The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome), and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100, when placentation was completed; and at Day 150, when the hydrops syndrome frequently develops. The NT fetuses were compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal cotyledon formation and vascularization of the chorioallantoic membranes was initiated normally in NT conceptuses, but fewer cotyledons successfully formed placentomes. By Day 100, the mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers were represented in surviving NT pregnancies at Day 150. The mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and 150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier than contemporary AI controls. Placentome, liver, and kidney overgrowth accompanied the hydrops syndrome at Day 150. The NT fetal overgrowth was not a consequence of in vitro embryo culture and showed no correlation with placental overgrowth. However, in vitro culture and incomplete reprogramming of the donor genome are epigenetic effects that may override genetic traits and contribute to the greater variability in placental and fetal development in the NT group compared with AI half-siblings.

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [³H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

Quality-control mechanisms in spermatogenesis are important to eliminate injured or abnormal cells, thereby protecting the organism from abnormal development in the next generation. The processes of spermatogenesis are highly sensitive to high temperatures; however, the mechanisms by which injured germ cells are eliminated remain unclear. Here, we found that heat shock proteins are not induced in male germ cells in response to thermal stress, although heat shock transcription factor 1 (HSF1) is activated. Using HSF1-null mice, we showed that apoptosis of pachytene spermatocytes was markedly inhibited in testes with a single exposure to heat and in the cryptorchid testes, indicating that HSF1 promotes apoptotic cell death of pachytene spermatocytes exposed to thermal stress. In marked contrast, HSF1 acts as a cell-survival factor of more immature germ cells, probably including spermatogonia, in testes exposed to high temperatures. These results demonstrate that HSF1 has two opposite roles in male germ cells independent of the activation of heat shock genes.

We investigated the effects of cadmium (Cd² ) on transcription of the cytochrome P450 side chain cleavage (P450scc) gene and on progesterone synthesis in stable granulosa cells. We used the stable porcine granulosa cell line, JC-410, genetically modified to express a luciferase genomic construct carrying 2320 base pairs (bp) of the P450scc gene promoter (P450scc-2320-LUC). A construct containing only the luciferase gene, pOLUC, was used as a promoterless control. At 1 μM, cadmium chloride (CdCl₂) increased transient expression of P450scc-2320-LUC in JC-410 cells by 2.6-fold after 24-h incubation. A similar pattern of stimulation by CdCl₂ was observed in cells transiently transfected with a luciferase genomic construct carrying 100 bp of the P450scc gene promoter P450scc-100-LUC, whereas no stimulation by CdCl₂ was observed in cells transfected with pOLUC. At 0.6, 1, and 2 μM, CdCl₂ stimulated the activity of the P450scc-2320-LUC promoter in a dose-related fashion by 1.58-, 3.19-, and 2.67-fold, respectively, after 24-h incubation. Northern blot analysis showed that CdCl₂ at 0.1, 1, 2, and 3 μM increased P450scc mRNA levels by 3.13-, 1.38-, 1.61-, and 1.57-fold, respectively, after 24-h incubation. After 48-h incubation, CdCl₂ at 0.6, 1, and 2 μM further increased P450scc mRNA levels by 3.43-, 2.08-, and 2.4-fold, respectively. At 1, 2, and 3 μM, CdCl₂ inhibited progesterone synthesis to 0.48-, 0.38-, and 0.29-fold, respectively. After 48-h incubation, CdCl₂ at 0.1 μM stimulated progesterone synthesis by 1.6-fold. We conclude that Cd² has a dual action in stable porcine granulosa cells: Low concentrations activate, whereas high concentrations inhibit, expression of the P450scc gene and progesterone synthesis. The stimulatory effect of Cd² appears to be mediated via a cis-acting element located 100 bp upstream of the P450scc gene transcription start site.

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1) whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2) whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1–100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.

