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Histopathologic changes of uterus following gossypol treatment during early pregnancy in rats.

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THE ANATOMICAL RECORD 213:72-76 (1985)
Histopathologic Changes of Uterus Follow ng
Gossypol Treatment During Early
Pregnancy in Rats
YASUKO RIKIHISA, YOUNG C. LIN, AND AMELIA WALTON
Division of Veterinary Biology and Clinical Studies, Virginia-Maryland Re) onal
College of Veterinary Medicine, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061
ABSTRACT
Intramuscular injections of gossypol acetic acid (25 mg in 10%EtOHi
kglday beginning on day 2 of diestrus) disrupted early pregnancy in rats as determined by light and electron microscopy. As in pregnant controls, in the uteri of
treated rats increased glandular secretion, stromal hyperemia, and decidual tissue
formation were noted at days 3-5 of pregnancy. At day 6, extreme hyperemia and
stromal hemorrhage had occurred around well-developed decidual tissue with foci of
denuded mucosal surface. There was extravasation of blood into the uterine lumen,
which was absent in controls. At days 5 and 6 of pregnancy, electron microscopy
revealed shorter and fewer microvilli on the uterine glandular cells in the treated
versus the control uterus. Luminal epithelial cells had not undergone the normal
changes of pregnancy. These results imply that gossypol administered under our
conditions neither prevented nor delayed implantation and formation of decidual
tissue in the rat uterine endometrium but continuing development of the endometrium was disrupted a t day 6 of pregnancy. This disruption of pregnancy may have
resulted from a luteolytic action by gossypol that would not permit full structural
differentiation in the rat uterus after implantation.
5'-diisopropyl3,
Gossypol [1,1t,6,6',7,7'-hexahydroxy-5,
3 '-dimethyl-(2,2'-binaphthalene)-8,8'-dicarboxaldehyde]
is a yellow phenolic compound (518.5 MW) found in the
cotton plant that is especially concentrated in the seed.
Gossypol was originally studied as a toxic compound in
cotton seed meal used for feeding livestock (Adams et
al., 1960). Later Chinese scientists demonstrated that
administration of gossypol to males in humans and rats
a t a low concentration (5-10 mg/kg) for several weeks
results in inhibition of spermatogenesis, which leads to
infertility without significant toxic effects (National Coordinating Group on Male Fertility, 1978). This finding
was subsequently confirmed in monkeys (Shandilya et
al., 1982), hamsters (Waller et al., 1981), mice (Coulson
et al., 1980; Shi and Zhang, 19801, and rabbits (Chang et
al., 1980). The antifertility effects of gossypol on males
are completely reversible (National Coordinating Group
on Male Fertility, 1978; Hadley et al., 1981).
In studies of antifertility induced by gossypol, administration of gossypol at 80 mgkg on each of the 3 days
prior to expected ovulation failed to inhibit ovulation in
the female rat (Hahn et al., 1980).Hahn et al. (1980) also
reported that in the female mouse administration of
gossypol a t 40-80 mgikg during days 1-13 of pregnancy
results in nonviable offspring (26-100% of the litters).
We have previously reported that intramuscular administration of gossypol to normally cycling female rats
induces an irregularity of the cyclic pattern for as long
as the treatment is continued (Lin et al., 1985).Furthermore, administration of gossypol from days 0 (day of
if-)
1985 ALAN
R. LISS, INC
sperm-positive vaginal smear) to 8 of pregnancy results
in the absence of implantation sites as determined by
laparotomy. Serum progesterone and estradiol 176 values in normally cycling and pregnant rats treated with
gossypol were significantly lower than control levels.
Supplementation with acornhination ofexogenousprogesterone and estradiol 17p eliminates the inhibitory effects of gossypol on the maintenance of pregnancy (Lin
et al., 1985).
