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Regular Article |
a Center for Animal Biotechnology and Genomics and the Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471
ABSTRACT
Endometrial glands are present in all mammalian uteri and produce secretions that are hypothesized to support conceptus (i.e., embryo/fetus and placental membranes) survival and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by chronic neonatal exposure to a progestin from birth, thereby producing an adult uterine gland knock-out (UGKO) phenotype. This study determined the long-term effects of neonatal progestin exposure on adult ovine reproductive tract structure and function. Neonatal ewes were exposed to norgestomet (Nor) from birth to 32 wk of age. Unexposed ewes served as controls. After puberty, adult Nor-treated (n = 6) and control (n = 6) ewes were repeatedly bred at estrus (Day 0) to intact rams of proven fertility. In contrast to a pregnancy rate of 80% for control ewes, pregnancy was never detected on Day 25 after mating (or thereafter) in bred UGKO ewes. Control and Nor-treated ewes were then bred and necropsied on Day 9. Similar numbers of hatched blastocysts were present in uterine flushings from control and Nor-treated ewes. Weights of the ovaries and cervices were not affected by treatment. No histoarchitectural differences between control and Nor-treated ewes were detected for ovaries, oviducts, cervices, or vaginae. However, uterocervical and uterine weight as well as uterine horn length were less for Nor-treated ewes. The uteri of Nor-treated ewes were devoid of endometrial glands and lacked the stromal delineation characteristic of intercaruncular endometrium in control ewes. Endometrial width, area, and lumenal epithelial length were decreased in uteri from Nor-treated ewes, but myometrial width and morphology were not affected. Expression of a number of mRNAs that are expressed predominantly in the endometrial epithelia was not different between uteri from control and from Nor-treated ewes. Collectively, these results indicate that neonatal exposure of ewes to a progestin from birth appears to only affect development of the uterus and not any extrauterine reproductive tract tissues. The infertility of the UGKO ewes appears to result from a lack of endometrial glands and, by extension, of their secretions that are required to support growth and development of peri-implantation conceptuses.
cervix, developmental biology, fallopian tubes, gene regulation, hormone action, oviduct, pregnancy, progesterone, uterus, vagina
INTRODUCTION
Inappropriate exposure of the female reproductive tract to steroids during developmentally critical periods can disrupt the organizational events necessary for growth and differentiation, lead to altered adult phenotypes, and decrease reproductive efficiency [1–4]. The age of an animal at the time of exposure to an endocrine disruptor has considerable importance in determining the extent of detrimental effects detected in the adult, because age is inversely related to the magnitude of effects on development and organization of a specific tissue [1]. The dosage and period of exposure to the endocrine disruptor is also directly related to effects on organ development and function [5]. In the rodent uterus, developmental changes during the neonatal period are important determinants of the embryotrophic potential and functional capacity of the adult uterus [1]. Similarly, inappropriate exposure of neonatal livestock to endocrine disruptors has permanent effects on adult uterine structure and function [6, 7].
In pigs, exposure of the developing porcine uterus to estrogen has differential effects on uterine weight, cell proliferation, and protein expression, which compromise uterine capacity in the adult [7, 8]. In cattle, neonatal exposure to progesterone and estradiol benzoate implants decreased uterine weight, endometrial and myometrial surface area, and protein content of uterine flushes on Day 12 of the estrous cycle in adult heifers [9, 10]. In sheep, uterine gland morphogenesis or adenogenesis is not initiated until after birth [11, 12]. A critical window of uterine gland development occurs between postnatal Days (PNDs) 13 and 56, because exposure of the neonatal ewe to a progestin during this period results in permanent epigenetic ablation of uterine gland morphogenesis [3, 13, 14]. The result of inappropriate, prolonged progestin exposure in the neonate is a uterine gland knock-out (UGKO) phenotype in adult ewes [3, 14].
