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The Normal Genital Tract of the Female California Sea Lion Zalophus californianusCyclic Changes in Histomorphology and Hormone Receptor Distribution.

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THE ANATOMICAL RECORD 292:1801–1817 (2009)
The Normal Genital Tract of the
Female California Sea Lion
(Zalophus californianus): Cyclic Changes
in Histomorphology and Hormone
Receptor Distribution
KATHLEEN M. COLEGROVE,1* FRANCES M.D. GULLAND,2
DIANE K. NAYDAN,3 AND LINDA J. LOWENSTINE1
1
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine,
University of California, Davis, California
2
The Marine Mammal Center, Marin Headlands, Sausalito, California
3
Veterinary Medical Teaching Hospital, School of Veterinary Medicine,
University of California, Davis, California
ABSTRACT
Changes in reproductive tract histomorphology, and estrogen (ERa)
and progesterone receptor (PR) expression throughout the breeding cycle
were evaluated in free-ranging stranded female California sea lions (Zalophus californianus). Hormone receptor expression in the ovaries, uterus,
cervix, and vagina was evaluated using an immunohistochemical technique with monoclonal antibodies. During a large portion of the cycle,
ovaries contained both a corpora lutea (CL) and follicles in varying stages
of development. In the periods of pupping and estrus during June and
July, and in the spring morphologic features of the endometrium suggested estrogen influence. There were areas of squamous differentiation
in the pseudostratified columnar epithelium of the cervix and vagina in
some animals during estrus and in the spring. Estrogen receptor immunohistochemical scores were highest during pupping and estrus and in
the spring and lowest during embryonic diapause. Cyclic changes in uterine PR expression throughout the cycle were minimal. Both ERa and PR
were expressed in epithelial and stromal cells throughout the cervix and
vagina, however, receptor expression was typically higher in the stroma.
Stromal cell hormone receptors may play an important role in epithelial
responses to circulating sex hormones. The results of this investigation add to the general knowledge of California sea lion reproduction and establish baseline information on reproductive tract hormone
receptors that will aid in determining the factors involved in urogenital
C 2009
cancer development in sea lions. Anat Rec, 292:1801–1817, 2009. V
Wiley-Liss, Inc.
Grant sponsor: National Institutes of Health (Postdoctoral
Training in Environmental Pathology); Grant number:
5T32ES007055-30; Grant sponsor: West Coast Center of
Excellence (NOAA Oceans and Human Health Initiative);
Grant number: AB133F05SE5112.
*Correspondence to: Kathleen M. Colegrove, Zoological Pathology Program, College of Veterinary Medicine, University of
Illinois, Loyola University Medical Center Building 101 Room
C 2009 WILEY-LISS, INC.
V
0745, 2160 South First Avenue, Maywood, IL 60153. Fax: 708216-5934. E-mail: kcolegrove@lumc.edu
Received 18 June 2008; Accepted 7 July 2009
DOI 10.1002/ar.21009
Published online 18 September 2009 in Wiley InterScience (www.
interscience.wiley.com).
1802
COLEGROVE ET AL.
Key words: California sea lion; embryonic diapause; estrogen
receptor; progesterone receptor; reproductive
tract; urogenital cancer; Zalophus californianus
Evaluation of the normal reproductive cycle in wild
marine mammals is often hindered by limited access,
low sample numbers of healthy individuals, and lack of
a thorough reproductive history. Currently, opportunistic
sampling of deceased stranded animals is the primary
means of studying normal anatomy, histomorphology,
and diseases of free-ranging animals (Gulland, 1999).
Gaining an understanding of normal reproductive physiology is crucial, however, for accurate evaluation of population demographics, causes of reproductive loss, and
the potential effects of environmental contaminant exposure. In all pinniped species, the annual reproductive
cycle includes synchronized parturition, estrus, embryonic diapause, placental pregnancy, and lactation. The
most detailed information on the reproductive cycle of
pinnipeds comes from investigations of wild northern fur
seals (Callorhinus ursinus) and harbor seals (Phoca
vitulina). Histologic evaluations of normal reproductive
tracts in both species have shown cyclic morphologic
changes corresponding to ovarian cycling and pregnancy
(Craig, 1964; Bigg and Fisher, 1974). During pinniped
embryonic diapause, conceptus development arrests at
the blastocyst stage with the zona pellucida intact. The
blastocyst remains unattached in the uterus throughout
diapause until implantation (Boshier, 1981). Circulating
reproductive hormones have been measured in fur and
harbor seals in several studies (Daniel, 1974; Reijnders,
1990; Browne et al., 2006), however, the physiology and
endocrine regulation of diapause and reproduction in
pinnipeds remains poorly understood.
Like other pinniped species, California sea lions (Zalophus californianus) have an annual reproductive cycle
that is highly synchronized. Females give birth to a single pup in June on breeding rookeries on the Channel
Islands of California and islands off of Baja California,
Mexico. On San Nicolas Island, California, the majority
of pups are born during the first week in June (Odell,
1975). Estrus and conception occur 3–4 weeks after parturition. Embryonic diapause lasts approximately 2
months, with implantation occurring by early October.
Placental gestation lasts 8–9 months till the following
June (Reeves et al., 1992). Relatively little is known
about seasonal sex hormone fluctuations and corresponding changes in reproductive tract histology in California
sea lions. A recent study investigated seasonal changes
in circulating serum progesterone and estrogen in freeranging and captive female sea lions. Progesterone concentrations increased in the fall irrespective of pregnancy status and progesterone concentrations were
higher in pregnant animals when compared with nonpregnant animals in the spring. In the majority of animals, there was no clear pattern in the estrogen
concentration (Greig et al., 2007).
Sex steroids exert their effects through interaction with
intracellular steroid hormone receptors located in target
tissues. Accordingly, estrogen (ERa), progesterone (PR),
and androgen receptors (AR) are located throughout the
reproductive tract of mammalian species, mediating the
effects of steroid hormones on reproductive organs. The
distribution of steroid hormone receptors in the human
female reproductive tract, in relationship to cyclic
changes and histomorphology, has been well documented
(Press et al., 1986). Similar studies have documented hormone receptor distribution in the reproductive tract of
animal species including the dog (De Cock et al., 1997;
Vermeirsch et al., 2000a; Vermeirsch et al., 2002), cat (Li
et al., 1992), mare (Watson et al., 1992), sow (Sukjumlong
et al., 2003), and cow (Kimmins and MacLaren, 2001). To
our knowledge, reproductive tract hormone receptors
have not been examined in any marine mammal species.
A high prevalence of aggressive urogenital carcinomas
has been documented in California sea lions stranding
along the central California coast. These tumors arise in
the cervix and vagina of females and the penis, prepuce,
and urethra of males. Widely disseminated metastases
are often found in thoracic and abdominal viscera of
affected animals (Gulland et al., 1996; Lipscomb et al.,
2000). Some sea lions dying from other causes have histologic evidence of early neoplasia in genital epithelium
consisting of epithelial hyperplasia, dysplasia, and carcinoma in situ. Differentiating between early neoplasia
and epithelial changes due to normal cyclic hormone
changes is sometimes difficult.
The purpose of this investigation was to describe the
normal cyclic changes in histomorphology and steroid
hormone receptor distribution in the genital tract of normal free-ranging female California sea lions throughout
the reproductive cycle. These observations will provide
additional baseline information on the normal physiology and endocrine regulation of reproduction in sea lions
and will allow for better differentiation between normal
and precancerous epithelium. Additionally, defining the
hormone receptor distribution in tissues prone to cancer
will aid in identifying factors, such as endogenous hormones or environmental contaminants, which may play
a role in cancer development.
MATERIALS AND METHODS
Animals
Formalin-fixed whole reproductive tracts and archived
paraffin-embedded tissues opportunistically sampled
from California sea lions that stranded live on the central California coast (37 420 N, 123 050 W to 35 590 N,
121 300 W) were examined. Adult female sea lions (N ¼
24) and cycling juvenile female sea lion (N ¼ 2) that
died during rehabilitation at The Marine Mammal Center (TMMC) in Sausalito, California were evaluated.
