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Immunoelectron microscopic localization of relaxin in endometrial gland cells of the pregnant guinea pig.

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THE ANATOMICAL RECORD 209:373-379 (1984)
lmmunoelectron Microscopic Localization of Relaxin in
Endometrial Gland Cells of the Pregnant Guinea Pig
RUBE J. PARDO, LYNN H. LARKIN, AND RANDALL H. RENEGAR
Department of Anatomy, University of Florida College of Medicine,
Gainesville, FL 32610
ABSTRACT
Endometrial gland cells in uteri from late-pregnant guinea
pigs (day 60 to parturition) resembled typical protein-secreting cells. Extensive
rough endoplasmic reticulum and well-developed Golgi complexes were evident. The most striking features of endometrial gland cells were accumulations
of large (= 0.5 ym in diameter), dense, membrane-bounded granules. The
granules were located in the supranuclear region of the cell and frequently
occurred in close proximity to the plasma membrane adjacent to the lumen of
the gland. Thin sections of endometrial gland cells treated with relaxin antiserum and either colloidal gold-protein A or colloidal gold-goat antirabbit IgG
demonstrated that the granules contained relaxin. These studies provide additional evidence that the uterus of the guinea pig produces relaxin and
support the hypothesis that uterin relaxin may play a n important role in
pregnancy and parturition in the guinea pig.
Previous studies from this as well as other
laboratories have demonstrated that the
uterus of the guinea pig is a source of relaxin
(Zarrow, 1947, 1948; Pardo et al., 1980; Nagao and Bryant-Greenwood, 1981; Pardo and
Larkin, 1982). Endometrial (uterine) gland
cells (EGC) have been shown to be the source
of uterine relaxin in pregnant and nonpregnant guinea pigs by light microscopic immunoperoxidase localization (Pardo et al., 1980;
Pardo and Larkin, 1982).
While previous studies have established
that EGC are sources of relaxin in the guinea
pig, several questions remain unanswered.
For example: 1)Is relaxin synthesized in EGC
or is it produced elsewhere and accumulates
in or on the EGC, possibly as receptor-bound
hormone? 2) If relaxin is produced by EGC,
is it stored in membrane limited granules
similar to the relaxin containing granules
described in granulosa lutein cells of the pig
(Kendall et al., 1978)? 3) How and in which
direction is relaxin released from the cellapically into the lumen of the gland, where
the hormone would have access to the uterine lumen, or basally into the stroma of the
uterus, where it might effect the closely situated uterine musculature, which is a known
target organ of relaxin (Porter, 1979)?
0 1984 ALAN R. LISS. INC.
The work reported in this communication
concerns investigations of the ultrastructure
of EGC of the guinea pig with the intent of
obtaining answers to the questions just
presented.
MATERIALS
Guinea pigs were obtained from a local
vendor and housed in the University of Florida Health Center Animal Resources Department. Animals had access to food and water
ad libitum with a photoperiod of 12 hours
light and 12 hours dark. Relaxin antiserum
(R19) was produced against electrophoretically pure porcine relaxin in New Zealand
white rabbits as described by Larkin et al.
(1977). The R19 antiserum has been shown
to 1)give a single line of identity between
partially purified uterine relaxin from guinea
pigs and the purified porcine relaxin standard (NIH-RXN-P1) in Ouchterlony plate assays (Pardo and Larkin, 1980),2) inhibit the
action of porcine (Larkin et al., 1979) and
guinea pig relaxins (Pardo, 1982) in vitro, 3)
produce parallel lines of displacement in radioimmunoassay using extracts of guinea pig
uteri and purified porcine relaxin (Pardo,
Received March 21, 1983; accepted January 30, 1984.
374
R.J. PARDO, L.H. LARKIN. AND R.H. RENEGAR
of phosphate-buffered saline (pH 7.4) after
each step of the procedure. After imrnunostaining, sections were counterstained with
a 2% aqueous uranyl acetate solution for 3
minutes. Both osmicated and nonosmicated
immunostained sections were examined in a
JEOL 100s electron microscope a t 60 or 80
kV.