Mature UGKO ewes are unable to exhibit normal estrous cycles because of insufficient production of luteolytic pulses of prostaglandin F2
by the uterus [3]. In addition, the UGKO ewes are unable to support establishment and/or maintenance of pregnancy [3, 4]. The nature of the pregnancy defect in UGKO ewes is unknown, but the progestin exposure regimen used to create the UGKO phenotype possibly has adverse effects on extrauterine reproductive tract structures and compromises gamete transport and survival [3]. Therefore, the objective of this study was to determine the effects of prolonged exposure of neonatal ewes to a progestin on reproductive tract structure and function in adults.
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).
Experimental Design
Rambouillet ewe lambs were assigned randomly at birth (n = 6 ewes/group) to be treated with norgestomet (Nor) or to remain untreated as controls. Ewes assigned to Nor treatment received a single Synchromate B (Sanofi, Overland Park, KS) implant within 12 h of birth and every 2 wk until 32 wk of age. Implants were inserted s.c. in the periscapular area and released approximately 6 mg of Nor (17-acetoxy-11ß-methyl-19-norpreg-4-ene-3,20-dione), a potent synthetic 19-norprogestin, during a 14-day period [15].
As adults, Nor-treated ewes were synchronized to estrus using two injections (0700 and 1900 h) of 10 mg of prostaglandin F2 (Lutalyse; Upjohn, Kalamazoo, MI) on Day 0. This regimen was repeated 9 days later. All ewes were then bred at estrus (Day 0) and at 12 and 24 h after detection of estrus to 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 25–35 after mating to detect the presence of a conceptus. The Nor-treated UGKO ewes were resynchronized to estrus using Lutalyse as described above and rebred. This experiment was repeated three times with the same UGKO ewes.
Control (n = 6) and Nor-treated UGKO ewes (n = 6) were synchronized to estrus with Lutalyse and bred to fertile, intact rams as described above. On Day 9 after mating, ewes were killed by i.v. injection of a saturated potassium chloride solution and necropsied. The entire female reproductive tract was excised. The ovaries, oviducts, bladders, and vaginae were dissected. Ovarian weights and number of corpora lutea (CL) on each ovary were recorded. Sections of the ovaries, oviducts (ampulla and isthmus), and vaginae (anterior and posterior) were fixed in fresh, 4% w/v paraformaldehyde in PBS (pH 7.2) for histology. Uterocervical weights were recorded. Each uterine horn was flushed with 10 ml of Dulbecco's modified Eagle medium/F-12 medium (Sigma Chemical Co., St. Louis, MO). The cervix was separated from the uterus, and weights of the individual cervices and uteri were recorded. Uterine horn lengths were measured, and the uterine horns were separated by cutting through the intercornual ligament to the uterine body. Three separate sections from each uterine horn (near the uterotubal junction, the midportion, and the uterine body) were fixed in 4% paraformaldehyde. After 24 h, fixed tissues were changed to 70% ethanol, dehydrated, and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO).
Uterine Flush Processing and Analysis
The uterine flushes were examined using a dissecting microscope for the presence of conceptuses. If present, stage of conceptus development was assessed and recorded. Volume of each uterine flush was recorded, and flushes were then clarified by centrifugation (3000 x g for 30 min at 4°C) and stored at -20°C. Uterine flush protein content was determined using a Bradford protein assay (Bio-Rad, Hercules, CA), with BSA as the standard. Total protein content of each uterine flush was calculated based on the recovered volume.
Histology and Morphometry
Paraffin-embedded tissues were sectioned (4–6 µm) and stained with hematoxylin-and-eosin as described previously [3]. For morphometry, stained sections from the midportion of the uterine horns of both control and Nor-treated ewes were photomicrographed at 164x magnification. Images were analyzed using Scion Image software (Scion Corporation, Frederick, MD). Measurements were standardized using the image of a stage micrometer at the same magnification. In each uterine cross-section, the length of the lumenal epithelium (LE), endometrial width, endometrial surface area, and myometrial width were measured using computer-assisted, image-analysis software. Morphometrical measurements were obtained from multiple cross-sections (n = 4) of the uterine wall from each ewe. For width determinations, multiple measurements (n = 3–9) were made from each uterine cross-section.