Animals dying during all months of the yearly reproductive cycle were represented. Causes of death included
domoic acid toxicity (N ¼ 14), trauma (N ¼ 4), leptospirosis (N ¼ 4), pneumonia (N ¼ 1), enteritis (N ¼ 1), and
peritonitis (N ¼ 1), pyelonephritis (N ¼ 1). Animals
selected for the study had no significant reproductive
1803
FEMALE SEA LION REPRODUCTIVE TRACT
tract lesions based on gross and histologic examination.
Age class was estimated by standard length (measured
from nose tip to tail tip), tooth dentin growth rings (Oosthuizen et al., 1998), and gross and histologic evaluation
of the uterus for evidence of previous pregnancy.
Histomorphology
A gross necropsy was performed on all animals within
24 hr of death. Routine tissue samples, including the
entire reproductive tract, were fixed in 10% neutral buffered formalin, processed routinely for paraffin-embedding, sectioned at 5 lm and stained with hematoxylin
and eosin for histologic examination. Sections examined
histologically included cross sections of both uterine
horns and midsagittal sections through both ovaries, longitudinal sections through the cervix, and transverse sections of the cranial and middle vagina. Ovaries were
evaluated for the presence of follicles, corpora lutea
(CLs), and corpora albicans (CAs). Primary follicles had
an oocyte surrounded by a single layer of cuboidal cells.
Follicles were defined as secondary follicles if they had
two or more layers of granulosa cells but without an
antrum. Tertiary (antral) follicles contained a fluid filled
cavity with multiple layers of granulosa cells surrounded
by the theca interna, and theca externa. Folliculogenesis
was defined as the presence of secondary and antral follicles within the ovary. In degenerating CLs, cells were
variably shrunken with vacuolated cytoplasm, and were
dissected by bands of fibrous connective tissue. Corpora
albicans were defined as structures containing hyalinized
eosinophilic fibrous connective tissue with no remaining
luteal cells (Senger, 2005). Atretic follicles were present
in most ovaries and were not quantified. The size, shape,
and degree of cytoplasmic vacuolation of stromal interstitial cells were determined for each ovary. Uterine surface
and glandular epithelia were evaluated based on the following features: shape and height of epithelial cells, cytoplasm vacuolation (low, moderate, heavy), secretion
(absent, low, moderate, high), tortuosity (low, moderate,
high), and the presence of subnuclear glycogen vacuoles
(SNV). Uterine stromal edema and vascularity were also
evaluated. In animals that were pregnant or post parturient at the time of death, the pregnant and nonpregnant
horns were evaluated separately. Epithelium of the cervix and vagina were evaluated for the following features:
thickness of the epithelium, height of epithelial cells,
cytoplasmic vacuolation, degree of invagination, gland
formation, and presence of goblet cells.
Immunohistochemistry
Immunohistochemistry was performed on serial sections of urogenital tissue from a subset of 15 females in
which tissues were best preserved using a strepavidin-biotin method. Females evaluated had died during the time
of pupping and estrus (N ¼ 4), diapause (N ¼ 3), fall (N
¼ 4), and winter to spring (N ¼ 4). After deparaffinizing,
endogenous peroxidase activity was blocked by incubating sections in 0.2% H2O2 in methanol for 30 min. Antigen retrieval was accomplished for ERa and PR by
heating sections to 95 C for 30 min in Citrate buffer (pH
¼ 6.2) (Dako Cytomation, Carpinteria, CA) in a rice
steamer. Sections were incubated in 3% normal goat serum for 30 min. Sections were incubated with a monoclo-
nal antibody to human ERa (1:125:1D5 Immunotech,
Marseille, Cedex 9, France) and a monoclonal antibody to
human PR (1:200:10A9 Immunotech, Marseille, Cedex 9,
France) overnight at 4 C in a moist chamber. For Ki67,
sections were incubated with the primary monoclonal
antibody (1:75: MIB-1 Dako Cytomation, Carpinteria,
CA). Slides were incubated with a biotinylated antimouse link reagent (Biocare Medical, Concord, CA) for 10
min and then incubated with streptavidin horseradish
peroxidase (Biocare Medical, Concord, CA) for 10 min.
Positive staining was visualized using 3-amino-9-ethylcarbazole (AEC) chromogen (Zymed Labs, San Francisco,
CA). After all steps, sections were rinsed in phosphatebuffered
saline
(PBS)
spiked
with
polyoxyethylenesorbitan monolaurate (TWEENV 20, SigmaAldrich, St. Louis MO). Sections of canine uterus known
to be positive for ERa and PR were included in each procedure as positive controls. Antibodies utilized have been
shown to be cross reactive to ERa and PR in an number
of animal species (Vermeirsch et al., 2000a; Vermeirsch
et al., 2000b; Martin de las Mulas et al., 2002; Vermeirsch et al., 2002; D’Haeseleer et al., 2006; D’Haeseleer et al., 2007). Negative controls were sections of sea
lion uterus incubated with omission of the primary
antibody.
Immunostaining of all sections was evaluated by a single person without prior knowledge of the animal from
which the tissue was sampled. Expression of ERa and
PR was scored using a semiquantititative grading system
identical to the grading system used in similar canine
studies (De Cock et al., 1997; Vermeirsch et al., 1999;
Vermeirsch et al., 2002). For each tissue examined, the
representative section was dividend into five regions of
approximately equal surface area and randomly selected
areas evaluated within those regions were selected prior
to observation of immunohistochemical stains. For each
of the five sections, 100 of 500 cells were evaluated. A
total score was calculated by the following formula TS ¼
PS þ IS, where TS is the total score, PS is the proportional score and IS is the intensity score. The proportional score corresponded to the percentage of 500 cell
nuclei that stained positive. The intensity score reflected
the subjectively evaluated intensity of positive, brown—
red staining (Table 1). Proliferation index was calculated
as a percentage of positively stained uterine epithelial
nuclei out of 500 total evaluated cells.
R
Serum Hormone Analysis
Serum archived at 80 C at the time of death was
available from 11 animals for steroid hormone analysis.
Serum samples were assayed for progesterone by enzyme
immunoassay (EIA) and estradiol by radioimmunoassay
(RIA) at the Clinical Endocrinology Laboratory, Department of Population Health and Reproduction, School of
Veterinary Medicine, University of California, Davis, CA
(Shille et al., 1979; Munro and Stabenfeldt, 1984).
Statistics
The Kruskal-Wallis, nonparametric test was used to
analyze the difference in total immunohistochemical
scores between the four different times of the reproductive cycle. The Spearman rank correlation test was used
to analyze potential correlations between receptor scores
1804
COLEGROVE ET AL.
TABLE 1. Immunohistochemistry grading scores
Intensity score
Score
Score
Score
Score
0
1
2
3
No staining
Weak staining
Moderate staining
Strong staining
Proportional score
Score
Score
Score
Score
Score
Score
0
1
2
3
4
5
No staining
<1% positively stained nuclei
1–9% positively stained nuclei
10–32% positively stained nuclei
33–65% positively stained nuclei
>65% positively stained nuclei
Total scores were calculated by adding the intensity and the proportional scores.
Fig. 1. Endometrial surface at a site of previous placental attachment
in a California sea lion (Zalophus californianus). Scale bar ¼ 2.0 cm.
and serum hormone levels. Statistical calculations were
performed using MedcalcV statistical software, Version
9.1.0.1 1993 (Medcalc, Mariakerke, Belgium). A P-value
< 0.05 was considered statistically significant.
R
RESULTS
Gross Morphology
The reproductive tract of female sea lions is similar to
other phocids and otariids. The oblong ovaries are
encased in a thick bursa that can become enlarged and
contain small amounts of mucous in adult animals. Long
arborizing oviduct fimbria extend over the surface of the
ovary. The uterus is bicornuate and in a previous investigation of 52 archived specimens the uterine horns
averaged between 11.0 and 12.0 cm in length after formalin fixation (Lowenstine, unpublished data). In multiparous adults, the uterine serosa is coarsely nodular,
corresponding to the numerous thick-walled blood vessels that remain after pregnancy. The endometrial surface ranges from light pink to tan with prominent
longitudinal folds extending along the length of the uterine horns. In post parturient animals, there is a well
demarcated, circumferential, yellow brown band of variable width visible on the endometrial surface, corresponding to the site of previous attachment of the
zonary placenta (Fig. 1). There are two internal cervical
ostia and the internal cervical mucosa extends into the
uterine horns such that there is no appreciable uterine
Fig. 2. Anatomic features of the female California sea lion (Zalophus californianus) reproductive tract. Formalin-fixed. (a) uterine horns;
(b) external uterine bifurcation, (c) internal cervical bifurcation; (d),
external cervical fold; e, urinary bladder; f, hymenal fold; g, urethral
protuberance; h, clitoris.
body (Fig. 2). The length of the cervical canal from the
bifurcation to the external cervical fold averaged 5.6 cm
in a previous investigation (Lowenstine, unpublished
data). There are no cervical rings and longitudinal folds
extending along the length of the cervix. There are small
transverse ridges in the cranial vagina and a prominent
hymeneal fold in the middle portion of the vagina. A triangular urethral tubercle lies along one side of the hymeneal fold directly above the urethral opening. It has
been postulated that the prominent hymeneal fold of
pinnipeds is an adaptation to prevent entry of water
FEMALE SEA LION REPRODUCTIVE TRACT
into the urogenital canal during diving (Harrison, 1969).