The following tests for specificity of immunolabeling were carried out: 1)substitution
of the relaxin antiserum (R19) with preimmune serum, 2) absorption of R19 antiserum
with purified porcine relaxin (NIH-RXN-P1)
prior to incubation, 3) omission of the primary antiserum (R19) and substitution with
phosphate buffered saline, and 4)dilution of
the R19 antiserum until no response was obtained. All of these tests gave results that
indicated the procedure specifically detected
relaxin.
Quantitative evaluation of immunogold
METHODS
staining was determined for grids treated
Adult female guinea pigs were housed with with R19 relaxin antiserum or preimmune
a male, and pregnancies were timed from the serum and protein A-gold. A minimum of ten
day on which sperm were found in a vaginal fields were photographed for each treatment
smear. Three animals on day 60 of pregnancy at a primary magnification of 10,000 or
and three animals that demonstrated marked 15,000x and enlarged 2.7 x during the printrelaxation of pelvic ligaments (day 63 to 65) ing process. Granules, randomly chosen by
were used in these studies.
application of a 1-cm2grid to each print, and
Uterine tissues were removed from anes- all nuclei (control area) in each field were
thetized guinea pigs and immersed in a 1% analyzed. Surface area for each organelle was
solution of glutaraldehyde (0.1 M cacodylate determined with a HIPAD DT-11 digitizer
buffer, pH 7.2) for 2 hours. Approximately (Houston Instrument, Austin, TX), interone-half the specimens were postfixed in 1% faced with a TRSSO model 1microcomputer
osmium tetroxide for 1hour. Both osmicated (Tandy Corporation, Fort Worth, TX). The
and nonosmicated specimens were dehy- number of gold particles present over the
drated through a series of cold alcohols and same compartment was counted and the denpropylene oxide and embedded in Araldite sity of labeling calculated (gold particles/
6005.
pm2). Density determinations for each orgaLight gold sections from the osmicated tis- nelle were considered as individual data
sues were placed on copper grids and stained points in a 2 x 2 factorial analysis of variwith uranyl acetate and lead citrate (Venable ance with location (granule or nucleus) and
and Coggeshall, 1965). Light gold sections treatment (R-19 relaxin antiserum or preimfrom nonosmicated tissues were placed on mune serum) as main effects.
nickle grids and processed for immunolabelRESULTS
ing after the technique of Roth et al. (1978).
Endometrial glands are simple, coiled, tuColloidal gold was prepared following the
technique of Frens (1973) and labeled with bular glands that extend from the lumen of
protein A as described by Roth et al. (1978). the uterus into the stroma, where their deepGrids containing the nonosmicated sections est portions lie adjacent to the inner circular
were first floated on a 10% solution of hydro- muscle layer of the uterus. Sections (1pm) of
gen peroxide for 10 minutes. Sections were plastic-embedded specimens viewed with the
treated with R19 relaxin antiserum (diluted light microscope revealed the tubular nature
1:lOO) for 1 hour and IgG-gold (diluted 1:10) of the glands and showed dense aggregates
or protein A-gold (diluted 150) for 2 hours. of granules within the cytoplasm of the EGC
Grids were washed with two 3-minute rinses (Fig. 1).The majority of the EGC exhibited
1982),and 4)produce no cross-reactivity with
other growth peptides such as insulin and
proinsulin in radioimmunoassay (unpublished observations). Specificity of the R19
antiserum to detect guinea pig uterine reIaxin has been discussed previously in articles concerning localization of the hormone
with the light microscope (Pardo et al., 1980;
Pardo and Larkin, 1982). Purified porcine relaxin (NIH-RXN-P1, 3,000 U/mg) was obtained from the National Pituitary Agency,
National Institute of Arthritis, Metabolism,
and Digestive Diseases. Goat antirabbit IgGlabeled colloidal gold (IgG-gold) was purchased from E-Y laboratories (San Mateo,
CA). Protein A was obtained from Sigma (St.
Louis, MO). Materials used in electron microscopy were purchased from either Ladd
Research Industries (Burlington, VT) or Polysciences, Inc. (Warrington, PA).
IMMUNOELECTRON LOCALIZATION OF RELAXIN
granules, and in most cells the granules were
accumulated in the supranuclear regions of
the cells near the lumen of the gland.