HOXA genes, essential regulators of differentiation in the embryo, are also essential for adult cyclic endometrial development and for endometrial receptivity. Mice deficient in Hoxa10 or Hoxa11 exhibit reduced fertility because of defects in implantation. We hypothesized that HOXC10, HOXC11, HOXD10, and HOXD11, paralogs of HOXA10 and HOXA11, might also be involved in endometrial development. Here, we showed that the expression of HOXC10, HOXC11, HOXD10, and HOXD11 was evident throughout the menstrual cycle in the endometrium by semiquantitative reverse transcription-polymerase chain reaction. In the secretory phase, expression of HOXC10, HOXC11, and HOXD11 decreased to 4% and HOXD10 decreased to 25% of the proliferative phase expression (P < 0.001, P < 0.001, P < 0.02, P < 0.01, respectively). In situ hybridization demonstrated expression of each of these HOX genes primarily in the stroma and confirmed the decreased expression in the secretory phase. HOXC10, HOXC11, HOXD10, and HOXD11 expression was not regulated by sex steroids in primary endometrial stromal cells or Ishikawa cells. The expression and regulation of HOXC and HOXD genes varies from that of HOXA10 and HOXA11. Whereas HOXA10 and HOXA11 are regulators of endometrial differentiation, HOXC and HOXD genes likely regulate endometrial proliferation. Paralogous HOX genes typically have a redundant function in development; a novel evolutionary divergence of paralogous Hox genes has resulted in HOXC and HOXD genes having distinct expression patterns, regulation, and likely also distinct functions from HOXA genes. A network of HOX genes may be involved in regulating multiple aspects of endometrial development, including both proliferation and differentiation.

Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h after reconstruction. Four activation treatments were evaluated at each time point: injection of sperm extract alone, or in combination with 6-dimethylaminopurine (6-DMAP), cytochalasin B, or 1% dimethylsulphoxide. There was no significant difference in normal cleavage rate or average nucleus number of embryos between equine oocytes activated <30 min or at 2 h after reconstruction. The combination of 6-DMAP with sperm extract significantly (P < 0.01) improved cleavage rate compared with the other three treatments. Activation with sperm extract and 6-DMAP 2 h after donor nucleus injection gave the highest cleavage (79%) and the highest cleavage with normal nuclei (40%). Sperm extract and 6-DMAP also effectively activated oocytes parthenogenetically, yielding 83% cleavage and 73% cleavage with normal nuclei. These results indicate that although nuclear remodeling occurs rapidly in equine cytoplasts, early activation does not improve embryonic development after reconstruction.

On the basis of results obtained in vitro, we previously proposed a model in which signals from the conceptus, namely interferon-tau (IFN-τ) and prostaglandin E₂, increase the expression of cyclooxygenase (COX)-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in immune and nonimmune cells of the bovine endometrium. Two experiments were conducted to verify the validity of this hypothesis in vivo. In experiment 1, the in vivo expression of COX-2 and GM-CSF during early pregnancy was monitored. Uteri from heifers were collected at different days (d) of the estrous cycle and pregnancy (P). In experiment 2, the effects of intrauterine infusions of IFN-τ on the expression of COX-2 and GM-CSF were analyzed. Immunohistochemistry was performed on uterine sections, and image analysis was used to evaluate the staining intensity in the conceptus, the luminal epithelium (LE), and the subepithelial stroma. In experiment 1, staining for COX-2 was maximal between d18P and d24P, both in the LE and in the conceptus, whereas staining for GM-CSF reached a plateau between d18P and d30P in the LE. In experiment 2, in response to IFN-τ, COX-2 was up-regulated in the LE of the ipsilateral horn, whereas GM-CSF was enhanced in both uterine horns. The current report supports the view that the conceptus, through its secretion of IFN-τ, stimulates maternal epithelial expression of COX-2 and GM-CSF during the peri-attachment period in the cow.

Progesterone (P₄) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1) examine the in vivo and in vitro effect of P₄ on the binding of OT to its receptor in the ovine endometrium and 2) determine whether the endometrial plasma membranes have high-affinity binding sites for P₄. Ovariectomized ewes were pretreated with a sequence of estradiol-17β (2 days) and P₄ (5 days) before being treated with estradiol-17β plus either vehicle (corn oil), P₄, or P₄ mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P₄/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P₄. Similarly, incubation of endometrial plasma membranes with P₄ (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P₄ was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P₄ and the progestin agonist promegestone (K_d 1.2 × 10⁻⁹ and 1.74 × 10⁻¹⁰M, respectively). Incubation of endometrial plasma membranes with P₄ (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P₄, RU 486, and OT but not by estradiol-17β, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P₄ on the binding of OT to OT receptors in the ovine endometrial plasma membrane.