Longitudinal folds extend along the caudal vagina and
the clitoris is sheathed in the clitoral fossa along the
ventral commissure of the vestibule. During estrus the
clitoris can become erect and protrude from the vestibule. Small, dark brown submucosal vestibular glands
(Bartholin’s glands) can be noted on cut sections of the
vestibule-vagina junction.
Histomorphology
Ovary. General histologic characteristics of the sea
lion ovary included prominent subsurface epithelial
structures and stromal interstitial cells. Similar to the
bitch, the subsurface epithelial structures are invaginations of the surface (germinal) epithelium that extend
into the superficial cortex forming cords and nests that
often run parallel to the ovarian capsule. These structures generally increased in prominence with age and
were occasionally cystic. Focal clusters of hyperplastic
surface epithelium could occasionally be found along the
ovarian capsule. Thick-walled hyalinized arterioles were
scattered throughout the medulla and in some females
there was segmental subintimal vascular mineralization.
Thick, folded, eosinophilic membranous structures were
often scattered in stroma. These structures were interpreted to be remnants of atretic follicles. Interstital cells
in the stroma surrounding atretic follicles were often
more prominent and clustered, with moderate to large
amounts of eosinophilic cytoplasm.
Cyclic changes in the presence of CLs and follicles,
and the appearance of stroma interstitial cells were
noted throughout the yearly reproductive cycle (Table 2).
During pupping and estrus in June and July, follicles
were present bilaterally in most animals. In some
females folliculogenesis was asymmetric, with greater
numbers of antral and larger preovulatory follicles in
one of the ovaries. One animal (sea lion 4) dying in early
July had a large cluster of polygonal cells with abundant
pale eosinophilic cytoplasm. These cells resembled the
lutenized cells of a CL suggesting recent ovulation and
early CL formation. In post parturient animals, CAs
were sometimes seen on the ovary from the gravid horn
and CLs on the contralateral ovary, suggesting ovulation
occurs in alternating ovaries each year. When folliculogenesis was present, ovarian interstitial cells were often
large, polygonal, and had foamy to vacuolated eosinophilic cytoplasm (Fig. 3A). During diapause, in August
and September, ovaries usually contained a CL. Interstitial cells were smaller than in estrus, and contained
granular cytoplasm, often with abundant light brown
granular pigment consistent with lipofuscin. Ovarian
morphology was highly variable during the fall, with
ovaries containing a CL and follicles in varying stages.
Implantation in sea lions is thought to occur during late
September and early October. During this period, all of
the sea lions examined had an active CL in one of the
ovaries and some follicular development. Folliculogenesis
was common in ovaries of animals dying from late
November till May regardless of pregnancy status, however, follicles rarely reached the size of the large, preovulatory follicles noted in July. During this time period,
folliculogenesis typically occurred in both ovaries, even
in the presence of a large CL (Fig. 3B). Some nonpregnant animals had a degenerating CL in one ovary sug-
1805
gesting prior fetal loss. In the spring, stromal interstitial
cells were often larger than in diapause and contained
foamy to vacuolated cytoplasm.
Uterus. Distinct changes in the morphologic appearance of the uterine mucosa were also noted throughout
the yearly reproductive cycle (Table 3). During the pupping and estrus, in late June and July, folliculogenesis
was accompanied by changes indicative of estrogen influence on the endometerium. Endometrial glands were
straight, epithelial cells of the surface lining and glands
were cuboidal to medium columnar, and there was variable edema and congestion of the stroma (Fig. 4A). By
late July, the endometrium was thicker and contained
more developed glands. Several animals had tall
columnar surface and glandular epithelium, increased
amounts of foamy cytoplasm, and moderate to high levels of proteinaceous glandular secretion. Glands in the
basal region of the endometrium appeared more tortuous
and epithelium in the glands was often more than one
cell layer thick. The endometrial stroma was edematous
and congested in several animals sampled during July.
Progestational changes were more common in animals
with a CL (sea lions 6, 7) or animals with evidence of
recent ovulation (sea lion 4). The endometrium was
thickest and contained numerous gland profiles during
embryonic diapause and in the fall. Endometrial glands
were tortuous and the surface and gland epithelial cells
were tall columnar with abundant foamy cytoplasm indicative of a response to progesterone secretion from the
CL (Fig. 4B). Animals dying during the winter and
spring commonly had similar endometrial features indicative of estrogen influence, regardless of pregnancy status. Surface and gland epithelial cells were cuboidal to
low columnar, had small amounts of pale cytoplasm, and
there were fewer gland profiles than in diapause or during the fall (Fig. 4C). Glands were often dilated and
filled with proteinaceous secretion and glands in the basal region of the endometrium appeared more tortuous.
Significant stromal infiltration by leukocytes was not
found at any time period of the cycle. Mitotic figures
were extremely rare in the epithelium of nonpregnant
uterine horns. The average proliferation index for the
surface and gland epithelium was 1.2% and 1.7%,
respectively in the nongravid horn.
In the pregnant uterine horn at the zone of placentation, the endometrium was thin and glands were widely
separated by pale eosinophilic highly congested and often
edematous stroma. Glands were lined by cuboidal to low
columnar epithelium with minimal cytoplasm and were
sometimes dilated by eosinophilic proteinaceous fluid.
The superficial epithelium in contact with the placenta
was composed of shallow folds of columnar to low cuboidal epithelial cells containing only small amounts of cytoplasm mixed with occasional syncytial trophoblastic cells.
Trophoblastic cells were only noted in the superficial zone
of endometrial-placental contact and did not invade
deeply into the endometrium. There was only mild proliferation of endometrial epithelium and no evidence of a
decidual reaction in any of the pregnant sea lions.
At parturition, the superficial portion of the mucosa is
partially torn away, leaving the basal glands intact. The
endometrial stroma in the zone of placentation of early
post-parturient sea lions was expanded by edema,
a
November 26
Pregnant left horn
Pregnant right horn
Pregnant left horn
Pregnant right horn
Pregnant left horn
Pregnant right horn
Post-parturient
right horn
17
18
19
20
21
22
23
24
May 10
May 12
April 11
April 19
March 5
January 19
February 18
January 12
December 16
CL, corpora lutea; CA, corpora albicans.
