Neutrophil depletion retards endometrial repair in a mouse model

The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair. This work was funded by the National Health and Medical Research Council of Australia (#143798, #241000) and by an Australian Postgraduate Scholarship to T.K.     

CXCR4, regulated by HIF1A,promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45⁺ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.     

Matrix metalloproteinases in endometrial breakdown and repair: functional significance in a mouse model

Considerable correlative evidence suggests an important role for matrix metalloproteinases (MMPs) in menstruation, a process which occurs naturally in very few species. In this study, MMP expression was examined in a mouse model of endometrial breakdown and repair and the functional importance of MMPs determined. In the model, progesterone support was withdrawn from mice in which endometrial decidualization had been induced; 24 h later, endometrial breakdown was complete, and the entire decidual zone had been shed. Re-epithelialization had occurred by 36 h, and the endometrium had undergone extensive restoration toward a predecidualized state by 48 h. Immunoreactive MMP9 and MMP7 colocalized with leukocyte subsets, particularly neutrophils, whereas MMP13 staining was always extracellular. MMP3 and MMP7 were abundant during re-epithelialization in close proximity to newly reforming epithelium. The functional importance of MMPs in these processes was examined using two MMP inhibitors, doxycycline and batimistat. Both inhibitors effectively reduced MMP activity, as assessed by in situ zymography, but did not have significant effects on endometrial breakdown or repair. This study demonstrates that although MMPs are present in abundance during endometrial breakdown and repair in this mouse model, they are not the key mediators of these processes.     

Active Role of the Predecidual-Like Zone in Endometrial Shedding in a Mouse Menstrual-Like Model

Cyclic shedding of the endometrium is unique to menstruating species. The status of the decidua in mouse menstrual-like models seems to differ from that of the predecidua in humans before endometrial breakdown. The aim of this study was to determine how this difference in decidual status is related to endometrial breakdown. A mouse menstruallike model was generated by pharmacological progesterone withdrawal. Histomorphological analysis and reticular fiber staining were used to evaluate endometrial status. In situ zymography was used to determine the localization of active collagenase and gelatinase. The functional endometrial layer containing the mature decidual-like zone (MDZ) and predecidual-like zone (PZ) underwent breakdown. The reticular fibers underwent disruption and fragmentation and became loose or disappeared at 12 h in the PZ, where active collagenase and gelatinase were limited. The reticular fibers were visibly reduced at 24 h in the MDZ, where active collagenase was detected. A few reticular fibers remained; however, the functional layer had sloughed into the lumen of the uterus. The results showed that reticular fibers of the PZ are actively degraded during endometrial shedding.Key words: mouse menstrual-like model, predecidual-like zone, reticular fiber, gelatinase, collagenase     

Progesterone withdrawal and RU-486 treatment stimulate apoptosis in specific uterine decidual cells

Progesterone secretion is required for the growth and differentiation of endometrial stromal cells to form decidual cells. For many cells where a growth factor supports cell growth and proliferation, withdrawal of the growth factor initiates apoptosis. This study determined the time course and tissue location of apoptosis in deciduomal tissue after withdrawal of progesterone or injection of the antiprogestin, RU-486. Total DNA was isolated from decidual tissues at intervals after experimental treatments and separated electrophoretically. Internucleosomal DNA fragmentation characteristic of apoptosis was measured by quantitating levels of the 200 bp fragment. Apoptotic cells in tissue sections were detected by direct immunoperoxidase detection of digoxigenin-labeled DNA. Decidual apoptosis reached maximal levels at 12 h after withdrawal of progesterone or injection of RU-486. Increased concentrations of apoptotic cells were observed at the periphery of the growing deciduoma and in the antimesometrial deciduoma near the luminal epithelium after both treatments. These results suggest the withdrawal of progestin initiates apoptosis in cells at the early stages of decidualization.     