Spermatogenesis is dependent on a small population of stem cells. Despite the biological significance of spermatogonial stem cells, their analysis has been hampered by their scarcity. However, spermatogonial stem cells can be enriched by selection with an antibody against cell-surface molecules. In this investigation, we searched for new antigens expressed on spermatogonial stem cells. Using the spermatogonial transplantation technique, we examined expression of the CD9 molecule, which is commonly expressed on stem cells of other tissues. Selection of both mouse and rat testis cells with anti-CD9 antibody resulted in 5- to 7-fold enrichment of spermatogonial stem cells from intact testis cells, indicating that CD9 is commonly expressed on spermatogonial stem cells of both species. Therefore, CD9 may be involved in the common machinery in stem cells of many self-renewing tissues, and the identification of a common surface antigen on spermatogonial stem cells of different species has important implications for the development of a technique to enrich stem cells from other mammalian species.

Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (5′-CANNTG-3′) located in the regulatory region of most gene promoters. The rat enhancer of split- and hairy-related protein 2 (SHARP-2) is a member of the bHLH protein family. To analyze the possible role of SHARP-2 in the rat ovary, the regulation of the expression of the SHARP-2 gene was examined, and the SHARP-2 protein was characterized. Northern blot analysis revealed that the level of SHARP-2 mRNA abruptly and temporarily increases as the result of the action of LH, i.e., eCG or hCG treatment alone or hCG after eCG treatment, in the rat ovary, as indicated by the treatment of primary cultured rat granulosa cells with hCG after FSH treatment or of mouse Leydig MA-10 cells with hCG or 8-bromoadenosine 3′,5′-cyclic monophosphate. An in situ hybridization analysis showed that eCG treatment increases the level of the SHARP-2 transcript in theca interna cells and that hCG treatment, after the administration of eCG, increases the level of the SHARP-2 transcript in granulosa cells. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into primary cultured granulosa cells and MA-10 cells revealed that the entire coding sequence of SHARP-2 fused to the GFP is localized in the nucleus. The transcriptional activity of SHARP-2 also was examined using transient DNA transfection experiments. When an expression vector encoding the full length of SHARP-2 was cotransfected with thymidine kinase promoter-luciferase reporter plasmids, with or without E box sequences, into MA-10 cells, the luciferase activity was decreased in an E box-dependent manner. We conclude that the level of SHARP-2 mRNA is regulated by gonadotropins and that SHARP-2 functions as a transcriptional repressor localized in the nucleus.

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 μm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 μg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360 000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

A series of experiments was performed to investigate the influence of progesterone at Days 2 and 3 of pregnancy on conceptus development and uterine capacity. In experiment 1, unilaterally hysterectomized-ovariectomized (UHO) white crossbred gilts were given no treatment, estradiol valerate (5 mg given on Days 11 and 12), or progesterone (200 mg/day on Days 2 and 3 after mating). On Day 105 of pregnancy, each fetus and its associated placenta were weighed, and the number of live and dead fetuses was recorded for each litter. Early progesterone treatment reduced (P < 0.05) litter size (a measure of uterine capacity in UHO gilts). In experiment 2, intact white crossbred gilts were mated, given no treatment or progesterone treatment on Days 2 and 3 of pregnancy, and farrowed. Progesterone treatment decreased (P < 0.05) pregnancy rates. In pregnant gilts, progesterone had no effect on the number of live or stillborn piglets at birth, and gestation length was decreased (P < 0.05). Progesterone treatment did not affect the number of large or small piglets. In experiment 3, intact gilts were mated at estrus and then received 1) no treatment or treatment with 2) 100 mg, 3) 200 mg, or 4) 400 mg mifepristone (also known as RU486) on Day 2 of pregnancy. On Day 11 of pregnancy, both uterine horns were flushed, the number and diameter of each conceptus was recorded, and the flushed material was assayed for total protein and acid phosphatase. The 400 mg mifepristone treatment decreased conceptus diameter (P < 0.05) and total protein (P = 0.06) in the uterine flushings. In experiment 4, UHO gilts were mated at estrus, injected with either corn oil (control) or mifepristone (400 mg) on Day 2 of pregnancy, and killed on Day 105 of pregnancy, and the number and weight of live fetuses and placentas was recorded. In contrast to the effect of progesterone treatment, mifepristone decreased uterine capacity by decreasing the number of small conceptuses. These data suggest that progesterone concentrations on Days 2 and 3 of pregnancy in swine influence the rate of conceptus development during early pregnancy and uterine capacity during later pregnancy.