Post parturient
left horn
October 31
16
25
26
October
October
October
October
12
13
14
15
10
11
19
23
August 8
August 11
September 4
September 14
July 30
8
9
10
11
7
July 9
July 22
Post-parturient
left horn
Post-parturient
right horn
5
6
July 7
June 23
July 6
Date collected
July 8
Post-parturient
left horn
Post-parturient
right horn
Condition
4
3
1
2
Sea lion #
Folliculogenesis
Folliculogenesis
CL
Degenerating CL
Secondary follicles
CL
Secondary follicles
Folliculogenesis CL
CA
Tertiary follicle CL
Folliculogenesis
CL
CL
Folliculogenesis
CL
CL
Folliculogenesis CL
CA
Folliculogenesis
Folliculogenesis CL
Folliculogenesis
Folliculogenesis CA
Secondary follicles
Folliculogenesis
Right ovary
Folliculogenesis CL
Folliculogenesis
Folliculogenesis
Folliculogenesis CL
Folliculogenesis
Secondary follicles
CL
Folliculogenesis CL
Folliculogenesis CL
Folliculogenesis CL
Tertiary follicle
CA
Folliculogenesis CL
Folliculogenesis
luteinized cells
Folliculogenesis
Folliculogenesis
Folliculogenesis
Folliculogenesis
Secondary follicles CA
Left ovary
Large size, polygonal, foamy cytoplasm with lipid vacuoles
Small size, cuboidal, granular cytoplasm with lipofuscin
Small size, cuboidal, granular cytoplasm with lipofuscin
Small size, spindle to cuboidal, granular cytoplasm with
lipofuscin
Moderate size, cuboidal, granular cytoplasm with lipofuscin
Moderate size, polygonal, granular cytoplasm
Moderate size, cuboidal, granular cytoplasm with lipofuscin
Small size, spindle to cuboidal, granular eosinophilic
cytoplasm with lipofuscin
Small size, spindle to polygonal, granular cytoplasm with
lipofuscin
Moderate size, cuboidal, foamy cytoplasm with lipid
vacuoles and lipofuscin
Moderate size, polygonal, foamy cytoplasm with lipid
vacuoles
Moderate size, polygonal, foamy cytoplasm with lipid
vacuoles
Large size, polygonal, foamy cytoplasm
Moderate size, polygonal, foamy to granular cytoplasm and
lipid vacuoles
Small size, cuboidal to spindle, granular eosinophilic
cytoplasm
Moderate size polygonal, foamy cytoplasm
Large size, polygonal, foamy cytoplasm with lipid vacuoles
and lipofuscin
Small size, cuboidal, granular eosinophilic cytoplasm
Moderate size, polygonal, foamy cytoplasm with lipid
vacuoles
Moderate size, cuboidal, granular cytoplasm
Moderate size, cuboidal, granular eosinophilic cytoplasm
Moderate size, cuboidal, granular eosinophilic cytoplasm
Large size, polygonal, pale eosinophilic cytoplasm
Large size, polygonal, pale eosinophilic cytoplasm
Moderate size, polygonal foamy vacuolated cytoplasm
Large size, polygonal, foamy vacuolated cytoplasm
Interstitial cells
TABLE 2. Histologic detail of the ovaries from 26 stranded California sea lions (Zalophus californianus)a
1806
COLEGROVE ET AL.
1807
FEMALE SEA LION REPRODUCTIVE TRACT
Fig. 3. A, B: Ovarian structures noted in California sea lions (Zalophus californianus) collected (A) in
July during pupping and estrus (Scale bar ¼ 500 lm) and (B) in the spring during pregnancy (Scale bar ¼
1.0 mm). Note the developing follicles (FOL), subsurface epithelial structures (SES), stromal interstitial
cells (INT), and the corpora lutea (CL). H&E.
TABLE 3. Histologic features of the uterus of 26 stranded California sea lions (Zalophus californianus)a
Sea
lion #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Condition
post-parturient
post-parturient
post-parturient
post-parturient
Pregnant
Pregnant
Post-parturient
Pregnant
Pregnant
Post-pregnant
Pregnant
Post parturient
Date
collected
Surface cell
height and shape
Gland cell
height and shape
June 23
July 6
July 7
July 8
July 9
July 22
July 30
August 8
August 11
September 4
September 14
October 10
October 11
October 19
October 23
October 31
November 26
December 16
January 12
January 19
February 18
April 11
April 29
March 5
May 10
May 12
Cuboidal
Cuboidal
Medium columnar
Tall columnar
Cuboidal
Tall columnar
Tall columnar
Cuboidal
Medium columnar
Medium columnar
Tall columnar
Tall columnar
Tall columnar
Medium columnar
Medium columnar
Tall columnar
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Cuboidal
Medium columnar
Medium columnar
Tall columnar
Cuboidal
Tall columnar
Medium columnar
Cuboidal
Medium columnar
Tall columnar
Tall columnar
Tall columnar
Tall columnar
Medium columnar
Medium columnar
Tall columnar
Low columnar
Low columnar
Low columnar
Cuboidal
Low columnar
Cuboidal
Low columnar
Cuboidal
Cuboidal
Cuboidal
Gland
cytoplasmic
vacuolation
Low
Moderate,
Moderate,
High
Low
High
Moderate,
Low
Moderate,
Moderate,
High
High
High
Moderate
Moderate,
High
Low
Low
Low
Low
Low
Low
Low, SNV
Low
Low
Low
SNV
SNV
SNV
SNV
SNV
SNV
Gland
secretion
Gland
tortuosity
High
High
Low
Low
Absent
Low
Low
Low
Moderate
Moderate
Low
Moderate
Moderate
Moderate
High
Low
High
High
High
High
High
Moderate
Moderate
Moderate
High
High
Low
Moderate
Moderate
High
Low
High
Moderate
Low
Moderate
High
High
High
High
Moderate
High
High
Moderate
Low
Moderate
Low
Moderate
Low
Moderate
Low
Low
Low
a
SNV, subnuclear glycogen vacuoles.
congested blood vessels, and dilated lymphatics. Endometrial glands and stromal cells were widely separated
by pale staining, homogenous connective tissue, and the
mucosa was infiltrated by small numbers of lymphocytes, plasma cells, and macrophages. Glands were lined
by cuboidal epithelium and contained eosinophilic proteinaceous material. Mitotic figures were occasionally
noted in the superficial epithelium. Later during involu-
tion, the stromal connective tissue became thicker and
more compact and there were increased numbers of lymphocytes, plasma cells, and hemosiderin laden- macrophages in the superficial mucosa. During diapause and
in early fall, the endometrium at the previous placentation site contained large numbers of hemosiderin-laden
macrophages, compact stroma, and numerous thickwalled hyalinized arterioles (Fig. 5).
1808
COLEGROVE ET AL.
Fig. 5. Histologic features of the endometrium at a site of previous
placental attachment in a California sea lion that died in early fall.
Note the hemosiderin-laden macrophages (HM) and the hyalinized
arterioles (HA). Scale bar ¼ 500 lm. H&E.
Fig. 4. A–C: Histologic features of the endometrium in California
sea lions (Zalophus californianus) collected (A) in July during estrus,
(B) during diapause, and (C) in the spring during pregnancy. Scale
bars ¼ 200 lm. H&E.
Cervix and vagina. The morphologic features of the
cervix and vagina varied by location and there was a
lack of keratinization in all regions. In the endocervical
canal, there were many long, complex epithelial infold-
ings and cervical glands were often filled with large
amounts of mucous (Fig. 6A). Epithelium ranged from
pseudostratified columnar to stratified squamous. The
endocervical stroma and smooth muscle were densely
cellular and compact, resembling uterine horn stroma
and myometrium. The ectocervix consisted of two portions. The prominent distal external cervical fold was
lined at the tip by segmental, thin cuboidal to non-keratinized stratified squamous epithelium with few invaginations. The fornix, at the cervix–vagina junction, and
ectocervix folds were lined predominately by pseudostratified columnar epithelium (Fig. 6B). There were many
shallow invaginations in this region, some of which
formed distinct glands. The epithelium of the cranial
vagina was similar to the fornix, however, with fewer
and less complex invaginations. Vaginal epithelium was
predominantly pseudostratified columnar up to the middle vagina at the hymeneal fold where the mucosa
transitions into non-keratinized stratified squamous epithelium (Fig. 6C). Subtle changes were observed in
epithelial morphology during different times of the
reproductive cycle. During pupping and estrus, the endocervical epithelium ranged from pseudostratified columnar to thicker, non-keratinized stratified squamous.
Pseudostratified columnar epithelial cells had clear
foamy cytoplasm and were interspersed with moderate
numbers of goblet cells. In some animals, stratified squamous epithelium partially filled cervical glands and
invaginations and in a few females the surface epithelium was entirely stratified squamous and cervical
glands were completely filled with squamous epithelium
(Fig. 7). Invaginations in the ectocervical and proximal
vaginal epithelium were more numerous, prominent,
and slightly deeper during June and July. Similar to the
endocervix, some animals had regions of ectocervical and
vaginal epithelium in which squamous epithelium was
present within invaginations and glands, in addition to
pseudostratified columnar epithelium. In a few animals,
the columnar epithelium of the ectocervix and proximal
vagina was nearly entirely replaced by well-differentiated stratified sqaumous epithelium. During diapause,
FEMALE SEA LION REPRODUCTIVE TRACT
1809
Fig. 7. Ectocervical epithelium with stratified squamous epithelium
in the deep aspects of the cervical glands. Scale bar ¼ 100 lm.
the fall, winter, and spring was predominantly pseudostratified columnar, with a prominent basal layer, and
foamy to pale eosinophilic cytoplasm. Goblet cells were
more numerous in the fall and spring; however, in general, goblet cell numbers were highly variably during all
times of the reproductive cycle. Several animals dying in
the spring had segmental stratified squamous epithelium in the cervical glands and surface epithelium similar to that which was noted in some animals dying
during pupping and estrus. Cervical and vaginal epithelium was generally thicker during pupping and estrus
and during the spring, ranging from three to eight celllayers thick. Thickness was greatest in epithelium with
squamous differentiation, with some invaginations containing up to 10–12 cell layers. In diapause and during
the fall epithelium was notably thinner, averaged three
to five cell-layers thick.
