BIOLOGY OF REPRODUCTION 64, 1608–1613 (2001)
Endometrial Glands Are Required for Preimplantation Conceptus Elongation
and Survival1
C. Allison Gray,3 Kristin M. Taylor,3 W. Shawn Ramsey,3 Jonathan R. Hill,4 Fuller W. Bazer,3
Frank F. Bartol,5 and Thomas E. Spencer2,3
Center for Animal Biotechology and Genomics,3 Department of Animal Science, Texas A&M University,
College Station, Texas 77843-2471
Department of Veterinary Anatomy and Public Health,4 Texas A&M University,
College Station, Texas 77843
Department of Animal and Dairy Sciences,5 Program in Cell and Molecular Biosciences, Auburn University,
Auburn, Alabama 36849-5415
ABSTRACT
Endometrial glands secrete molecules hypothesized to support conceptus growth and development. In sheep, endometrial
gland morphogenesis occurs postnatally and can be epigenetically ablated by neonatal progestin exposure. The resulting stable adult uterine gland knockout (UGKO) phenotype was used
here to test the hypothesis that endometrial glands are required
for successful pregnancy. Mature UGKO ewes were bred repeatedly to fertile rams, but no pregnancies were detected by
ultrasound on Day 25. Day 7 blastocysts from normal superovulated ewes were then transferred synchronously into Day 7 control or UGKO ewes. Ultrasonography on Days 25–65 postmating
indicated that pregnancy was established in control, but not in
UGKO ewes. To examine early uterine-embryo interactions, four
control and eight UGKO ewes were bred to fertile rams. On
Day 14, their uteri were flushed. The uterus of each control ewe
contained two filamentous conceptuses of normal length. Uteri
from four UGKO ewes contained no conceptus. Uteri of three
UGKO ewes contained a single severely growth-retarded tubular
conceptus, whereas the remaining ewe contained a single filamentous conceptus. Histological analyses of these uteri revealed
that endometrial gland density was directly related to conceptus
survival and developmental state. Day 14 UGKO uteri that were
devoid of endometrial glands did not support normal conceptus
development and contained either no conceptuses or growthretarded tubular conceptuses. The Day 14 UGKO uterus with
moderate gland development contained a filamentous conceptus. Collectively, these results demonstrate that endometrial
glands and, by inference, their secretions are required for periimplantation conceptus survival and development.
conceptus, developmental biology, early development, embryo,
female reproductive tract, gamete biology, implantation, pregnancy
INTRODUCTION
Endometrial glands are present in all mammalian uteri
and develop after birth in sheep [1–5], pigs [6, 7], rats [8],
This work was supported by NRI Competitive Grants Program/USDA
grant 98-35203-6322 to T.E.S. and, in part, by NIH grant P30 ES09106.
2
Correspondence: Thomas E. Spencer, Center for Animal Biotechnology
and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University,
College Station, TX 77843-2471. FAX: 979 862 2662;
e-mail: [email protected]
1
Received: 14 September 2000.
First decision: 17 October 2000.
Accepted: 10 January 2001.
Q 2001 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
and mice [9]. Uterine glands secrete a variety of molecules,
collectively termed histotroph, that are hypothesized to be
essential for maternal support of conceptus (embryo/fetus
and associated extraembryonic placental membranes) survival and growth in mammals [10–15]. Available evidence
supports the idea that secretions of endometrial epithelia
influence conceptus development, onset of pregnancy recognition signals, and conceptus growth in species that display synepitheliochorial placentation [16, 17]. Components
of uterine histotroph include enzymes, cytokines, growth
factors, ions, hormones, glucose, transport proteins, and adhesion molecules [14, 18]. Specific functional roles for histotroph components in ovine conceptus development are
not known. However, two endometrial gland secretory
products, leukemia inhibitory factor (LIF) and calcitonin,
are required for implantation in rodents [19, 20].