Comprehensive analysis of leukocytes, vascularization and matrix metalloproteinases in human menstrual xenograft model

In our previous study, menstrual-like changes in mouse were provoked through the pharmacologic withdrawal of progesterone with mifepristone following induction of decidualization. However, mouse is not a natural menstruation animal, and the menstruation model using external stimuli may not truly reflect the occurrence and development of the human menstrual process. Therefore, we established a model of menstruation based on human endometrial xenotransplantation. In this model, human endometrial tissues were transplanted subcutaneously into SCID mice that were ovarectomized and supplemented with estrogen and progestogen by silastic implants with a scheme imitating the endocrinological milieu of human menstrual cycle. Morphology, hormone levels, and expression of vimentin and cytokeratin markers were evaluated to confirm the menstrual-like changes in this model. With 28 days of hormone treatment, transplanted human endometrium survived and underwent proliferation, differentiation and disintegration, similar to human endometrium in vivo. Human CD45+ cells showed a peak of increase 28 days post-transplantation. Three days after progesterone withdrawal, mouse CD45+ cells increased rapidly in number and were significantly greater than human CD45+ cell counts. Mouse CD31+ blood vascular-like structures were detected in both transplanted and host tissues. After progesterone withdrawal, the expression levels of matrix metalloproteinases (MMP) 1, 2, and 9 were increased. In summary, we successfully established a human endometrial xenotransplantation model in SCID mice, based on the results of menstrual-like changes in which MMP-1, 2 and 9 are involved. We showed that leukocytes are originated from in situ proliferation in human xenografts and involved in the occurrence of menstruation. This model will help to further understand the occurrence, growth, and differentiation of the endometrium and the underlying mechanisms of menstruation.     

Uterine blood flow during the development and regression of the decidual cell reaction in ovariectomized, steroid-treated mice

Uterine blood flow was assessed in mice by measuring organ uptake of intravenously injected [14C]butanol. In ovariectomized mice, injection of 100 ng oestradiol-17 beta increased blood flow 5-fold over that of untreated controls. The injection of oestradiol-17 beta in progesterone-treated mice also increased uterine blood flow at the time of maximal sensitivity to a decidual stimulus, but not 4 days later. Absolute values of blood flow increased during development of the decidual cell reaction in proportion to the increase in uterine weight, reaching maximal values 96 h after decidual induction. When progesterone injections were stopped 72 h after decidual induction, a rapid decrease in absolute and relative blood flow values preceded the decrease in uterine weight. This decrease in uterine blood flow occurred within 12 h of removing a subcutaneous implant containing progesterone. These results are consistent with the view that increased uterine blood flow during decidual development may be necessary to support the rapid increase in uterine weight at implantation and the subsequent decrease in both relative and absolute uterine blood flow on withdrawal of progesterone may promote decidual regression in the mouse.     

Induction of epithelial cell apoptosis in the uterus by a mouse uterine ischemia-reperfusion model: possible involvement of tumor necrosis factor-alpha

Menstruation in primates is preceded by a period of intense vasoconstriction, with resultant ischemia-reperfusion. Although apoptosis is involved in endometrial breakdown, the relationship between ischemia-reperfusion and apoptosis in the female genital tract has not been determined. To investigate the relationship between ischemia-reperfusion and apoptosis in the uterus, we analyzed a uterine ischemia-reperfusion model using BDF1 and C57BL/6 mice. Ischemia was induced by clamping the uterine horn and uterine artery for 5 to 30 min, followed by 6, 12, 24, or 48 h of reperfusion (n = 4 for each group). The number of TUNEL-positive endometrial cells increased with the duration of ischemia and reached a maximum at 24 h of reperfusion, but then tended to decrease at 48 h. Transmission electron micrographs of endometrial cells revealed a typical nuclear condensation, confirming the occurrence of apoptosis. The mRNA expression level of the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) in the uterus increased after reperfusion. Ischemia-reperfusion-induced endometrial apoptosis was markedly decreased in TNF-R p55-deficient mice, confirming the essential role of TNFalpha in the induction of apoptosis by ischemia-reperfusion (n = 4). Our results suggest that ischemia-reperfusion and subsequent TNFalpha expression may be critical factors in inducing endometrial cell apoptosis. Our mouse model could be suitable for investigating ischemia-related uterine injury in humans, particularly in menstruation.     