In Situ Hybridization
We previously identified and cloned several predominantly epithelial-expressed mRNAs by analysis of normal and UGKO endometrium using mRNA differential display-polymerase chain reaction (DD-PCR) or PCR-based suppression subtractive cDNA hybridization (SSH) [14]. The mRNAs for DD54, SSH82, SSH117, SSH133, and SSH179 were localized in uterine tissue sections (5 µm) by in situ hybridization analysis as described previously [14]. Deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized ovine cDNAs using in vitro transcription with -35S-uridine triphosphate. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Eastman Kodak, Rochester, NY), stored at 4°C for 3 days to 1 wk, and developed in Kodak D-19 developer. Slides were then counterstained with Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ), dehydrated through a graded series of alcohol to xylene, and protected with a coverslip.
Photomicroscopy
Photomicrographs were taken using a Zeiss Axioplan2 photomicroscope (New York, NY) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu, Japan). Photomicrographs of in situ hybridization slides were taken under bright-field and dark-field illumination. 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-and-white prints were made using a Kodak DS8650 color printer.
Statistical Analyses
All quantitative data were subjected to least-squares ANOVA using general linear models procedures of the Statistical Analysis System [16]. Data are presented as least square means and SEM. The initial statistical model for analyses of morphometry included variation caused by treatment, ewe, uterine section within ewe, and appropriate interactions. Given that uterine section was not a significant source of variation, the final model was a simple one-way ANOVA.
RESULTS
Effects of Nor Exposure on Fertility
All control and Nor-treated ewes attained puberty and exhibited behavioral estrus. Normal control ewes bred to rams were fertile, with an average pregnancy rate on first breeding of 80% as determined by ultrasound. However, UGKO ewes bred to the same fertile rams as control ewes were consistently unable to establish or maintain pregnancy to Day 25.
Gross Observations and Measurements
All ewes had complete and intact reproductive tracts. Examination of the gross morphology of the entire reproductive tract revealed no differences between Nor-treated and control ewes, except for uteri (Fig. 1). The ovaries from both Nor-treated and control ewes contained follicles and CL characteristic of Day 9 cyclic ewes. In contrast, uteri were affected by Nor exposure. Uterine horns from control ewes were long, curved, and substantial in size and appearance, whereas uterine horns from Nor-treated ewes appeared immature, with short uterine horns that exhibited little curvature. The uterocervical weights, uterine weights, and uterine horn lengths were reduced in Nor-treated ewes (Table 1). However, weights of the ovary and cervix were not affected by Nor treatment.
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Histoarchitecture and Morphometry
Careful histoarchitectural evaluation of ovarian, oviductal, ampullar and isthmus, cervical, and anterior and posterior vaginal tissues detected no differences between Nor-treated and control ewes (Figs. 2 and 3). All ewes possessed oviducts with considerable crypt formation. The epithelium of the cervix and vagina appeared appropriately differentiated.
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Uterine gland development was extensive in the intercaruncular endometrium of control uteri (Fig. 4). However, endometrial glands were absent from uteri of Nor-treated ewes. In the aglandular uteri of UGKO ewes, a ruffled and undulating LE was apparent in endometrium between caruncular nodules that represented intercaruncular endometrial areas. In Nor-treated uteri, the overall size of the uterine lumen appeared greater, whereas the number of endometrial folds was markedly lower. Endometrial width, area, and LE length were decreased in Nor-treated ewes (Table 2), but Nor exposure had no effect on myometrial histoarchitecture or width.
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Uterine Flushes
Uterine flushes from both control and Nor-treated ewes on Day 9 after mating contained morphologically normal hatched blastocysts (data not shown). Pregnancy rates were not different (P > 0.1) between control (n = 4/6) and Nor-treated ewes (n = 2/5). Total protein content of uterine flushes from control (0.84 mg) and Nor-treated (0.82 mg) ewes was not different (P > 0.1).