In cattle and mares, free insulin-like growth factor 1 (IGF-1) is higher in the future dominant follicle (F1) than in the future largest subordinate follicle (F2) before deviation in diameter or selection is manifested between the two follicles. The effect of IGF-1 on other follicular-fluid factors and on the destiny of F2 were studied in two experiments in each species, using a total of 40 heifers and 42 mares. An injection of IGF-1 was made into F2 at the expected beginning of deviation (heifers, F1 ≥ 8.5 mm; mares, F1 ≥ 20.0 mm; Hour 0). In heifers, follicular fluid was taken from F2 at Hours 3, 6, 12, or 24; each heifer was sampled only once. In mares, sequential F2 samples were taken from each mare at Hours 0, 6, and 24 or at Hours 12 and 24. Transvaginal ultrasound guidance was used for treatment and sample collection. In heifers, IGF-1 treatment of F2 stimulated the secretion of estradiol (P < 0.05) between Hours 3 and 6 and androstenedione (P < 0.05) between Hours 3 and 12. In F2 of control heifers, estradiol decreased (P < 0.05) and androstenedione did not change significantly. In mares, IGF-1 treatment of F2 did not affect the concentrations of estradiol during the 24-h posttreatment period; androstenedione decreased (P < 0.04) in the IGF-1 group and increased (P < 0.006) in the controls. Compared with control mares, the IGF-1 group had higher (P < 0.04) activin-A at Hours 12 and 24 and higher (P < 0.0006) inhibin-A at Hour 24. After ablating F1 at Hour 24 in mares, F2 became dominant and ovulated in more mares (P < 0.0002) in the IGF-1 group (12/14) than in the control group (2/14). These results are consistent with reported temporal relationships among follicular factors during deviation in both species and indicate that IGF-1 plays a key role in controlling the temporal relationships; however, no indication was found that IGF-1 stimulated estradiol production in mares during the 24 h after treatment.

Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression. To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait. This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57. Tsp57 encodes a basic protein with a mass of 56.8 kDa. The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human. The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively. Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development. Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development. In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI–VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids. Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids. Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARα null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.

Sry in some varieties of Mus musculus domesticus fails to form normal testis when introduced into the C57BL/6J (B6) strain. We studied the developmental pattern of pre-Sertoli cells that express Sox9 by immunofluorescence and the profile levels of Sox9 transcripts by semiquantitative reverse transcriptase polymerase chain reaction and in situ hybridization in developing gonads of B6-Y^tir mice. Sox9-positive cells (pre-Sertoli cells) appeared in all B6.Y^tir genital ridges at 11.5 and 12.5 days postcoitum (dpc). However, at 13.5 dpc, Sox9-positive cells were not detected only in 50% of the B6.Y^tir gonads compared with 100% of B6 gonads. Although pre-Sertoli cells formed the seminiferous cords after 14.5 dpc in the medial region of the B6.Y^tir gonad, the cranial and caudal regions formed ovarian tissue. Further, B6.Y^tir ovaries have lower levels of Sox9 than ovotestes at all fetal stages. These results suggest that although the pre-Sertoli cell lineage forms in B6.Y^tir genital ridges, its further differentiation into Sertoli cells is apparently prevented. The cause may be the low levels of Sox9 and down-regulation of its product. Results suggest that inhibitory signals of Sox9 acting along the whole genital ridge or only at its cranial and/or caudal regions underlie formation of B6.Y^tir ovaries or ovotestes, respectively. Furthermore, our results suggest that infertility of B6.Y^tir females may be due to the abnormal presence of Sox9 transcripts in their ovaries.