In preparing the uterus for the implantation, each of
the cell types has a different function to perform. The
luminal epithelium provides the initial surface to which
the trophoblast attaches; the glands provide secretion,
probably to assist in the nourishment of the embryo
before the formation of the chorioallantoic placenta; and
the stromal cells differentiate into specialized decidual
cells that are especially important in animals in which
the blastocyst embeds in the wall of the endometrium.
In each case differentiation follows a period of rapid cell
division in the tissue. It is at these stages we have used
light and electron microscopy to focus on the histopathological changes in the uterus that follow gossypol treatment in rats.
MATERIALS AND METHODS
Animals
Adult female Sprague-Dawley rats (Charles River
Breeding Laboratory, Wilmington, MA) were housed in
Received January 10, 1985; accepted April 5, 1985
UTERINE HISTOPATHOLOGY AFTER GOSSYPOL
73
a temperature-controlled (19-22 "C) and humidity-con- packed and appeared darker (Figs. l b , 2b). On day 6,
trolled (50-60%) vivarium, and exposed to a photoperiod extreme hyperemia and stromal hemorrhage had ocregimen of 14 hours of light and 10 hours of darkness curred around well-developed decidual tissue. There
(lights on a t 0600 hours). The rats had free access to were foci of denuded luminal epithelium and extravasalaboratory chow and water. To define the stages of the tion of blood into the lumen. No inflammatory reaction
estrous cycle, vaginal smears were prepared and exam- was noted (Fig. 3b). At higher magnification in plastic
ined from all the experimental animals between 8:30 sections on day 6 of pregnancy, structures of scattered
and 1O:OO A.M. daily. Only the animals exhibiting a t decidual cells and red blood cells that had infiltrated the
least three consecutive, regular, 4-day cycles were used stroma were evident in treated rats (Fig. 4b), whereas
in these experiments. All of the rats were caged with the control endometrium at the same stage of pregnancy
males of proven fertility (one female with one male to a had a n organized distribution of decidual cells and no
cage) on the day of proestrus and separated on the next hemorrhage (Fig. 4a).
morning. The presence of sperm in a morning vaginal
Ultrastructural Observations
smear was used as a n indication of conception (desigTransmission electron microscopy of specimens from
nated day 0 of pregnancy).
pregnant control rat uteri at days 5 and 6 revealed that
Gossypol Treatment
the luminal epithelium had undergone autolytic degenGossypol acetic acid was obtained from the Southern eration (Fig. 5a), whereas a t the same stage in the pregRegional Research Center (USDA, New Orleans, LA). nant rat treated with gossypol the uterine epithelium
Gossypol dissolved in the vehicle (10% ethanol in 0.85% was intact except for the areas of massive hemorrhage
NaCl) was administered intramuscularly to 20 normally in the stroma at day 6 (Fig. 5b). The small blood vessels
cycling rats at 25 m g k g in 500 pl daily from day 2 of in the stroma beneath the uterine epithelium were endiestrus. Parenteral rather than oral administration was gorged with blood. Endothelial linings were absent in a
used to ensure that the entire dosage was received by few areas, and many red blood cells were seen in the
every animal every time. The 20 control animals re- stromal connective tissue. Blood vessels in the deeper
ceived 500 pl of vehicle only.
stroma appeared intact. The uterine glands had long,
Treated and control pregnant rats, five from each tightly packed microvilli a t the same stage in control
group, were sacrificed on days 3 , 4 , 5 , or 6 of pregnancy, rats (Fig. 6a), whereas in treated rats, the uterine glands
respectively. Uteri were excised and immediately fixed had short, sparse microvilli (Fig. 6b), which suggests
in a mixture of 5% glutaraldehyde, 2.5% paraformalde- that they were inactive.
hyde, and 0.03% trinitrophenol in 0.1 M cacodylate
DISCUSSION
buffer (pH 7.4) at room temperature for 2 hours and
washed in 0.1 M cacodylate buffer overnight.