Establishment of pregnancy requires that the preimplantation ovine conceptus enter a progesterone-stimulated uterus and develop sufficiently to synthesize and release interferon tau (IFNt), the pregnancy recognition signal [21]. After pregnancy recognition, maintenance of pregnancy requires reciprocal communication between the conceptus
and endometrium during implantation and synepitheliochorial placentation [22]. Throughout gestation, both conceptus and maternal endometrial secretions affect uterine
fluid composition. Prior to implantation, these secretions
are hypothesized to influence conceptus survival, development and production of IFNt [16, 23]. Specific components, such as mitogenic uterine-derived growth factors, can
directly induce proliferation of ovine trophoblast and myoblast cells in vitro [24]. Species that display delayed implantation serve to illustrate the importance of uterine histotroph in conceptus development [25–27]. Delayed implantation extends gestation by arresting conceptus development at the blastocyst stage. In marsupials, the end of
this developmental arrest is marked by increased endometrial secretory activity that directly promotes conceptus development [26, 28, 29]. In ruminants, similar direct embryotrophic effects of endometrial secretions during early pregnancy are not well defined.
Ovine uterine glandular development can be inhibited
epigenetically by neonatal progestin exposure for 8 wk
from birth, resulting in adult ewes that lack endometrial
glands and display the uterine gland knockout (UGKO)
phenotype [17, 30, 31]. Mature UGKO ewes cannot support
pregnancy [17] and do not express a number of glandular
epithelial genes that may be required for conceptus development and growth [31]. We hypothesize endometrial
glands and their secretions are essential for normal concep-
1608
ENDOMETRIAL GLANDS ARE ESSENTIAL FOR CONCEPTUS DEVELOPMENT
tus development. Therefore, these studies were designed to
identify the stage at which early pregnancy loss occurs in
UGKO ewes and to begin elucidation of uterine factors and
conditions responsible for pregnancy failure.
MATERIALS AND METHODS
Animals
Experimental and surgical procedures complied with the
Guide for Care and Use of Agriculture Animals and were
approved by the Institutional Agricultural Animal Care and
Use Committee of Texas A&M University (Animal Use
Protocol 7-286).
The UGKO ewes were produced by implanting Rambouillet ewe lambs with a single Synchromate B (Sanofi,
Overland Park, KS) implant within 12 h of birth and every
2 wk thereafter for a total of 8 wk. Implants were inserted
s.c. in the periscapular area and released approximately 6
mg of norgestomet (17a-acetoxy-11b-methyl-19-norpreg4-ene-3,20-dione), a potent synthetic 19-norprogestin, over
a 14-day period [4]. Control ewes did not receive implants.
Natural Breeding
At approximately 17 mo of age, adult UGKO (n 5 18)
ewes were given two i.m. injections (0700 and 1700 h) of
10 mg prostaglandin F2a (Lutalyse; Upjohn, Kalamazoo,
MI). These injections were repeated 9 days later to synchronize estrus. All ewes were bred at estrus (Day 0) and
at 12 h and 24 h postestrus by intact rams of proven fertility. Ewes were monitored daily for estrous behavior using
vasectomized rams. Ewes not returning to estrus were subjected to transabdominal ultrasound on Days 25 and 35
postmating to confirm pregnancy by detection of the conceptus or placentome formation. This experiment was repeated three times.
Embryo Transfer
Donor ewes (n 5 5) were synchronized to estrus by administering controlled internal drug releasing (CIDR) progesterone pessaries (InterAg, New Zealand). The CIDRs
were inserted on Day 0 and replaced on Day 7. Donor ewes
were superovulated using FSH by administering 2.5 ml
Folltropin-V (Vetrapham Canada Inc., London, ON, Canada) i.m. on Day 8 (0700 h), followed by 1.25 ml FolltropinV twice daily (0700 h and 1700 h) for 3 days (Day 8, 1700
h; Day 10, 1700 h). On Day 8 (0700 h), donor ewes received 250 IU (i.m.) Pregnecol (Horizon Technology Pty
Limited, North Ryde, Australia) and 10 mg Lutalyse i.m.