Serum Hormone Analysis
Serum estradiol was higher in the three animals collected during the pupping and estrus period compared to
animals sampled during diapause and early fall (Table
4). Pregnant animals sampled during winter and spring,
had estradiol concentrations that were as high, or
higher, than those noted during pupping and estrus.
Progesterone concentrations were variable but during
the winter and spring were higher in pregnant animals
than in a single nonpregnant female sampled.
Immunohistochemistry
Fig. 6. A–C: Histologic features of the (A) internal cervix (Scale bar
¼ 500 lm), (B) cervix and proximal vagina at the fornix (Scale bar ¼
1.0 mm), and (C) middle vagina (Scale bar ¼ 200 lm). H&E.
the endocervical epithelium was typically thinner and
pseudostratified columnar with foamy cytoplasm. Similar to diapause, cervical, and vaginal epithelium during
Positive dark red nuclear staining for both ERa and
PR was observed in surface and glandular epithelial cells
of the uterus, uterine stromal cells, uterine smooth muscle cells, epithelial cells throughout the cervix and vagina,
stromal cells in the cervix and vaginal submucosa, distal
urethral epithelial cells, and epithelium of the ovary. In
all tissues, staining was multifocal, with positively
stained cells interspersed with negative cells. In the ovaries, scattered surface and subsurface epithelial cells
exhibited moderately intense positive ERa staining (Fig.
8). PR expression was typically absent or weak.
1810
COLEGROVE ET AL.
TABLE 4. Serum estradiol (pg/ml) and progesterone (ng/ml) concentrations in 11
stranded female California sea lions (Zalophus californianus)
Sea
lion#
4
5
6
10
14
15
16
17
19
24
25
Condition
Post-parturient
Pregnant
Post-parturient
Pregnant
Date collected
Serum estradiol
(pg/ml)
Serum progesterone
(ng/ml)
July 8
July 9
July 22
September 4
October 19
October 23
October 31
November 26
January 12
March 5
May 10
10.2
5.6
10.7
3.4
4.9
3.5
4.5
14.4
27.5
10.4
4.7
3.2
0.2
12.3
2.3
2.7
5.8
9.1
2.8
5.8
10.4
0.7
In the uterus, the total ERa score was highest in the
uterine glands and lowest in the surface epithelium
throughout all stages of the reproductive cycle. Positive
surface epithelial cells were typically clustered and the
epithelial cells lining the base of the surface crypts often
exhibited more intense staining. In the endometrium,
ERa staining was most intense in the basal glands. In
the stroma, receptor staining was more intense in stromal cells surrounding blood vessels. Cyclic changes were
seen in the total uterine ERa score during the four different general time periods of the reproductive cycle;
however, these differences were not statistically significant (Fig. 9A). The cyclic changes in stromal ERa staining between time periods were most dramatic. Total ERa
scores in all layers of the uterus were highest during the
spring and the pupping and estrous periods (Fig. 10A).
Scores were lowest during embryonic diapause. In pregnant animals, ERa staining was similar in the pregnant
and non-pregnant horns and there was no appreciable
difference in staining between pregnant and non-pregnant animals. Except for the uterine gland epithelium
score, the total PR scores were higher than total ERa
scores in all layers of the uterus, cervix, and vagina.
Progesterone receptor staining was most intense in the
uteri in which columnar epithelium predominated (Fig.
10B). There was little variation in total uterine PR
scores throughout the reproductive cycle (Fig. 11A).
In the cervical and vaginal epithelium, intensity of
ERa staining was higher in the basal and parabasal
layers. Intensity of PR varied greatly among layers of
the epithelium. Both ERa and PR positive nuclei were
randomly scattered throughout the cervical and vaginal
stroma. In all time periods except for the fall, ERa scores
were higher in the cervical and vaginal stroma, than in
the overlying epithelium; however, this difference was
not statistically significant (Fig. 9B). The mid-vaginal
epithelium had the lowest total ERa scores during all
time periods. The lowest ERa scores occurred during embryonic diapause. Except for the middle vagina stroma
(P < 0.05) the difference between total ERa scores were
not significantly different during different time periods
of the reproductive cycle. In regions of stratified squamous cervical and vaginal epithelium, ERa expression
was high in the underlying stroma but usually low to
moderate in the epithelium (Fig. 12A). Progesterone receptor expression was moderate to high in both the epithelium and underlying stroma (Fig. 12B). Differences
in total PR scores were not statistically significant in dif-
Fig. 8. Immunohistochemical localization of ER a in the nuclei of
ovarian subsurface epithelial structures. Solid arrow ¼ positive staining White arrow ¼ negative staining. Scale bar ¼ 100 lm.
ferent layers of the cervix or vagina or during different
times of the reproductive cycle (Fig. 11B). Similar to the
ERa staining patterns, the intensity of PR staining was
often higher in the stroma than in the overlying epithelium. Estrogen receptor score was statistically positively
correlated with serum estradiol concentrations for the
uterine stroma and myometrium, endocervix epithelium,
cervical stroma, and the cranial vagina epithelium and
stroma (P < 0.05). There was no statistically significant
correlation between ERa score and serum progesterone
concentrations. Progesterone receptor scores were not
significantly correlated with either serum estradiol or
progesterone concentrations.
DISCUSSION
The primary goals of this study were to better define
the normal changes in reproductive tract morphology and
sex hormone receptor distribution that occur throughout
the yearly reproductive cycle in female California sea
lions. Although the number of animals examined was relatively low, general patterns in ovarian, uterine, and cervico-vaginal morphology were noted in the animals
examined. In most mammals, the reproductive cycle is
FEMALE SEA LION REPRODUCTIVE TRACT
1811
Fig. 9. A, B: Average total immunohistochemistry scores for ER a in the (A) uterus and (B) cervix and
vagina. Error bars indicate standard error.
divided into a follicular and luteal phase. The follicular
phase is short and corresponds to the period from regression of the CL to ovulation, when the primary reproductive hormone is estradiol. The luteal phase corresponds
to the presence of ovarian CLs and the primary reproduc-
tive hormone is progesterone (Senger, 2005). Sea lions,
however, have a complex reproductive cycle that includes
a period of embryonic diapause and an extended period in
which the ovaries can contain both developing follicles
and a CL.
1812
COLEGROVE ET AL.
Fig. 10. A, B: Immunohistochemical localization of hormone receptors in glandular epithelial (G) and
stoma cell (S) nuclei in the endometrium. (A) Intense nuclear staining of ER a during estrus. (B) High PR
expression in the progestational endometrium. Scale bars ¼ 100 lm.
Cyclic Histomorphologic Changes Throughout
the Reproductive Cycle
During the period of pupping and estrus in June and
July, ovaries commonly contained developing follicles
and the uterine epithelium exhibited characteristics of
estrogenic influence. In other species, estrogen concentration peaks just prior to ovulation (Senger, 2005) and
in this study two of three animals evaluated had relatively high serum estradiol concentrations in July. Similar peaks in total serum estrogen also were reported in
several captive sea lions sampled in July (Greig et al.,
2007), further suggesting that an ovulatory peak in
estrogen occurs during July. During mid to late July, in
several females, a CL in the ovary and morphologic
changes in the uterus were consistent with increasing
progesterone concentrations. These animals likely had
already ovulated prior to death. Similarly, marked progestational changes have been noted in uterine epithelium of fur seals shortly after ovulation (Craig, 1964).
Subnuclear vacuoles are considered indicative of
increased secretory activity of endometrial glands and,
similar to sea lions, have been noted in fur seals and
harbor seals starting in estrus and early diapause (Bigg
and Fisher, 1974; Boshier, 1981; Roth et al., 1995).