In Situ Hybridization
In uteri from control ewes, expression of DD54, SSH82, SSH117, and SSH133 mRNAs was detected in both endometrial LE and superficial glandular epithelium (GE) of the intercaruncular endometrium (Fig. 5). The expression of SSH82 mRNA was also detected in the stratum compactum of the intercaruncular endometrium. Although SSH82 mRNA was detected in the caruncular LE, this mRNA was not detected in the caruncular stroma of control uteri. In uteri from Nor-treated ewes, expression of the same mRNAs was detected specifically in the endometrial LE. The SSH82 mRNA was not detected in the stroma of Nor-treated uteri. Overall, the relative intensity of endometrial epithelial mRNA expression was not different between Nor-treated and control ewes.
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DISCUSSION
We recently found that exposure of neonatal ewes to a progestin for 28 days from birth specifically ablated endometrial gland development without affecting the histoarchitecture of ovaries, oviducts, cervices, and vaginae in the ewes on PND 28 [17]. In that study, an increase in lumen size of the cervix and vagina as well as keratinization of the vaginal epithelium were observed on PND 28, Nor-exposed ewes [17]. In the present study, differences in the cervix and vagina were not detected, suggesting that the previously observed differences in these tissues on PND 28 ewes resulted from a direct influence of the 19-norprogestin. Collectively, results from this and previous studies indicate that development of female reproductive structures derived from the mullerian duct and urogenital sinus in sheep are organizationally complete at birth, with the exception of the uterus. However, vaginal development in rodents [18, 19] and humans [20] is clearly not complete until early postnatal life, suggesting that the ontogeny of female reproductive tract structures is species specific.
In the present study, profound effects of neonatal Nor exposure on gross morphology and histoarchitecture of the adult uterus were observed. Uterocervical weights, uterine horn lengths, and uterine wet weights were lower in Nor-exposed adult ewes. Similar decreases in uterine wet weight were detected in adult cows exposed neonatally to a combination of progesterone and estradiol benzoate [9, 10]. Uteri from Nor-exposed ewes completely lacked uterine glands and intercaruncular endometrial areas and showed marked reductions in endometrial folds. An increase in lumen size was observed, which most likely results from the absence of endometrial folds stemming from agenesis of the intercaruncular endometrial areas. In addition, endometrial width, endometrial area, and LE length were dramatically reduced in Nor-treated as compared to control ewes. Effects of Nor on uterine structure appeared confined to the endometrium, because no histoarchitectural or morphometrical differences in the myometrium were detected between control and Nor-exposed ewes. Indeed, the myometrium is differentiated into layers at birth and appears to grow only slightly from birth to PND 56 [12].
As expected from previous studies [3, 13, 14], exposure of the developing neonatal uterus to a progestin for 32 wk inhibited endometrial gland morphogenesis and produced an UGKO phenotype. The uteri of UGKO ewes was essentially glandless, with only a ruffled LE and compact stroma in the area that contained numerous coiled and branched endometrial glands in uteri of control ewes. The appearance of LE undulations has been reported for the fetal Day 145 ovine uterus, and these undulations likely represent primordial GE buds [11]. Besides the absence of endometrial glands, no distinct differences in histology or morphology of the myometrium were observed. Uteri from UGKO ewes had less endometrial area and width, perhaps because of the lack of stratum spongiosum in the lower portion of the intercaruncular stroma of control uteri. Therefore, the endometrium of the UGKO uterus appears to consist of only caruncular areas that contain LE supported by dense stroma or stratum compactum. These observations and those from studies of Nor-exposed, PND 28 ovine uteri [17] suggest that delineation of the stratum compactum and stratum spongiosum during postnatal intercaruncular endometrial morphogenesis requires the differentiation and development of endometrial glands. The idea that the stratum compactum of the caruncular and intercaruncular endometrium is different in normal uteri is supported by differential expression of SSH82 mRNA. Given that the stroma of UGKO endometrium lacks SSH82 mRNA, this stroma likely has a caruncular stromal phenotype. Collectively, results of the present study clearly indicate that a lack of endometrial glands during prepubertal uterine morphogenesis disrupts the stromal-epithelial interactions required for normal uterine stromal growth and development [21].