(1700 h). On Days 11–12 (1700–0700 h), donor ewes were
mated by fertile rams. Recipient ewes were also synchronized using CIDRs inserted on Day 0 and removed on Day
9. On Day 8 (1700 h), recipient ewes received 10 mg Lutalyse i.m., followed by 400 IU Pregnecol (Horizon Technology Pty Limited) i.m. on Day 9 (1700 h).
On Day 7 postmating, the uteri of donor ewes were
flushed with 20 ml Medium 199 with Hanks salts, L-glutamine, and 25 mM Hepes buffer (Gibco BRL, Grand Island, NY) to obtain blastocysts. Blastocysts (grade 1 or 2)
were transferred into recipient control (n 5 7) and UGKO
(n 5 5) ewes by laparoscopy. Control ewes received one
blastocyst and UGKO ewes received two blastocysts. In
addition to daily observation for estrous behavior using vasectomized rams, ultrasonography was performed every 5–
7 days from Day 25 to approximately Day 65 postmating
to determine pregnancy status.
1609
Day 14 Postmating Uterine Flush
The UGKO (n 5 8) and normal control (n 5 4) ewes
were given two i.m. injections (0700 h and 1700 h) of 10
mg Lutalyse. Injections were repeated 9 days later to synchronize estrus. All ewes were bred at estrus and at 12 h
and 24 h postestrus by fertile rams. On Day 14 postmating,
the uterine lumen of each ewe was flushed with 20 ml Dulbeccos minimum essential medium/F-12 (Sigma, St. Louis,
MO) medium. Conceptus morphology was analyzed using
a dissecting microscope, and length of conceptus was measured. All ewes were then ovariohysterectomized. Both
ovaries and a portion (;1.0 cm) from the middle region of
each uterine horn were fixed in 4% paraformaldehyde in
PBS (pH 7.2). After 24 h, fixed tissues were changed to
70% ethanol for 24 h and then dehydrated and embedded
in Paraplast-Plus (Oxford Labware, St. Louis, MO). Tissues
were then sectioned (4–6 mm) and stained with hematoxylin and eosin as described previously [17]. Endometrial
gland density was determined by counting the number of
gland cross sections present per uterine cross section for
both UGKO (n 5 8) and control (n 5 4) ewes. Multiple
uterine cross sections (n 5 4) were counted per ewe.
Western Blot Analysis
Uterine flushes (2 ml aliquant) obtained from normal
control and UGKO ewes on Day 14 postmating were concentrated using Centricon-3 columns (Amicon, Inc., Beverly, MA). Protein content was determined using a Bradford
protein assay (Bio-Rad, Hercules, CA) with BSA as the
standard. Uterine flush proteins (20 mg) were denatured,
separated on a 12% SDS-PAGE gel, and transferred to nitrocellulose membranes as described previously [32]. Membranes were blocked for 1 h at room temperature with 5%
milk-Tris-buffered saline with Tween-20 (TBST) and then
incubated with mouse monoclonal antiovine IFNt (HL129)
[33] as primary antibody or mouse IgG at 20 mg/ml, as a
negative control, overnight at 48C. Following primary antibody incubation, membranes were rinsed for 30 min with
TBST and then incubated with goat antimouse IgG-horseradish peroxidase-conjugated secondary antibody (KPL,
Bethesda, MD) for 1 h at room temperature. Membranes
were again rinsed with TBST for 30 min before detection
by enhanced chemiluminescence (Amersham Pharmacia
Biotech, Piscataway, NJ) and Kodak X-OMAT AR film.
Photomicroscopy
Photomicrographs of hematoxylin and eosin-stained tissues were taken using a Zeiss Axioplan 2 photomicroscope
(New York, NY) fitted with a Hamamatsu chilled 3CCD
color camera (Hamamatsu, Japan). Digital images were
captured and assembled using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA) and a MacIntosh PowerMac G3
computer (Apple Computer, Cupertino, CA). Black-andwhite prints were made using a Kodak DS8650 color printer.