Histologic evaluations in fur seals during diapause
suggested luteal regression and a corresponding uterine
estrogenic appearance (Craig, 1964). There was no morphologic evidence of luteal regression in any of the sea
lions during diapause and progestational uterine
changes dominated. Similar to the sea lions examined
during this study, small follicles were noted throughout
diapause in harbor seals, with follicle numbers decreasing by implantation (Bigg and Fisher, 1974). Uterine secretory activity was moderate throughout diapause and
in early fall and proteins or molecules elaborated by the
uterus are thought to play an important role in control
of diapause and blastocyst implantation. Uterine contents have been shown to vary significantly in the periimplantation period in a number of species exhibiting
delayed implantation and uterine secretion is thought to
activate the blastocyst in fur seals (Daniel, 1971, 1972).
Recent molecular studies utilizing differential gene
expression and mouse knockout models have shown that
sets of genes involved in cell cycle, cell signaling, and
energy metabolism regulation are likely involved in blastocyst activation and implantation (Dey et al., 2004;
Hamatani et al., 2004). Further analysis of uterine protein content and gene expression studies will likely be
needed to completely understand the physiology and regulation of embryonic diapause in pinnipeds.
Several studies in northern fur seals found an
increase in both estradiol and progesterone prior to implantation (Daniel, 1974). A more recent study found no
significant increase in hormones during the peri-implantation period (Browne et al., 2006). Sea lions sampled
during September and October, in this study, had relatively low serum estradiol concentrations; however,
Greig et al. (2007) found increases in total serum estrogen in several California sea lions sampled in October
and November. Frequent sampling of normal sea lions
during diapause and implantation is needed to definitively determine if implantation associated hormonal
surges occurs in sea lions. As free and conjugated estrone are thought to be the primary circulating estrogen
in fur seals (Browne et al., 2006), determination of estrone as well as estradiol concentrations may be helpful
in elucidating changes in circulating sex hormones.
Pregnancy was not observed in females prior to late November; however, it was impossible to differentiate
between animals in which early pregnancy was not detectable and non-pregnant animals. Accordingly, the histologic appearance of the ovaries and uterine epithelium
was variable in the fall. The presence of a CL and the
predominance of progestational changes in uterine epithelium, however, suggest either pregnancy or the existence of a pseudopregnant state, as has been suggested
to occur in other pinnipeds species (Reijnders, 1990;
Boyd, 1991; Greig et al., 2007).
FEMALE SEA LION REPRODUCTIVE TRACT
1813
Fig. 11. A, B: Average total immunohistochemistry scores for PR in the (A) uterus and (B) cervix and
vagina. Error bars indicate standard error.
Ovarian Morphology During Pregnancy
The finding of concurrent follicular activity and CLs in
the ovary of pregnant sea lions is similar to studies in
northern fur seals and harbor seals in which folliculogenesis was observed in the ovary contralateral to the
ovary containing the CL during pregnancy (Craig, 1964;
Bigg and Fisher, 1974). Unlike fur and harbor seals,
however, some sea lions had follicular activity in the
ovary containing the CL during pregnancy. Waves of follicular activity during pregnancy have also been noted
in other pinnipeds species (Harrison, 1969). Follicular
development during pregnancy varies among species. In
species such as rats and primates, development of large
antral follicles is suppressed during the luteal stage by
1814
COLEGROVE ET AL.
Fig. 12. A, B: Region of squamous metaplasia in the cervix. Weak immunohistochemical staining in
the epithelium and moderate staining in the stroma for (A) ERa and (B) PR. Solid arrows ¼ strong positive
staining; Grey arrows ¼ weak positive staining. Scale bars ¼ 100 lm.
the negative feedback of progesterone on GnRH secretion by the hypothalamus. Sea lions appear to be similar
to cattle and horses, in which, follicle development to
near ovulatory size can occur during both the follicular
and luteal stages of the cycle (Fortune, 1994). The role
of folliculogenesis in estrogen production during pregnancy in sea lions is unknown. Rising levels of estradiol
have been noted in other pinniped species during late
gestation (Reijnders, 1990; Boyd et al., 1999) and in
three pregnant and recently post-parturient sea lions in
this study, serum estradiol was as high or higher than
the concentrations observed during the estrous period.
In fur seals, steroidogenic enzymes have been localized
to granulosa and theca cells of preovulatory follicles
indicating the capability for estradiol synthesis with follicle development (Browne et al., 2006). Additionally,
steroidogenic enzymes have been identified in the CL of
fur seals, spotted seals (Phoca largha), ribbon seals
(Phoca fasciata), and Stellar sea lions (Eumetopias jubatus) and the placenta of spotted seals, ribbon seals, and
Stellar sea lions (Ishinazaka et al., 2001; Ishinazaka
et al., 2002; Browne et al., 2006) indicating that the CL
and placenta of pinnipeds may produce estrogen during
pregnancy. In northern fur seals, regression of the CL
begins during mid-gestation and progresses until parturition (Craig, 1964; Boyd et al., 1999). No morphologic
evidence of CL regression was found in pregnant sea
lions, in this study. The high estrogen receptor scores in
pregnant and post parturient sea lions and morphology
of the endometrium further suggests some estrogen
influence during the later half of pregnancy. Craig
(1964) described similar estrogenic features of the fur
seal uterus during late gestation. In contrast, ERa
expression is low to nondetectable in epithelial cells of
the uterus in pregnant dogs and cows (Vermeirsch et al.,
2000b; Kimmins and MacLaren, 2001).
Interstitial cells are a feature of the ovary in some
species and are prominent in the horse and cat
(McEntee, 1990). The function of these cells is poorly
understood, however, patterns of steroidogenic enzymes
expression in cats suggests the cells are differentiated
from theca interna and are associated with atretic follicles (Perez et al., 1999). The clustering of interstitial
cells around atretic follicles in sea lions supports this
theory. The increase in interstitial cell size and cytoplasmic vacuolation noted in this study during estrus and in
the winter and spring suggests secretory and potentially
hormone-responsive activity. Browne et al. (2006) found
high expression of CYP 17 and cytochrome b5 in fur seal
interstitial cells and hypothesized that, like some other
species that undergo delayed implantation (MondainMonval et al., 1983; Stoufflet et al., 1989), androgens
may play an important role in regulating reproductive
events in pinnipeds. Future studies should include evaluation of circulating androgen levels, androgen receptor
expression, and ovarian steroidogenic enzyme expression
to better determine the role of androgens and interstitial
cells in sea lion reproductive biology.
Estrogen and Progesterone Receptor
Expression
Similarities between the ERa expression in sea lions
and canines included ovarian surface and subsurface
epithelial structures expression, clustering of ERa positive uterine surface epithelial cells and a higher expression of ERa in the basal uterine glands (De Cock et al.,
1997). In the bitch, uterine ER a expression increases in
the late secretory period of the estrous cycle and is highest during proestrous. Receptor levels then decrease during the late proliferative phase of estrus with rising
progesterone concentrations (De Cock et al., 1997; Vermeirsch et al., 1999). The highest estrogen receptor
scores in sea lions were noted during the pupping and
estrus period, which likely encompassed the sea lion
proestrus stage. Rising progesterone concentrations
decrease estrogen receptor expression in humans and
other species with and without high concurrent levels of
estradiol (Lessey and Gorell, 1981; Boomsma et al.,
1982; Li et al., 1992). Although serum progesterone varied greatly in this study, the high levels of uterine estrogen receptor noted in animals collected during the
FEMALE SEA LION REPRODUCTIVE TRACT
winter, spring, and estrus suggest that progesterone is
lower during these time periods than during diapause
and in the fall. Accordingly, Greig et al. (2007) found low
levels of progesterone during April and May in both
pregnant and nonpregnant sea lions. Progesterone receptor expression remained high in the uterus throughout the reproductive cycle. In most species, PR mRNA
levels and protein synthesis are increased by estrogen
via ER-mediated gene transcription and decreased by
progesterone (Critchley and Healy). The relatively consistent expression of PR throughout the sea lion reproductive cycle is unusual, as cyclic changes in receptor
expression similar to that observed with ERa have been
noted in other species (Lessey and Gorell, 1981; Watson
et al., 1992; Vermeirsch et al., 2000a; Kimmins and
MacLaren, 2001). Low sample size may account for absence of statistically significant differences in hormone
receptor expression during differing times of the sea lion
reproductive cycle.