Interleukin-10 (IL-10) is an anti-inflammatory and immune-deviating cytokine expressed in the endometrium and placenta. IL-10 null mutant (IL-10−/−) mice have been employed to examine the role of IL-10 in regulating immune events in early pregnancy and its significance in implantation and pregnancy success. The inflammatory response elicited in endometrial tissue by insemination was amplified in IL-10−/− mice, with a 66% increase in leukocytes in the endometrial stroma on Day 3 of pregnancy. Despite this, no evidence of abnormal type 1/type 2 skewing was seen in T-lymphocytes from lymph nodes draining the uterus. On Day 18 of gestation, IL-10−/− females mated with IL-10−/− males had 15% more implantation sites and 27% more viable fetuses than pregnant wild-type (IL-10 / ) mice. Placental weight was unaffected, but fetal weight and the fetal:placental weight ratio were higher in IL-10−/− pregnancies. Similar data were obtained in allogeneic pregnancies when IL-10−/− females were mated with major-histocompatibility complex (MHC) disparate IL-10−/− males. Pups delivered by IL-10−/− mothers had increased birth weight and followed an altered growth trajectory, with growth impairment evident from early postnatal life into adulthood, which was reflected in alterations in body composition at 14 wk of age. This study shows that although IL-10 is not essential for maternal immune tolerance or successful pregnancy irrespective of MHC disparity in the fetus, maternal IL-10 is a determinant of growth trajectory in progeny in utero and after birth.

Establishment of pregnancy in the pig is accompanied by a localized uterine acute inflammatory response and increase in uterine blood flow. Following rapid trophoblast elongation on Day 12 of pregnancy there is an increase in tissue kallikrein activity and release of bradykinin into the uterine lumen, suggesting the kallikrein-kininogen-kinin system is active in the porcine uterus. The present study investigated endometrial expression and presence of the various factors of the kallikrein-kininogen-kinin system. Endometrial L- and H-kininogen gene expression as well as presence of kininogens in the uterine flushings was evaluated throughout the estrous cycle and early pregnancy in the pig. The possible involvement of plasma kallikrein and Factor XII, activators of the kallikrein-kininogen-kinin system, were evaluated through analysis of gene expression in endometrial and conceptus tissues. Gene expression for plasma kallikrein, Factor XII, and H-kininogen were detected in endometrium but not early conceptus tissues. Factor XII and H-kininogen gene expression were similar across the days of the estrous cycle and early pregnancy. Endometrial plasma kallikrein gene expression was low but increased on Day 15 of the estrous cycle, whereas expression was similar across the days of early pregnancy. In comparison to cyclic gilts, endometrial L-kininogen gene expression increased fourfold on Days 15 and 18 of pregnancy. Both L- and H-kininogen were detected in the uterine flushings of cyclic and pregnant gilts. Presence of L- and H-kininogen in the porcine uterus and endometrial gene expression of plasma kallikrein and Factor XII provide evidence that the kallikrein-kininogen-kinin system is biologically active during establishment of pregnancy in the pig.

Various molecular interactions not operating in other cell types are most likely required for mammalian oocytes to develop into fully competent eggs. This study seeks to initiate analyses of the potential oocyte-specific functions of regulators of G1/S progression—CDK4, CDK6, D-type cyclins, and p27—by first determining their expression patterns in growing and maturing mouse oocytes and in mouse embryos early after fertilization. Western blot and immunofluorescence analyses on isolated oocytes were employed to evaluate both their levels and their localization. The data show that 1) mouse oocytes contain significant amounts of all studied regulators; 2) their amounts and localization undergo dramatic changes as the oocytes grow, meiotically mature, and transit into embryogenesis; and 3) some regulators (CDK4, CDK6, cyclin D2, and p27) appear in unusual, most likely posttranslationally modified, forms. These data distinguish G1/S regulators as the potential players in molecular processes that are important for oocytes to function normally.

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.