Statistical Analyses
Data for pregnancy rate, conceptus length, and endometrial gland density were subjected to one-way leastsquares ANOVA, using general linear models procedures
of the Statistical Analysis System [34]. Data are presented
as least square means with SEM.
1610
GRAY ET AL.
TABLE 1. Relationship of endometrial gland density and conceptus number, morphology, and length in Day 14
bred control and UGKO ewes.
Treatment
Control
UGKO
UGKO
UGKO
a
Number
of ewes
Conceptus number
in uterine flush
from each ewe
Conceptus
morphology
Conceptus length
(mm 6 SEM)
Endometrial
glandular
densitya (6SEM)
4
1
3
4
2
1
1
0
Filamentous
Filamentous
Tubular
No embryo
97 6 15
82
763
—
181 6 37
621
67 6 37
75 6 37
Number of endometrial gland cross sections per uterine section.
RESULTS
Pregnancy was not detected in any of the UGKO ewes
following repeated matings with rams of proven fertility.
The same ewes were then used to determine the ability of
UGKO ewes to support an embryo transferred from a normal control ewe and to eliminate the possibility of a fertilization or embryo transport defect, Day 7 sheep embryos
were transferred from normal, superovulated donor ewes
into synchronized UGKO and control ewes on Day 7 after
onset of estrus. Ultrasonography of ewes on Days 25–65
postmating indicated that five of seven control ewes established and maintained pregnancy. Pregnancy was detected
in zero of five UGKO ewes. Indeed, pregnancy rate was
higher (P , 0.01) in normal control as compared to UGKO
ewes.
Control (n 5 4) and UGKO (n 5 8) ewes were then
bred at estrus and their uteri flushed on Day 14. The uterine
flush from each control ewe contained two filamentous conceptuses of normal length (97 6 15 mm; Table 1). Uterine
flushes from four of the UGKO ewes did not contain a
conceptus. Uterine flushes from the other four UGKO ewes
FIG. 1. Western blot analyses of oIFNt in concentrated uterine flushes
obtained from Day 14 pregnant UGKO and control ewes (A), a doseresponse test using recombinant oIFNt (roIFNt) (B), and a negative control
using mouse IgG (C). Each lane in blot A represents a single flush from
an individual ewe (20 mg per lane) and is labeled by conceptus morphology (N, no embryo; T, tubular conceptus; E, elongated conceptus).
Each lane in blot B represents different concentrations of roIFNt (1000,
500, 250, or 125 ng). Blot C contains one lane of 500 ng roIFNt and one
lane of a uterine flush obtained from a control ewe containing an elongated conceptus. Immunoreactive protein was detected using a mouse
monoclonal antibody directed against oIFNt. Positions of prestained molecular weight standards (31023) are indicated.
contained either a filamentous conceptus of approximately
normal length (82 mm; n 5 1) or severely growth-retarded
tubular conceptuses (7 6 3 mm length; n 5 3). Indeed,
conceptus length was greater (P , 0.01) in uterine flushes
recovered from normal control as compared to UGKO
ewes.
Immunoreactive IFNt was detected in uterine flushes obtained from all control ewes on Day 14 postmating (Fig.
1). The IFNt was also present in the uterine flush from the
UGKO ewe that contained a filamentous conceptus. However, IFNt was not detected in uterine flushes from UGKO
ewes lacking a conceptus or containing a tubular conceptus.
Uteri of all control ewes exhibited extensive, normal
glandular development in the intercaruncular endometrium
(Fig. 2a). As expected, none of the UGKO ewes displayed
uterine glandular development characteristic of controls.
However, among UGKO ewes, uterine glands were most
often either absent (Fig. 2b) or sporadically distributed at
very low density (Fig. 2c) or were distributed somewhat
more regularly, albeit at lower density than in controls, as
observed in the one UGKO ewe in which a filamentous
conceptus was found (Fig. 2d). Endometrial glandular density was directly related to state of conceptus development
on Day 14 postmating (Table 1). Uteri of UGKO ewes with
the more glandular endometria contained more developmentally advanced conceptuses. Endometrial glandular
density was greater (P , 0.10) in uteri of control ewes than
that of UGKO ewes containing either a tubular conceptus
or no conceptus. However, endometrial glandular density
was not different (P . 0.10) in uteri of control ewes as
compared to the one UGKO uterus from which a filamentous conceptus was recovered.