Urogenital carcinoma frequently develops in the cervix
and vagina of free-ranging female sea lions and dysplastic lesions are commonly found on histologic evaluation
of stranded adult animals (Gulland et al., 1996). In order
to better understand the factors involved in cancer development, characterization of the normal morphology, and
hormone receptor distribution of the cervix and vagina
was essential. The varying types of epithelium noted in
the cervix and vagina further suggests that complex factors are likely involved in tumor development. Sea lions
appear to be more similar to cows than to canines, in
that the cranial portion of the vagina is lined by pseudostratified columnar epithelium. Of particular interest
were the two squamous-columnar transition zones noted
in the cervix and the middle vagina. In humans, the cervical transformation zone is located where columnar
endocervical epithelium transitions to the squamous epithelium of the ectocervix and vagina. This zone shifts at
puberty in response to hormonal influences, trauma, and
infectious agents. Transformation zone epithelium is
more susceptible to human papillomavirus (HPV) infection and estrogen-induced squamous metaplasia that can
lead to cervical carcinoma (Elson et al., 2000). High levels of ER and PR are expressed in transformation zone
epithelium in humans (Remoue et al., 2003). Both ERa
and PR are expressed throughout the sea lion cervix and
vagina surrounding these epithelial transition zones, and
similar to dogs, there was little variation in receptor
expression in the cervix and vagina throughout the cycle
(Vermeirsch et al., 2002). Differences between receptor
distribution at these transformation zones and other
regions of the cervix and vagina were not noted; however,
more detailed evaluation of this region in a larger sample
population is needed. Infection with a gammaherpesvirus, Otarine Herpesvirus-1, has been associated with sea
lion urogenital cancer (Lipscomb et al., 2000; Buckles
et al., 2006). Given the susceptibility of the human transformation zone to viral infection, determining the specific
regions of the sea lion reproductive tract susceptible to
viral infection may be important in determining factors
involved in carcinogenesis (Elson et al., 2000).
The lower ERa expression in regions of cervical and
squamous differentiation was an unexpected finding, as
in most species, estradiol mediates epithelial proliferation and squamous differentiation. Progesterone receptor
expression remained high in these areas, however, and
1815
both ERa and PR were often highly expressed in the
underlying stroma. Recent studies utilizing receptor
knockout mouse models have demonstrated that hormone-induced epithelial proliferation can be mediated by
stromal receptors in a paracrine manner (Cunha et al.,
2004). Similar to previous findings in harbor seals, squamous differentiation was most often noted during late
estrus, further suggesting hormone involvement (Bigg
and Fisher, 1974). Because of lack of epithelial dysplasia,
these areas of squamous differentiation were not
regarded as preneoplastic lesions. These squamous
changes could potentially lead to cancer development;
however, as cervical carcinogenesis in humans can begin
as squamous metaplasia of uterine gland epithelium
(Elson et al., 2000). The high expression of hormone
receptors throughout the reproductive tract stroma and
the cyclic variation throughout the cycle, suggests that
stromal cells play a key role in mediating epithelial hormone responses in sea lions.
All of the sea lions in this study were free-ranging,
stranded animals that died or were euthanized during
rehabilitation due to severe illness. Several animals (N
¼ 6) were moderately to severely underweight at the
time of death. The significance of illness and malnutrition to the data presented in this study is unknown.
Significantly lower serum progesterone and estradiol
concentrations have been noted in starved rats when
compared with nonstarved rats (Baranowska et al.,
2001). Lower ERa and PR expression was noted in malnourished ewes when compared with ewes fed maintenance feed requirements (Sosa et al., 2006). The
underweight sea lions evaluated had ER a and PR scores
similar to animals with normal body condition scores
that died during the same time period, indicating that if
malnutrition affected hormone receptor expression the
effect was likely not detected.
Although sample numbers were small and immunohistochemical evaluation was partially subjective, the data
presented in this study adds to the general understanding of the reproductive cycle in female California sea
lions. Significant changes in uterine morphology were
noted to occur throughout the reproductive cycle. Histologic evaluation of the entire reproductive tract should
be included in routine postmortem evaluation of
deceased individuals to further the understanding of
normal sea lion reproductive biology and natural history.
Additional characterization of the potential relationship
between hormone receptors and cervico-vaginal squamous differentiation is needed to determine whether
this change can lead to tumor formation.
ACKNOWLEDGMENTS
The authors thank Dr. Dennis Wilson and Dr. Chuck
Mohr for advice and critical review of the manuscript.
The authors are especially thankful to Dr. Alan Conely
for his helpful comments on the manuscript and to the
histology laboratory at the VMTH. Alejandro Vico, Irwin
Liu, and Coralie Munro at the Clinical Endocrinology
Laboratory, School of Veterinary Medicine, University of
California, Davis, CA are acknowledged for assistance
with serum progesterone and estradiol measurements.
We thank Denise Greig, Tracey Goldstein, and Elizabeth
Wheeler for help in sample organization and to the staff
and volunteers of The Marine Mammal Center.
1816
COLEGROVE ET AL.
LITERATURE CITED
Baranowska B, Chmielowska M, Wolinska-Witort E, Roguski K,
Wasilewska-Dziubinska E. 2001. The relationship between neuropeptides and hormones in starvation. Neuro Endocrinol Lett
22:349–355.
Bigg M, Fisher HD. 1974. The reproductive cycle of the female harbor seal off southeastern Vancouver Island. In: Harrison RJ, editor. Functional anatomy of marine mammals. London: Academic
Press. p 329–347.
Boomsma RA, Jaffe RC, Verhage HG. 1982. The uterine progestational response in cats: changes in morphology and progesterone
receptors during chronic administration of progesterone to estradiol-primed and nonprimed animals. Biol Reprod 26:511–521.
Boshier DP. 1981. Structural changes in the corpus luteum and
endometrium of seals before implantation. J Reprod Fertil Suppl
29:143–149.
Boyd IL. 1991. Changes in plasma progesterone and prolactin concentrations during the annual cycle and the role of prolactin in
the maintenance of lactation and luteal development in the
Antarctic fur seal (Arctocephalus gazella). J Reprod Fertil 91:
637–647.
Boyd IL, Lockyer C, Marsh HD. 1999. Reproduction in marine
mammals. In: Reynolds JE, Rommel SA, editors. Biology of
marine mammals. Washington, DC: Smithsonian Institution
Press. p 218–286.
Browne P, Conley AJ, Spraker T, Ream RR, Lasley BL. 2006. Sex
steroid concentrations and localization of steroidogenic enzyme
expression in free-ranging female northern fur seals (Callorhinus
ursinus). Gen Comp Endocrinol 147:175–183.
Buckles EL, Lowenstine LJ, Funke C, Vittore RK, Wong HN, St
Leger JA, Greig DJ, Duerr RS, Gulland FM, Stott JL. 2006. Otarine Herpesvirus-1, not papillomavirus, is associated with endemic
tumours in California sea lions (Zalophus californianus). J Comp
Pathol 135:183–189.
Craig AM. 1964. Histology of the reproduction and the estrus cycle
in the female fur seal, Callorhinus ursinus. Bull Fish Res Board
Can 21:773–811.
Critchley H, Healy DL. 1998. Effects of estrogen and progesterone
on the endometrium. In: Fraser IS, Jansen, RP, Lobo RA, Whitehead MI, editors. Estrogens and progestogens in clinical practice.
New York: Churchhill Livingstone. p 145–171.
Cunha GR, Cooke PS, Kurita T. 2004. Role of stromal-epithelial
interactions in hormonal responses. Arch Histol Cytol 67:
417–434.
D’Haeseleer M, Cornillie P, Simoens P, van den Broeck W. 2006.
Localization of oestrogen receptors within various bovine ovarian
cell types at different stages of the oestrous cycle. Anat Histol
Embryol 35:334–342.
D’Haeseleer M, Simoens P, Van den Broeck W. 2007. Cell-specific
localization of progesterone receptors in the bovine ovary at different stages of the oestrous cycle. Anim Reprod Sci 98:271–281.
Daniel JC, Jr. 1971. Growth of the preimplantation embryo of the
northern fur seal and its correlation with changes in uterine protein. Dev Biol 26:316–328.