Hormonal regulation of estrogen and progestin receptors in decidual cells

Total estrogen receptor (Re) and total progestin receptor (Rp) were measured in the cytosol and nuclear fractions from hamster deciduomal tissue and decidual cell cultures. Correlation of serum steroid (estradiol and progesterone) and deciduomal receptor profiles revealed a significant loss of Re during the first four days of decidualization that was not attributable to changes in serum steroid levels. A decidual cell-tissue culture system was used to study the receptor's recovery response to progesterone withdrawal. Decidual cells were plated and grown in Ham's F12/Dulbecco's modified Eagle's medium with 5% horse serum supplemented with insulin, transferrin, selenium and progesterone (10 ng/ml). Within 48 h of culture large, multinucleate decidual cells were observed by phase microscopy. At 72 h of culture in medium containing progesterone, only Rp was detectable in decidual cells. Re was not detectable (less than 200 fmol/mg DNA) in either cytosol or nuclei from cells maintained in the presence of progesterone. However, when progesterone was deleted from the medium, cytosol Re recovered progressively from 8 h to 16 h of culture. Progesterone withdrawal also caused parallel increases in cytosol and nuclear Rp, and estradiol treatment (2 ng/ml) in combination with progesterone withdrawal further enhanced Rp levels in decidual cell cultures. These results with cultured decidual cells demonstrate that progesterone down-regulates Re and Rp, Re recovers rapidly upon progesterone withdrawal, and the Re system is competent to respond to estrogen action in terms of Rp induction. We used the density-shift method to determine that progestin increases the turnover of nuclear Re in hamster decidual cells within 3 h. Hamster decidual cells were isolated from the endometrium and cultured in progesterone-free medium containing normal amino acids (1H, 12C, 14N) for 2 days. Confluent monolayers of cells were exposed to 1 nM estradiol (E2) for 1 h to maximize the amount of occupied Re in the nuclear fraction. Then, at time 0, cells were transferred to medium supplemented with dense (2H, 13C, 15N) amino acids and either 1 nM E2 or E2 plus 100 nM progesterone. After Re was labeled with dense amino acids for 1, 3, 6 and 9 h, nuclear Re was extracted with 10 mM pyridoxal -5' phosphate and labeled with 125I-iodoestradiol (5 nM). Two radioactive peaks representing preexisting and newly synthesized Re were separated by sucrose density-gradient centrifugation. The halflife of nuclear Re in decidual cells was 3.7 h when cells were treated with E2 alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of label-retaining perivascular cells in a mouse model of endometrial decidualization, breakdown, and repair

The human endometrium is incredibly dynamic, undergoing monthly cycles of growth and regression during a woman's reproductive life. Endometrial repair at the cessation of menstruation is critical for reestablishment of a functional endometrium receptive for embryo implantation; however, little is understood about the mechanisms behind this rapid and highly efficient process. This study utilized a functional mouse model of endometrial breakdown and repair to assess changes in endometrial vasculature that accompany these dynamic processes. Given that adult endometrial stem/progenitor cells identified in human and mouse endometrium are likely contributors to the remarkable regenerative capacity of endometrium, we also assessed label-retaining cells (LRC) as candidate stromal stem/progenitor cells and examined their relationship with endometrial vasculature. Newborn mouse pups were pulse-labeled with bromodeoxyuridine (BrdU) and chased for 5 wk before decidualization, endometrial breakdown, and repair were induced by hormonal manipulation. Mean vessel density did not change significantly throughout breakdown and repair; however, significantly elevated endothelial cell proliferation was observed in decidual tissue. Stromal LRC were identified throughout breakdown and repair, with significantly fewer observed during endometrial repair than before decidualization. A significantly higher percentage of LRC were associated with vasculature during repair than before decidualization, and a proportion were undergoing proliferation, indicative of their functional capacity. This study is the first to examine the endometrial vasculature and candidate stromal stem/progenitor cells in a functional mouse model of endometrial breakdown and repair and provides functional evidence suggesting that perivascular LRC may contribute to endometrial stromal expansion during the extensive remodeling associated with this process.     