DISCUSSION
Results of the present study provide the first direct evidence to support the hypothesis that endometrial glands
and, by extension, their secretions are required for conceptus survival and growth. Together, control and UGKO ewes
presented a continuum of endometrial phenotypes, ranging
from intensely glandular to effectively aglandular. Thus, a
novel opportunity to establish relationships between state
of conceptus development and endometrial histoarchitecture was created using the ovine UGKO model. Uterine
gland density was positively related to conceptus survival
and state of conceptus development on Day 14 postmating.
However, regardless of endometrial phenotype, none of the
UGKO ewes were able to support pregnancy to Day 25.
Data indicate that the UGKO phenotype, characterized by
extreme reduction in or absence of endometrial glands, constitutes a uterine lesion that compromises periimplantation
conceptus growth and survival.
Results of embryo transfer experiments indicate that early stages of ovine conceptus development, i.e., prior to Day
11, may not be entirely dependent upon the uterine envi-
ENDOMETRIAL GLANDS ARE ESSENTIAL FOR CONCEPTUS DEVELOPMENT
1611
FIG. 2. Histological comparison of Day
14 pregnant uteri from control and UGKO
ewes. The uteri were fixed in paraformaldehyde, sectioned (5 mm), and stained
with hematoxylin and eosin. a) Control
uterus that contained two normal Day 14
conceptuses. b) A UGKO uterus that failed
to support conceptus development. c) A
UGKO uterus that contained only a single
tubular conceptus. d) A UGKO uterus that
contained a single filamentous conceptus.
The UGKO phenotype was produced by
exposing neonatal ewe lambs to 19-norprogestin from birth for 8 wk. L, Lumenal
epithelium; G, glandular epithelium; S,
stroma; M, myometrium. Original magnification 3166.
ronment. Averill et al. [35] transferred 2-cell- through morula-stage ovine embryos into the oviducts of pseudopregnant rabbit does. Five days later, morula- and blastocyststage conceptuses were recovered and transferred into the
uteri of Day 6 nonpregnant ewes, wherein they developed
normally [35]. Consistently, uterine flushes obtained from
UGKO ewes on Days 6 and 9 postmating contained developmentally normal blastocysts (unpublished results).
This suggests that compaction and blastocyst differentiation
by ovine conceptuses are not dependent upon histotroph in
the ovine uterine environment. In addition, preliminary
studies in our laboratory indicate that conceptuses recovered from UGKO ewes are capable of implanting in the
uteri of normal recipient control ewes, supporting the hypothesis that the pregnancy loss defect in UGKO ewes occurs due to an inappropriate uterine environment rather than
a developmental defect in the conceptus (unpublished results).
In sheep, blastocysts hatch from the zona pellucida on
Days 8–9 and then begin to expand and grow to tubular
form by Day 11. Tubular conceptuses elongate rapidly on
Day 13 to the filamentous state, reaching 150–190 mm in
length by Day 15 [36–38]. During the preimplantation period from Days 4 to 15 [39, 40] conceptuses are free-floating in the uterine lumen where they are bathed in and presumed to be supported by uterine histotroph [41]. Fléchon
et al. [42] demonstrated that trophoblastic vesicles derived
from Day 12 conceptuses survived but were unable to undergo elongation in vitro. However, when these vesicles
were transferred into the uterus, they elongated and produced IFNt over the ensuing 5-day period [42]. In the present study, distinct differences in conceptus development
were detected between UGKO and control ewes by Day 14
postmating, suggesting that a critical window of conceptus
development occurs during the periimplantation period that
requires endometrial secretions. Growth-retarded conceptuses recovered from uteri of UGKO ewes were equivalent
developmentally to tubular conceptuses normally found on
Days 11–12. This suggests that endometrial secretions be-
gin to impact conceptus growth and development on Day
11 of pregnancy.