Daniel JC, Jr. 1972. Blastokinin in the northern fur seal. Fertil
Steril 23:78–80.
Daniel JC, Jr. 1974. Circulating levels of oestradiol-17beta during
early pregnancy in the Alaskan fur seal showing an oestrogen
surge preceding implantation. J Reprod Fertil 37:425–428.
De Cock H, Ducatelle R, Logghe JP. 1997. Immunohistochemical
localization of estrogen receptor in the normal canine female genital tract. Domest Anim Endocrinol 14:133–147.
Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H.
2004. Molecular cues to implantation. Endocr Rev 25:341–373.
Elson DA, Riley RR, Lacey A, Thordarson G, Talamantes FJ, Arbeit
JM. 2000. Sensitivity of the cervical transformation zone to estrogen-induced squamous carcinogenesis. Cancer Res 60:1267–1275.
Fortune JE. 1994. Ovarian follicular growth and development in
mammals. Bio Reprod 50:225–232.
Greig DJ, Mashburn KL, Rutishauser M, Gulland FMD, Williams
TM, Atkinson S. 2007. Seasonal changes in circulating progester-
one and estrogen concentrations in the California sea lion (Zalophus californianus). J Mammal 88:67–72.
Gulland FM. 1999. Stranded seals: important sentinels. J Am Vet
Med Assoc 214:1191–1192.
Gulland FM, Trupkiewicz JG, Spraker TR, Lowenstine LJ. 1996.
Metastatic carcinoma of probable transitional cell origin in 66
free-living California sea lions (Zalophus californianus), 1979 to
1994. J Wildl Dis 32:250–258.
Hamatani T, Daikoku T, Wang H, Matsumoto H, Carter MG, Ko
MS, Dey SK. 2004. Global gene expression analysis identifies
molecular pathways distinguishing blastocyst dormancy and activation. Proc Natl Acad Sci USA 101:10326–10331.
Harrison RJ. 1969. Reproduction and reproductive hormones. In:
Andersen HT, editor. The biology of marine mammals. New York:
Academic Press. p 253–348.
Ishinazaka T, Suzuki M, Yamamoto Y, Isono T, Harada N, Mason
JI, Watabe M, Tsunokawa M, Ohtaishi N. 2001. Immunohistochemical localization of steroidogenic enzymes in the corpus
luteum and the placenta of the ribbon seal (Phoca fasciata) and
steller sea lion (Eumetopias jubatus). J Vet Med Sci 63:955–959.
Ishinazaka T, Suzuki M, Mizuno AW, Harda N, Mason JI, Ohtaishi
N. 2002. Immunohistochemical localization of steriodogenic
enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca larghna) during late pregnancy.
J Vet Med Sci 64:329–333.
Kimmins S, MacLaren LA. 2001. Oestrous cycle and pregnancy
effects on the distribution of oestrogen and progesterone receptors
in bovine endometrium. Placenta 22:742–748.
Lessey BA, Gorell TA. 1981. Hormonal regulation of cytoplasmic
estrogen and progesterone receptors in the beagle uterus and oviduct. Mol Cell Endocrinol 21:171–180.
Li W, Boomsma RA, Verhage HG. 1992. Immunohistochemical analysis of estrogen and progestin receptors in uteri of steroid-treated
and pregnant cats. Bio Reprod 47:1073–1081.
Lipscomb TP, Scott DP, Garber RL, Krafft AE, Tsai MM, Lichy JH,
Taubenberger JK, Schulman FY, Gulland FM. 2000. Common
metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus. Vet Pathol 37:609–617.
Martin de las Mulas J, Van Niel M, Millan Y, Ordas J, Blankenstein MA, Van Mil F, Misdorp W. 2002. Progesterone receptors in
normal, dysplastic and tumourous feline mammary glands. Comparison with oestrogen receptors status. Res Vet Sci 72:153–161.
McEntee K. 1990. Reproductive pathology of domestic animals. San
Diego, CA: Academic Press.
Mondain-Monval M, Bonnin M, Scholler R, Canivenc R. 1983. Plasma
androgen patterns during delayed implantation in the European
badger (Meles meles L.). Gen Comp Endocrinol 50:67–74.
Munro C, Stabenfeldt G. 1984. Development of a microtitre plate
enzyme immunoassay for the determination of progesterone.
J Endocrinol 101:41–49.
Odell DK. 1975. Breeding biology of the California sea lion, Zalophus californianus. Rapports et Proces-Verbaux des Reunions
Conseil International pour l’Exploration de la Mer 169:374–378.
Oosthuizen WH, Greyling FJ, Bester MN. 1998. Estimation of age
from stained sections of canine teeth in the Cape fur seal (Arctocephalus pusillus pusillus). SADJ 53:47–51.
Perez JF, Conley AJ, Dieter JA, Sanz-Ortega J, Lasley BL. 1999.
Studies on the origin of ovarian interstitial tissue and the incidence of endometrial hyperplasia in domestic and feral cats. Gen
Comp Endocrinol 116:10–20.
Press MF, Nousek-Goebl NA, Bur M, Greene GL. 1986. Estrogen receptor localization in the female genital tract. Am J Pathol
123:280–292.
Reeves RR, Steward BS, Leatherwood S. 1992. The Sierra club
handbook of seals and sirenians. San Francisco, CA: Sierra Club
Books.
Reijnders PJ. 1990. Progesterone and oestradiol-17 beta concentration profiles throughout the reproductive cycles in harbour seals
(Phoca vitulina). J Reprod Fertil 90:403–409.
Remoue F, Jacobs N, Miot V, Boniver J, Delvenne P. 2003. High
intraepithelial expression of estrogen and progesterone receptors
FEMALE SEA LION REPRODUCTIVE TRACT
in the transformation zone of the uterine cervix. Am J Obstet
Gynecol 189:1660–1665.
Roth TL, Munson L, Swanson WF, Wildt DE. 1995. Histological
characteristics of the uterine endometrium and corpus luteum
during early embryogenesis and the relationship to embryonic
mortality in the domestic cat. Biol Reprod 53:1012–1021.
Senger PL. 2005. Pathways to pregnancy and partuition. 2nd ed.
Ames, Iowa: Current Concepts.
Shille VM, Lundstrom KE, Stabenfeldt GH. 1979. Follicular function in the domestic cat as determined by estradiol-17 beta concentrations in plasma: relation to estrous behavior and
cornification of exfoliated vaginal epithelium. Biol Reprod 21:
953–963.
Sosa C, Abecia JA, Forcada F, Vinoles C, Tasende C, Valares JA,
Palacin I, Martin GB, Meikle A. 2006. Effect of undernutrition on
uterine progesterone and oestrogen receptors and on endocrine
profiles during the ovine oestrous cycle. Reprod Fertil Dev
18:447–458.
Stoufflet I, Mondain-Monval M, Simon P, Martinet L. 1989. Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic
diapause and implantation in the mink (Mustela vison). J Reprod
Fertil 87:209–221.
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Sukjumlong S, Kaeoket K, Dalin AM, Persson E. 2003. Immunohistochemical studies on oestrogen receptor alpha (ER alpha) and
the proliferative marker Ki-67 in the sow uterus at different
stages of the oestrous cycle. Reprod Domest Anim 38:5–12.
Vermeirsch H, Simoens P, Hellemans A, Coryn M, Lauwers H.
2000a. Immunohistochemical detection of progesterone receptors
in the canine uterus and their relation to sex steroid hormone levels. Theriogenology 53:773–788.
Vermeirsch H, Simoens P, Lauwers H. 2000b. Immunohistochemical
detection of the estrogen receptor-alpha and progesterone receptor
in the canine pregnant uterus and placental labyrinth. Anat Rec
260:42–50.
Vermeirsch H, Simoens P, Lauwers H, Coryn M. 1999. Immunohistochemical detection of estrogen receptors in the canine uterus
and their relation to sex steroid hormone levels. Theriogenology
51:729–743.
Vermeirsch H, Van den Broeck W, Simoens P. 2002. Immunolocalization of sex steroid hormone receptors in canine vaginal and
vulvar tissue and their relation to sex steroid hormone concentrations. Reprod Fertil Dev 14:251–258.
Watson ED, Skolnik SB, Zanecosky HG. 1992. Progesterone and
estrogen receptor distribution in the endometrium of the mare.
Theriogenology 38:575–580.
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