小鼠月经生理模型的探讨

目的减少Finn CA于1984年首次报道的小鼠月经模型的观察时间点,以期为月经生理学研究提供一种较廉价且易操作的月经模型。方法应用成年雌性去势C57BL/6小鼠,给予续贯性激素处理,最末次激素处理后4～6h,实验组小鼠宫腔内注射花生油以诱导子宫内膜蜕膜化反应,对照组小鼠给予同样激素处理但无宫腔油剂注射。分别于油剂处理后31～35h(T3组)、56～70h(T4组)处死小鼠,称量子宫湿重,制作H&E组织切片,运用图像分析软件CAST2,计算全子宫横截面积(TUA)与子宫内膜横截面积(EA)。结果H&E染色子宫组织切片示在单纯雌激素作用下宫内膜呈单层立方上皮,核浆比较高,内膜基质疏松;雌孕激素联合处理后,分泌细胞易见,腺腔内可见分泌物。激素撤退后实验组T3观察到子宫内膜剥离,T4组示子宫内膜修复。对照组子宫内膜始终完整。子宫湿重在激素撤退后,实验组下降较慢。激素撤退后实验组T3的TUA继续上升而EA则维持原水平,T4组TUA与EA均明显下降。结论此模型在子宫内膜剥落期和早期修复期组织学特征与人类子宫内膜有一定的相似性。     

Antiprogestins as a model for progesterone withdrawal

The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The withdrawal of progesterone is the initiating factor for breakdown of the endometrium. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. By administration of the antiprogestin mifepristone, it is possible to mimic progesterone withdrawal and study local events in early pregnancy decidua that may play a role in the process of early pregnancy failure. Our data indicate that antagonism of progesterone action at the receptor level results in an up-regulation of key local inflammatory mediators, including NF-kappaB, interleukin-8 (IL-8), monocyte chemotactic peptide-1 (MCP-1), cyclooxygenase 2 (COX-2) and others in decidua. Bleeding induced by mifepristone in the mid-luteal phase of the cycle is associated with changes in the endometrium similar to those that precede spontaneous menstruation including up-regulation of COX-2 and down-regulation of PGDH. Administration of antagonists of progesterone provide an excellent model to study the mechanisms involved in spontaneous and induced abortion as well as providing information which may help devise strategies for treating breakthrough bleeding associated with hormonal contraception.     

Induction of overt menstruation in intact mice

The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply.     

New insights into human endometrial aminopeptidases in both implantation and menstruation

The endometrium cycle involves proliferation of endometrial epithelial cells in preparation for implantation of fertilized ovum. With ovulation, the endometrium secretes nutrients such as peptides and amino acids into the endometrial cavity. The histological evidence of ovulation in normal menstrual cycle includes subnuclear glycogen vacuoles surrounded by placental leucine aminopeptidase (P-LAP) in endometrial epithelial cells. P-LAP is an essentially involved in intracellular trafficking of glucose transporter (GLUT) 4 which is primarily important for glucose uptake in skeletal muscles and fat tissues. On the other hand, glucose influx from blood into endometrial epithelial cells is not mainly mediated by GLUTs, but by coincident appearing progesterone just after ovulation. Progesterone increases permeability of not only plasma membranes, but also lysosomal membranes, and this may be primarily involved in glucose influx. Progesterone also expands the exocytosis in the endometrium after ovulation, and endometrial secretion after ovulation is possibly apocrine and holocrine, which is augmented and exaggerated exocytosis of the lysosomal contents. The endometrial spiral arteries/arterioles are surrounded by endometrial stromal cells which are differentiated into decidual/pre-decidual cells. Decidual cells are devoid of aminopeptidase A (APA), possibly leading to enhancement of Angiotensin-II action in decidual cell area due to loss of its degradation by APA. Angiotensin-II is thought to exert growth-factor-like effects in post-implantation embryos in decidual cells, thereby contributing to implantation. Without implantation, angiotensin-II constricts the endometrial spiral arteries/arterioles to promote menstruation. Thus, P-LAP and APA may be involved in homeostasis in uterus via regulating glucose transport and vasoconstrictive peptides.     

