapid utilization of existing glycogen stores. Furthermore,
the size of the pouch sample actually observed in relation to the complete
extent of the epithelial surface introduces the factor of sampling error.
The "spikes" observed on the surface of the cells that compose the distal
layer of the epithelium are presumably microvilli, and were described by
Rauther (1925) in his excellent and complete report. Such surface modifications are typical of structures concerned with ionic regulation, such as
the tubules of the kidney (Forster, 1961). Moreover, Rauther described
the intercellular bridges which we have also observed. These processes undoubtedly serve as an aid in maintaining the integrity of the epithelium
during the stresses induced by the growth of the embryos. The presence
of separations observed in the pouch epithelium of the post-brooding
animals may be a measure of the stresses resulting from the vigorous
movements of the older young in the pouch and their subsequent release.
The presence of lipid in the cells of the pouch epithelium was noticed
by Rauther (1925) and attributed by him to the resorption of yolk material. Our studies support this, although they do not rule out the possible
utilization of some lipid by the epithelial cells.
The origin of the PAS-positive material found between the chorion and
the epithelium may be the presumed to be gland cells. This indecision
concerning the gland cells results from the fact that Rauther did not describe gland cells in the pouch epithelium of his animals. However, this
may be a species difference. The empty or partially filled profiles observed are probably gland cells and appear to be a source of this PASpositive material. Since the material stains with neither toluidine blue nor
alcian blue, it could be composed principally of neutral mucopolysaccharide, mucoprotein, glycoprotein, or a mixture of these substances. That
it is not derived from the perivitelline or pouch fluid is indicated by the
observation that the precipitated materials from these sources are meta-
chromatic and alcian-blue positive. Our observations on the epithelium
do not preclude the possibility that each epithelial cell contributes to the
formation of this product.
The work reported in this paper is incomplete. However, the questions
posed by this study encompass those of transport, its control by the cells,
and by the organism itself. Therefore, we are continuing the physiolo~ical
and morphological studies of the pouch, and have begun the study of the
neurosecretory apparatus and pituitary of this fish in the hope of clarifying the question of the control of transport in the pouch e9ith::lium.
ACKNOWLEDGEMENTS
The authors wish to express their thanks to Dr. C. E. Lane, Professor
60
Bulletin of Marine Science of the Gulf and Caribbean
[14(1)
of Marine Science, for his ideas and encouragement
in this study and to
Mr. Robert Johnson, Radiation Officer, Institute of Marine Science, for the
use of isotopes and counting equipment. Thanks are also due Mr. Robert
Still, of Bob's Live Bait Company, Key Biscayne, Florida, who provided
the experimental animals.
SUMARIO
FISOLOGiA
DE LA BOLSA
INCUBATRIZ
DEL
Hippocampus
CABAL LITO
DE MAR
MACHO,
erectus
El caballito de mar macho carga los huevos y los j6venes en una bolsa
incubatriz que provee un ambiente control ado para su desarrollo.
Se ha encontrado que durante el periodo de incubaci6n, la concentraci6n
de sodio en el fluido dentro de la bolsa aumenta, mientras el sodio del
serum se mantiene relativamente constante. La bolsa provee condiciones
i6nicas y osmoticas 6ptimas para el desarrollo y sirve como camara de
adaptaci6n osm6tica para el joven.
La concentraci6n
de calcio en el fluido de la bolsa decrece durante
el periodo de incubaci6n. Calcio radio activo es tornado por los embriones
en la bolsa.
La relaci6n anat6mica entre el tejido paterno y los embriones y la
histologia del epitelio de la bolsa es descrito.
Estudios histoquimicos de glic6geno, Hpidos y fosfoHpidos son dados
para animales en los varios estados de cria y no criando.
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