When EGC were examined in the electron
microscope, dense cytoplasmic granules were
the most striking feature (Fig. 2). The granules (upper range of = 1.0 Fm in diameter)
were composed of a dense, homogeneous material and were enclosed by a single membrane. In most sections the limiting
membrane was obscured by the granule contents, however, the membrane could be discerned in fortuitous sections of granules or
in forming granules (Figs. 2, 3). The Golgi
complex appeared to be quite active (Fig. 3)
and numerous stacks of Golgi cisternae were
noted (Fig. 2). Rough endoplasmic reticulum
was present throughout the cytoplasm.
Smooth endoplasmic reticulum was noted
mainly in the vicinity of the Golgi complex.
The luminal surfaces of EGC were characterized by microvilli and well-developed junctional complexes (Fig. 2).
Sections stained with either protein A-gold
or IgG-gold demonstrated that the granules
contained relaxin (Fig. 4). Infrequently, relaxin-containing granules were noted at the
luminal surface of the cell (Fig. 412). Since the
specimen in Figure 4c was neither osmicated
nor stained with lead, the membranes are
not well defined, and it is difficult to determine if the granule is actually in the process
of being released from the cell into the lumen
of the gland.
Quantitative evaluation demonstrated that
the density of immunolabeling over granules
was significantly greater (P < .01) than labeling over nuclei or granules from grids
treated with preimmune serum (Table 1).
DISCUSSION
Work reported in this article supports the
contention that relaxin is produced and
stored in EGC of the guinea pig uterus. This
conclusion is drawn because of the following:
TABLE I. Density of immunostaining ouer granules
and nuclei in guinea pig endometrial gland cells
(protein-A gold technique)
R-19 relaxin antiserum
Preimmune serum
'Particles/Frn2area
*P < .01.
Endometrial
granules'
Nucleus'
258.34*
34.76
23.55
8.39
375
1)The typical endometrial gland cell resembled a protein secreting cell in that it exhibited a n active Golgi complex, significant
amounts of rough endoplasmic reticulum,
and membrane-bounded dense granules. 2)
The granules were shown to contain relaxin
with immunolocalization techniques. 3) The
presence of a highly active Golgi complex
with forming granules indicated that the
granule contents most likely were synthesized in EGC. 4) It does not seem likely that
relaxin in the EGC was receptor bound, since
relaxin was not detected in association with
plasma membranes or cytoplasmic vesicles,
which would have been expected if relaxin
was bound to plasma membrane receptors on
the surface of the cell or that had been internalized (Duello et al., 1983).
Although profiles demonstrating the actual mechanism of granule release from the
cell were not observed in this study, both
light and electron microscopic studies indicated that the granules were located in close
proximity to the plasma membrane that
formed the luminal border of the cell. These
observations strongly suggest that the granules are released into the lumen of the gland
rather than into the stroma at the base of
the EGC. It seems most likely that the secretory process is either very rapid or is of a n
episodic nature and has not been arrested
and/or detected a t the precise moment of
granule release. If relaxin is released into
the lumen of the endometrial glands, it would
then have free access to the uterine lumen,
where it might act locally to influence placental detachment, softening of the uterine
cervix, or softening of fetal membranes. The
effect relaxin may have on these processes
has been discussed previously (Pardo et al.,
1980; Nagao and Bryant-Greenwood, 1981;
Pardo and Larkin, 1982). Support for a local
effect of relaxin was provided by MacLennan
et al. (1980) and Perezgrovas and Anderson
(19821, who demonstrated that topical application of relaxin caused softening of the uterine cervix in women and cows, respectively.