Although pregnancy was detected in control ewes mated to fertile rams, establishment and maintenance of pregnancy to Day 25 of gestation or beyond was not detected in any of the UGKO ewes bred to the same fertile rams. The failure of pregnancy to be established does not appear to have resulted from a defect in gamete transport or fertilization, because morphologically normal hatched blastocysts were found in the uterine flushes from UGKO ewes on Day 9 after mating. As observed previously [3], the hypothalamic-pituitary-ovarian axis appears to be unperturbed in adult UGKO ewes, because Nor-treated and control ewes were equally responsive to exogenous prostaglandin F2 with behavioral estrus. Previously, we determined that circulating levels of progesterone in metestrus and diestrus were not different between UGKO and control ewes [3]. Therefore, failure of conceptus development in UGKO ewes between Days 9 and 25 of early pregnancy must result from the absence of uterine glands and/or their secretions.
In addition to a lack of endometrial glands, defects in pregnancy in UGKO ewes could also result from alterations in endometrial LE cell function. In this study, uteri from Day 9 control and UGKO ewes were analyzed for expression of a number of predominantly epithelial-specific genes cloned previously by this laboratory and regulated by progesterone and uterine progesterone receptor expression during the estrous cycle and early pregnancy [14]. In situ hybridization analyses failed to detect specific differences in the relative expression and cell type-specific patterns for expression of these selected mRNAs in the endometrial LE of Nor-treated and control ewes. Similarly, we failed to detect differences in uterine cell type-specific patterns of estrogen receptor , progesterone receptor, or oxytocin receptor expression between Day 9 or 15 postestrus normal and UGKO ewes [3]. Although definitive studies remain to be conducted, these results strongly suggest that failure of pregnancy establishment in UGKO uterus is caused by the absence of substances that are synthesized and secreted or transported into the uterine lumen by endometrial glands.
Uterine secretions, collectively termed histotroph, are hypothesized to be essential for support of conceptus (i.e., embryo/fetus and associated extraembryonic placental membranes) survival and growth during gestation in livestock [22–24] as well as in human and nonhuman primates [25–27]. Uterine histotroph is a complex mixture of many enzymes, cytokines, growth factors, ions, hormones, glucose, transport proteins, and adhesion molecules [24–26, 28, 29]. In this study, no differences were detected in total uterine flush protein content, conceptus morphology, or number of ewes pregnant between control and Nor-treated ewes on Day 9 after mating. Averill et al. [30] demonstrated that two cell- through morula-stage ovine embryos transferred into a rabbit oviduct underwent normal development for 5 days and continued to develop normally for an additional 16 days when transferred to a Day 6 nonpregnant ewe [30]. Results from the present study lend strong support to the concept that endometrial glands and their secretions are not required for conceptus development through the blastocyst stage.
In summary, exposure of neonatal ewe lambs from birth to 32 wk of age to a synthetic progestin epigenetically ablated endometrial gland morphogenesis without altering development of the uterine myometrium or other adult female reproductive tract structures. Neonatal exposure to Nor did not appear to affect endometrial gene expression patterns, suggesting that defects in pregnancy establishment in UGKO ewes stem from a lack of endometrial glands and their secretions. Available results indicate that the UGKO ewe is an excellent model to 1) determine the role of endometrial glands in uterine function; 2) study the long- and short-term effects of endocrine disruptors administered during the neonatal period; and 3) identify specific components of uterine endometrial gland secretions required for peri-implantation conceptus development and survival.
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FOOTNOTES
First decision: 5 October 2000.
1 Supported by National Research Initiative Competitive Grants Program/U.S. Department of Agriculture grant 98-35203-6322 and, in part, by National Institutes of Health 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; tspencer{at}ansc.tamu.edu
Accepted: October 11, 2000.
Received: August 17, 2000.