The present study demonstrated that daily intramuscular injection of gossypol acetic acid in the manner
Light and Electron Microscopy
described neither prevents nor delays the formation of
For light microscopy, fixed uteri were dehydrated, decidual tissue, but the continuing development of the
cleared, and embedded in paraffin. Sections were depar- endometrium is disrupted by early abortion.
afinized and hydrated for either hematoxylin and eosin
Lin et al. (1985) demonstrated that gossypol has a
staining or the technic for the periodic acid-Schiff reac- luteolytic action in early pregnancy. The serum progestion according to McManus (1948).
terone level of rats treated with similar dosages of gosFor electron microscopy, specimens of the fixed uteri sypol is 70% lower than controls a t day 6 of pregnancy,
were postfixed for 1hour in 1% osmium tetroxide in the whereas estrogen levels are 30% lower. Estrogen and
same buffer. After block stain in 1%uranyl acetate in progesterone should be administered to rats that have
maleate buffer (pH 5.21, tissues were dehydrated in a been ovariectomized 4 days postinsemination for imgraded series of ethanol and propylene oxide, and plantation to occur (Jackson, 1966). The 70% lower conembedded in PolyBed 812 (Polysciences, Inc., Warring- centration of the serum progesterone level may not
ton, PA). Thin sections (60-90 nm) were then stained inhibit decidual tissue formation, but may inhibit its
with uranyl acetate and lead citrate and examined with subsequent development. Ergotoxin alkaloids also tera JEM 100 CXII electron microscope.
minate early stages of pregnancy. These chemicals appear to inhibit progesterone biosynthesis (Shelesnyak et
RESULTS
al., 1963). Lack of delay in decidual response also supLight Microscopic Observations
ports our endocrinologic data. Delayed implantation and
In pregnant controls a t day 3, increased glandular decidual response normally occur in a uterus mainsecretion and stromal hyperemia had become evident. tained by progesterone but lacking sufficient estrogenic
At day 4, the lumen had become surrounded by wavelike stimulation (Mayer, 1963; Nutting and Meyer, 1963).
projections and focal closures had occurred (Fig. la). At
Prolactin has been reported to have luteotrophic acday 5 closure of the lumen had occurred in all parts. The tions in rodents (Horrobin, 1973);however, the effect of
stroma was extremely edematous and hyperemic (Fig. gossypol on prolactin secretion has not been reported. It
2a). Small foci of decidual tissue containing mitotic fig- is not known whether the abortion that took place in
ures were present beneath the uterine epithelium. At rats in our study is completely a secondary effect of
day 6, the decidual mass had become larger and in- gossypol, which has its primary effect on ovarian horcreased numbers of mitotic figures were present (Fig. mone production, or whether gossypol also has a direct
3a). In treated rats, similar changes of the endometrium effect on the uterus. Gossypol inhibits rapidly growing
were noted at days 3-5; however, in the stroma just cells such as Chinese hamster ovary (CHO) and HeLa
beneath the luminal epithelium, cells were closely cell cultures (Wang and Rao, 1984). Thus, it is possible
Fig.1. A section of the uterus from a control rat at day 4 of pregnancy
(a) compared to the uterus from a gossypol-treated rat at day 4 of
pregnancy (bj. The uterine lumen is surrounded by wavelike projections and focal closures have occurred. Glandular secretion is active.
In the treated rat uterus, however, the endometrium is thinner and
less edematous than in the control. PAS stain. Bar = 100 pm.
Fig.2.A section of the uterus from a control rat at day 5 of pregnancy
(a) compared to the uterus from a gossypol-treated rat at day 5 of
pregnancy (b).In (a) complete closure of the lumen has occurred; the
stroma is more edematous and hyperemic than at day 4. A small focus
of decidual tissue containing mitotic figures (arrows) is evident. In (b)
the uterine lumen is surrounded by wavelike projections. The endometrium is thinner than in the control. Stromal cells are condensed
beneath the epithelium. PAS stain. Bar = 100 pm.