Additional evidence, which indicates relaxin
may function as a local hormone, has been
L
m
R
S
Abbreviations
lumen of uterine gland
uterine musculature
rough endoplasmic reticulum
endometrial stroma
376
R.J. PARDO, L.H. LARKIN, AND R.H. RENEGAR
IMMUNOELECTRON LOCALIZATION OF RELAXIN
377
Fig. 3. Golgi complex of endometrial gland cell. A limiting membrane can be seen surrounding the forming granules (arrows). Note the accumulation of rough endoplasmic reticulum
located around the Golgi complex and in adjacent endometrial gland cells. ~ 2 7 , 6 0 0 .
published that demonstrates the presence of
relaxin in preovulatory follicles (BryantGreenwood et al., 1980; Matsumoto and
Chamley, 1980). Recent studies have indicated that relaxin may act to facilitate rup-
Fig. 1. Endometrial gland. The coiled, tubular nature
of the gland is demonstrated. These glands extend from
the uterine lumen (not shown) through the endometrial
stroma to lie adjacent to the uterine musculature. Onepm section stained with toluidine blue. ~ 3 3 0 .Inset.
Higher magnification of portion of endometrial gland
(indicated by "1. Note accumulations of granules in the
supranuclear regions of the cells. Few granules are noted
in stromal portions of the gland cells. One-pm section
stained with toluidine blue. ~ 8 5 0 .
Fig. 2. Endometrial gland cells. Numerous dense
granules are seen in the luminal portions of the cytoplasm of the gland cells. Stacks of Golgi cisterne (arrows)
are evident. Microvilli and dark-staining junctional complexes are noted at the luminal surfaces of the cells.
X 12,600.Inset. Higher magnification of dense granules.
Note the homogeneous texture of the granules and the
single limiting membrane. ~ 2 4 , 9 0 0 .
ture of the follicle wall a t ovulation (Too et
al., 1982).
Previous observations that relaxin produced by the uterus causes relaxation of pelvic ligaments and can be detected in serum
(Zarrow, 1948; Nagao and Bryant-Greenwood, 1981) indicate that relaxin produced
by EGC reaches the systemic circulation. Relaxin may reach the systemic circulation by
movement from the uterine cavity through
the uterine luminal epithelium. However,
evidence is not available at this time to support this hypothesis, and immunolabeling
studies with the light (Pardo et al., 1980;
Pardo and Larkin, 1982) and electron microscopes (unpublished observation) have not
detected relaxin in luminal epithelial cells.
Relaxin may not have been found because
the amount of hormone may be too low to be
detected with the techniques employed, or
relaxin may be bound to a carrier molecule
such that antigenic sites are not exposed to
antibodies applied to sectioned material.
378
R.J. PARDO, L.H. LARKIN, AND R.H. RENEGAR
Fig. 4. Immunolabeling of granules. a) Incubated with
relaxin antiserum (R19) and IgG-gold. While some nonspecific binding is present, the granules are heavily labeled. ~ 2 8 , 0 0 0 .b) Incubated with preimmune serum
and IgG-gold. Note diminished labeling over granules.
The nucleus is present in the lower portion of the micro-
graph. ~ 2 8 , 0 0 0 .c) Incubated with relaxin antiserum
(R19) and protein A-gold. Note labeled granule in close
proximity to plasma membrane bordering the lumen of
the gland. ~ 2 8 , 0 0 0d)
. Incubated with preimmune serum
and protein A-gold. Note diminished labeling over granules. ~ 2 8 , 0 0 0 .
Relaxin-containing granules of EGC are
considerably larger than relaxin-containing
granules of pig granulosa lutein cells ( = 0.5
pm to 0.2 pm, respectively) (Kendell et al.,
1978). The significance of this difference in
size of relaxin-containing granules between
the two cell types is unclear at this time but
most likely relates to the fact that the lutein
cell is an endocrine cell, while the endometrial gland cell is an exocrine cell.
The findings presented here are consistent
with the hypothesis that uterine relaxin may
play an important role in pregnancy and/or
parturition in the guinea pig. The detection
of relaxin in the uterus of the guinea pig,
coupled with the fact that the guinea pig can
complete pregnancy and parturition in the
absence of a functioning ovary (Herrick,
19281, would lend emphasis to the uterine
relaxin’s potentially important function.
IMMUNOELECTRON LOCALIZATION OF RELAXIN
ACKNOWLEDGMENTS
The authors wish to thank Mr. George Papadi for his technical assistance.
This work was supported primarily by
grant HD-15576 and in part by grant HD08552 from the NIH.
Preliminary results of this investigation
were presented a t the First International
Symposium on Relaxin held in Florence, Italy, in October, 1982.
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