REFERENCES
and other cytokines in early pregnancy. Reprod Fertil Dev 1997; 9:355–380[CrossRef][Medline]This article has been cited by other articles:
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  J. Kim, G. Song, H. Gao, J. L. Farmer, M. C. Satterfield, R. C. Burghardt, G. Wu, G. A. Johnson, T. E. Spencer, and F. W. Bazer Insulin-Like Growth Factor II Activates Phosphatidylinositol 3-Kinase-Protooncogenic Protein Kinase 1 and Mitogen-Activated Protein Kinase Cell Signaling Pathways, and Stimulates Migration of Ovine Trophectoderm Cells Endocrinology, June 1, 2008; 149(6): 3085 - 3094. [Abstract] [Full Text] [PDF]
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 K. Hayashi, A. R O'Connell, J. L Juengel, K. P McNatty, G. H Davis, F. W Bazer, and T. E Spencer Postnatal uterine development in Inverdale ewe lambs Reproduction, March 1, 2008; 135(3): 357 - 365. [Abstract] [Full Text] [PDF]
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T. E Spencer, O. Sandra, and E. Wolf Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches Reproduction, February 1, 2008; 135(2): 165 - 179. [Abstract] [Full Text] [PDF]
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C K Wang, R S Robinson, A P F Flint, and G E Mann Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels Reproduction, August 1, 2007; 134(2): 365 - 371. [Abstract] [Full Text] [PDF]
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 C. A. Gray, C. A. Abbey, P. D. Beremand, Y. Choi, J. L. Farmer, D. L. Adelson, T. L. Thomas, F. W. Bazer, and T. E. Spencer Identification of Endometrial Genes Regulated by Early Pregnancy, Progesterone, and Interferon Tau in the Ovine Uterus Biol Reprod, February 1, 2006; 74(2): 383 - 394. [Abstract] [Full Text] [PDF]
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K. Hayashi and T. E. Spencer Estrogen Disruption of Neonatal Ovine Uterine Development: Effects on Gene Expression Assessed by Suppression Subtraction Hybridization Biol Reprod, October 1, 2005; 73(4): 752 - 760. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D Carpenter, T. H Welsh Jr, R. C Burghardt, L. J Spicer, and T. E Spencer The IGF system in the neonatal ovine uterus Reproduction, March 1, 2005; 129(3): 337 - 347. [Abstract] [Full Text] [PDF]
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T. E Spencer, G. A Johnson, F. W Bazer, and R. C Burghardt Implantation mechanisms: insights from the sheep Reproduction, December 1, 2004; 128(6): 657 - 668. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D. Carpenter, and T. E. Spencer Neonatal Estrogen Exposure Disrupts Uterine Development in the Postnatal Sheep Endocrinology, July 1, 2004; 145(7): 3247 - 3257. [Abstract] [Full Text] [PDF]
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 T. E. Spencer and F. W. Bazer Uterine and placental factors regulating conceptus growth in domestic animals J Anim Sci, January 1, 2004; 82(13_suppl): E4 - 13. [Abstract] [Full Text] [PDF]
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G. A. Johnson, R. C. Burghardt, F. W. Bazer, and T. E. Spencer Osteopontin: Roles in Implantation and Placentation Biol Reprod, November 1, 2003; 69(5): 1458 - 1471. [Abstract] [Full Text] [PDF]
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B. J. Tarleton, T. D. Braden, A. A. Wiley, and F. F. Bartol Estrogen-Induced Disruption of Neonatal Porcine Uterine Development Alters Adult Uterine Function Biol Reprod, April 1, 2003; 68(4): 1387 - 1393. [Abstract] [Full Text] [PDF]
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K. D. Carpenter, C. A. Gray, S. Noel, A. Gertler, F. W. Bazer, and T. E. Spencer Prolactin Regulation of Neonatal Ovine Uterine Gland Morphogenesis Endocrinology, January 1, 2003; 144(1): 110 - 120. [Abstract] [Full Text] [PDF]
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C. A. Gray, F. F. Bartol, B. J. Tarleton, A. A. Wiley, G. A. Johnson, F. W. Bazer, and T. E. Spencer Developmental Biology of Uterine Glands Biol Reprod, November 1, 2001; 65(5): 1311 - 1323. [Abstract] [Full Text] [PDF]
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