Transforming growth factor-beta1 attenuates expression of both the progesterone receptor and Dickkopf in differentiated human endometrial stromal cells

TGFbeta1 is thought to be intimately involved in cyclic tissue remodeling and inflammatory events associated with menstruation. Menstruation is initiated by progesterone withdrawal; however, the underlying mechanisms are not well understood. In the present study, we have tested the hypothesis that locally produced TGFbeta1 may influence expression of progesterone receptor (PR) or the Wnt antagonist Dickkopf-1 (DKK) with consequential impact on regulation of menstruation. Endometrial stromal cells (ESC) were isolated from endometrial biopsy samples collected from patients undergoing gynecological procedures for benign indications. Treatment of differentiated ESC with TGFbeta1 (10 ng/ml) significantly inhibited the expression of mRNAs encoding PR and DKK. TGFbeta1 also attenuated the protein expression of PR and secretion of DKK proteins in culture supernatants. Neutralization of endogenous TGFbeta1 signaling abolished the TGFbeta1-induced effects, significantly increased expression of PR, and increased DKK protein release levels to that of differentiated ESCs, confirming the specificity of the TGFbeta1 effect. Additionally, in vitro decidualization of ESCs significantly augmented DKK protein release. Moreover, although TGFbeta1 was capable of signaling via the Sma- and mothers against decapentaplegic (MAD)-related protein (SMAD) pathway, the inhibitory effect on DKK was SMAD independent. Conversely, the inhibitory effect of TGFbeta1 on PR was dependent on SMAD signal transduction. In conclusion, these results suggest that local TGFbeta1 signaling can potentiate progesterone withdrawal by suppressing expression of PR and may coordinate tissue remodeling associated with menstruation by inducing Wnt-signaling via inhibition of DKK, which we found to be up-regulated as a consequence of decidualization of ESCs.     

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

Temporal relationships among uterine pituitary adenylate cyclase-activating polypeptide, decidual prolactin-related protein and progesterone receptor mRNAs expressions during decidualization and gestation in rats

Pituitary adenylate cyclase-activating peptide (PACAP), a novel compound with vasoactive intestinal polypeptide-like activity, was recently shown to be localized in the neuronal endings of the human uterus. The purpose of the present study was to assess the functional presence of PACAP mRNA in the decidual endometrium and its relationship to the expression levels of decidual prolactin-related protein (dPRP) and the progesterone receptor mRNAs during decidualization and pregnancy in Sprague-Dawley rats. PACAP was constitutively and temporally expressed in the decidual endometrium and gravid uterus. The time-dependent correlated expression levels of PACAP, dPRP and the progesterone receptor were induced by the neurogenic reproductive signals, i.e. the vagino-cervical/deciduogenic stimuli of decidualization and by the normal equivalent stimuli of mating/blastocyst implantation of gestation. Correlation among the mRNA expression levels of PACAP, dPRP and the progesterone receptor and the coordinated inhibitory actions of the anti-progesterone (RU-486) suggest that there is also correlated time-dependent steroid regulation of the mRNA levels of PACAP, dPRP and the progesterone receptor in the decidual and pregnant uteri. One possible functional meaning for the time-related localization of endometrial/uterine PACAP could be to facilitate endometrial blood flow and increase the availability of metabolic substrates to the developing deciduoma or embryo. The study demonstrates the potential importance of PACAP expression in the regulation of the maternal feto-placental component and suggests a prominent reproductive role for the neuropeptide in mammalian pregnancy.