The amount of IFNt secreted by the ovine trophectoderm is directly related to stage of conceptus development
but is not dependent upon a synchronous uterine environment [16]. Filamentous in vivo-derived ovine conceptuses
produced approximately 16 times more IFNt than normal
tubular stage conceptuses [16]. Results from the present
study support these findings. The IFNt was easily detected
in the uterine flushes obtained from Day 14 pregnant control and UGKO ewes with elongated conceptuses, whereas
IFNt was not detected in uterine flushes containing growthretarded tubular conceptuses. The IFNt may be present in
uterine flushes containing tubular conceptuses but at levels
below detectable limits of the antibody. This suggests that
the filamentous conceptus present in the one UGKO ewe
was functionally equivalent to normal control conceptuses
at the same developmental stage.
In addition to providing nutrition for the conceptus, uterine histotroph contains growth factors and cytokines that
promote cell division, proliferation, morphogenesis, and
differentiation [18]. The endometrium also expresses proteins that may be important mediators of conceptus-maternal interactions and implantation. For example, osteopontin
(OPN), a 70-kDa acidic component of the extracellular matrix [43], and glycosylated cell adhesion molecule-1
(GlyCAM-1), a sulfated glycoprotein in the mucin family
[32], are both expressed by the endometrial glands and present in uterine luminal fluid on and after Day 13 of pregnancy that represents the onset of implantation in sheep
[39]. By interacting with or modulating activation of endometrial cell surface integrins, such as the avb3 heterodimer thought to mediate critical cell-cell interactions in
human implantation [44] and serving as a selectin receptor
ligand, these uterine proteins may promote conceptus elongation and stabilize interactions between conceptus trophectoderm and endometrial lumenal epithelium that are essential for implantation [32, 43]. Thus, patterns of conceptus development and the process of implantation might be
1612
GRAY ET AL.
expected to be compromised in the UGKO uterus should
OPN, GlyCAM-1, or other adhesion-promoting molecules
fail to be expressed appropriately.
This study demonstrates that the UGKO ewe is an excellent model for understanding developmental aspects of
uterine biology and uterine function in adults. In humans,
endometrial gland morphogenesis occurs with each menstrual cycle, thereby increasing the opportunity for dysgenesis, dysplasia, and dysfunction. Survival of the human embryo prior to implantation appears to depend upon endometrial gland secretions [15]. It is likely that the human
conceptus does not receive direct hematotrophic nutrition
from the maternal circulation until the end of the first trimester of pregnancy [45]. Therefore, alterations in human
endometrial gland formation and function may lead to infertility and early pregnancy loss. Decreased expression of
cell-surface and secretory proteins within the uterine environment are correlated with abnormal uterine gland morphology that adversely affect uterine receptivity during the
periimplantation and early placentation stages of gestation
[44, 46, 47]. Perhaps these documented human reproductive
defects can be related to defects in UGKO ewes wherein
the absence of epithelial-specific gene expression [31] and
histotroph production results in failure of conceptus development beyond the preimplantation period.
The present study confirms that administration of a synthetic 19-norprogestin to neonatal ewe lambs disrupts normal endometrial gland development and results in adult
ewes that are unable to establish or maintain pregnancy
[17]. The absence of endometrial glands results in 1) defects in conceptus development during elongation and 2)
an inability of ewes to maintain pregnancy beyond the preimplantation period. Variations in UGKO phenotype observed for some ewes offer a unique opportunity to investigate the role of endometrial glands and their secretions
during various stages of gestation and to define specific
components of histotroph required for normal periimplantation conceptus development in sheep and, perhaps, other
mammals. In future studies, techniques of genomics and
proteomics will be employed to compare differences in endometrial gene expression and histotroph production between control and UGKO ewes.
8.
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ACKNOWLEDGMENT
Thanks are due to Mr. Todd Taylor of the Texas A&M Sheep and Goat
Center for animal husbandry and assistance with embryo transfer.
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