The function of cyclin B1 (CB1) and cyclin B2 (CB2) during porcine oocyte maturation was investigated by injecting oocytes with their antisense RNAs (asRNAs). At first, protein levels of both cyclin Bs were examined by immunoblotting, revealing that immature oocytes had only CB2, at a level comparable to 1/20 to 1/40 of that detected in first metaphase oocytes. Both cyclin B syntheses were started around germinal vesicle breakdown (GVBD); CB1 and CB2 peaked at the second metaphase and first metaphase, respectively. We obtained a porcine CB2 cDNA fragment, which was 88% homologous with human CB2, by reverse-transcriptase polymerase chain reaction (RT-PCR) using total RNAs of immature porcine oocytes and a primer set of human CB2. Specific asRNAs of CB1 and CB2 were prepared in vitro. Then one, the other, or both were injected into the cytoplasm of immature oocytes. CB1 asRNA inhibited CB1 synthesis specifically; the injected oocytes underwent first meiosis normally but could not arrest at the second meiotic metaphase. CB2 asRNA inhibited CB2 synthesis specifically, but had almost no effect on the maturation of injected oocytes. When both CB1 and CB2 asRNAs were injected, synthesis of both cyclin Bs was inhibited, and GVBD was significantly suppressed but occurred slowly. These results suggest that CB1 is the principal molecule for regulation in mammalian oocyte maturation, whereas CB2 has only an accessory role. They also show that in porcine oocytes, cyclin B synthesis is not necessary for GVBD induction itself, but synthesis of at least one cyclin B, CB1 or CB2, is necessary for GVBD induction in a normal time course.

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5′ regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.

The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) α- and β-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-β loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-α immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-β were absent. The cellular localization of PDGF-A within the epithelium and the α-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-α system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR α- and β-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-α mRNAs were seen by reverse transcription–polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-β were almost not detectable. During the first days of life, PDGF-B and PDGFR-β genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-β, and PDGFR-α were constant with the only exception of a progressive decrease of PDGFR-α in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-β −/− mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

Estrogen induces proliferation of uterine epithelium through a paracrine action of estrogen receptor (ERα) in the underlying stroma. In ovariectomized mice primed with progesterone, estrogen stimulates proliferation in both the epithelium and the stroma. We set out to test whether a paracrine mode of action is involved in estrogen-induced proliferation of the uterine stroma. Epithelial and mesenchymal tissues derived from uteri of neonatal ERα null mice (ERαKO) or wild-type mice were separated and recombined in all four possible configurations (ERα or ERα⁻ epithelium with ERα or ERα⁻ mesenchyme) and grafted into female athymic mice. After 5 wk, hosts were ovariectomized and challenged with hormone treatment, and cellular proliferation was monitored by thymidine autoradiography. Results showed that, although the full response of the epithelium was dependent on an ERα-positive mesenchyme, stromal cell proliferation was independent of tissue ERα. This latter observation suggests that the response of the stroma was due to a systemic factor induced in the ERα-positive hosts. To test this possibility, pieces of whole uterus from neonatal wild-type or ERαKO mice were grafted into syngeneic wild-type or ERαKO hosts. In these whole-uterus grafts, estradiol stimulated ERαKO uterine stroma when they were grown in wild-type hosts but not when grown in ERαKO hosts. The epithelium of whole-uterus ERαKO grafts did not respond to estrogen, regardless of the host phenotype. These observations suggest that treatment of progesterone-primed mice with estradiol stimulates production of a systemic factor that is capable of inducing uterine stromal cell proliferation and that this systemic factor is produced by an ERα-dependent mechanism.

The possible participation of capsaicin-sensitive sensory nerves in the modulation of neurogenic contractions was studied in nonpregnant and term pregnant rat uteri. Neurogenic contractions were elicited by electric field stimulation (40 V, 1–70 Hz, 0.6 msec) in intact uteri and uteri that were previously exposed to capsaicin in vitro. In capsaicin pretreated preparations obtained both from nonpregnant and term pregnant rats, a dose-dependent increase in the amplitude of uterine contractions was detected. Prior systemic treatment of the rats with capsaicin (130 mg/kg, s.c.) abolished the effect of in vitro capsaicin administration on the amplitude of neurogenic contractions. Use of a specific antagonist of calcitonin gene-related peptide revealed that depletion of this peptide, which normally elicits uterine smooth muscle relaxation, may be responsible for the increased responsiveness of the uterus to low-frequency stimulation. Experiments on the localization of calcitonin gene-related peptide in uterine tissue specimens exposed to capsaicin revealed dose-dependent depletion of calcitonin-gene related peptide-immunoreactive nerves innervating blood vessels and the myometrium. The findings indicate that capsaicin-sensitive afferent nerves, by the release of sensory neuropeptides, significantly contribute to the modulation of uterine contractility both in nonpregnant and term pregnant rats. It is suggested that uterine sensory nerve activation may be part of a trigger mechanism leading to preterm contractions evoked by, for example, inflammation.