Fig. 3.A section of the uterus from a control rat (a)compared to the
uterus from a gossypol-treated rat ( b j at day 6 of pregnancy. In the
control, the decidual mass has become much larger and protrudes into
the lumen (arrow), whereas in the gossypol-treated rat, there is extreme hyperemia and stromal hemorrhage around well-developed decidual tissue, foci of denuded mucosal surface, and extravasation of
blood (arrow) into the uterine lumen. PAS stain. Bar = 200 pm.
Fig.4.A plastic section of control rat uterus (a) compared to a plastic
section of the uterus from a gossypol-treated rat (b) at day 6 of pregnancy. In the treated rat uterus, scattered decidual cells and red blood
cells flooded (arrows) in the stroma are evident. Toluidine blue stain.
Bar = 100 rrm.
Fig. 5. A transmission electron micrograph of the uterine epithelium
of a control rat (a) at day 6 of pregnancy compared to a transmission
electron micrograph of the uterine epithelium of a gossypol-treated rat
(b) at day 6 of pregnancy. In the control, microvilli are very irregular.
Some epithelial cells are disintegrating (arrow). There are numerous
fat droplets around nuclei, a characteristic of the epithelial cells at this
stage of pregnancy (Enders and Schlafie, 1967). In the treated rat
uterus, epithelial cells are stretched by the stromal hemorrhage be-
neath them. Bar
=
5 pm.
Fig. 6. An electron micrograph of the uterine gland of a control rat
(a)at day 6 of pregnancy having relatively long, tightly packed microvilli on the apical surface (arrow)of the secretory cells compared to an
electron micrograph of the uterine gland of a gossypol-treated rat (b)
at day 6 of pregnancy with sparse, short microvilli (arrow). Bar = 2
pm,
76
Y. RIKIHISA, Y.C. LIN, AND A. WALTON
that the rapid growth of decidual cells may be directly
affected.
rats, the blastocyst attaches to the uterine epithelium at day 5 of pregnancy (Enders and Schlafke, 1967).
In our studies stromal cell differentiation and decidual
growth proceeded as is normal for the pregnant uterus
the G O s s difference was the masup to day 5*
sive
in the uterus, which Occurred
at day of
pregnancy in the rats treated with gossypol. Ultrastructural study revealed that the autolytic degeneration of
uterineepithelium, which normally occurs at day 6 of
pregnancy (Enders and Schlafke, 1967), did not Occur.
Microvilli of the uterine glands are much sparser and
shorter in the treated rats.
for the bleeding process that occurred is
The
unclear. The Pattern Of hemorrhage is rather similar to
that of serotonin-induced interruption of pregnancy. In
rats, vascular leakage owing to the widening of spaces
between endothelial cells in
vessels is suggested to be the cause Of the bleeding with serotonin
(Mitchell and Hammer, 1983). In gossypol-treated rats,
were dispersed at the sites Of
as in menstrual bleeding without autolysis. By contrast,
the hemorrhage in Our treated rats is different from
artificial implantation of glass beads into rat uterus,
where hemorrhage is observed in the implantation cavity at day 6, but there is no bleeding into the decidual
tissue itself (Blandau, 1949). This suggests that the fertilized ova were not dead at the time of abortion. The
lack of leukocyte infiltration also suggeststhat the hemorrhage is not a reaction to dead or abnormal ova.
Since the deleterious effects of gOSSYPOl on mainknance of pregnancy are almost completely prevented by
exogenous progesterone
and estrogen administration
et al.,
gossYpo1
is not cytotoxic to the
blastocyst or endometrium. The ability Of gOSSYPOl to
terminate the early stage of pregnancy in rats suggests
its possible application as a postcoital antifertility agent.
ain
1985)7
ACKNOWLEDGMENTS
This work was supported through NIH grant HD
17627 and the Southern Regional Research Center,
United States Department Of Agiculture, New Orleans,
LA.
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