Tumor necrosis factor-α (TNFα) has been shown to be a potent stimulator of prostaglandin (PG) F_2α synthesis in bovine endometrial stromal cells. The aims of the present study were to determine the effect of interferon-τ (IFNτ) on TNFα-stimulated PGF_2α synthesis and the intracellular mechanisms of TNFα and IFNτ action in the stromal cells. When cultured bovine stromal cells were exposed to TNFα (0.006–0.6 nM) for 24 h, the production of PGF_2α and cyclooxygenase (COX)-2 gene expression were stimulated by TNFα (0.06–0.6 nM, P < 0.05). Moreover, a specific COX-2 inhibitor (NS-398; 5 nM) blocked the stimulatory effect of TNFα on PGF_2α production (P < 0.05). Although IFNτ (0.03–30 ng/ml) did not stimulate basal PGF_2α production in the stromal cells, it suppressed TNFα action in PGF_2α production dose dependently (P < 0.05). Moreover, the stimulatory effect of TNFα (0.6 nM) on COX-2 gene expression was completely blocked by IFNτ (30 ng/ml; P < 0.05), although the gene expression of COX-2 was not influenced by IFNτ. The overall results indicate that the stimulatory effect of TNFα on PGF_2α production is mediated by the up-regulation of COX-2 gene expression and suggest that one of the mechanisms of the inhibitory effect of IFNτ on luteolysis is the inhibition of TNFα action in PGF_2α production in the stromal cells by the down-regulation of COX-2 gene expression stimulated by TNFα.

The oviduct contributes to the reproductive environment by secreting various factors, including a family of glycoproteins termed oviductins. Although many studies have demonstrated that ovarian hormones modulate oviductin gene expression in several mammalian species, there has been controversy surrounding the regulation of golden hamster oviductin. The current study was undertaken to investigate the transcriptional and translational modifications of hamster oviductin during the estrous cycle. First, we verified that hamster oviductin mRNA expression remains constant throughout the estrous cycle by semiquantitative reverse transcription polymerase chain reaction. We then developed a polyclonal antibody against recombinant hamster oviductin (rhaOv_m). The anti-rhaOv_m antibody was subsequently used in conjunction with quantitative immunocytochemistry to investigate the oviductin levels in the hamster oviduct during the estrous cycle. Quantification of immunolabeling revealed a high, consistent level of glycoprotein throughout the estrous cycle. Therefore, it appears that the production of oviductin is not regulated differentially during the estrous cycle. Size variations in hamster oviductin expression were also investigated by Western blot analysis. The oviduct contains several forms of oviductin at each stage of the estrous cycle, the native glycosylated form(s) of 160–350 kDa, and several precursor forms of 70–100 kDa. Although variations in the intensities of the polydispersed band were not evident during the estrous cycle, additional bands ranging from 90 to 100 kDa were detected in the estrus, metestrus, and diestrus 1 stages. The results from the present investigations suggest that whereas ovarian hormones do not appear to influence the hamster oviductin mRNA and protein expressions, glycosylation of hamster oviductin appears to be differentially regulated during the estrous cycle.

We have cloned the rat homologue of the ring-H2 protein Goliath involved in Drosophila development. The rat Goliath mRNA (1.85 kb) was translated as a major ubiquitous protein species of 28-kDa and three larger isoforms (50, 46, and 36 kDa) expressed mainly in liver, lung, stomach, heart, and thymus and barely detectable in other tissues (kidney, skeletal muscle, brain, testis, intestine, and spleen). By immunohistochemistry on rat testis sections, we localized the protein in interstitial tissue and seminiferous tubules. In tubules, Goliath was expressed mainly in postmeiotic germ cells and to a much lesser extent in Sertoli cells. In the interstitium, Goliath was exclusively present in Leydig cells. Using a series of immunolabeling, cellular fractionation, and electron microscopy experiments, we established that Goliath is present in mitochondria of the R2C Leydig cell line. Using short-term hypophysectomized animals, we showed that Goliath is regulated by LH/hCG in Leydig cells but not in germ cells. This regulation in Leydig cells concerned only the 50-kDa isoform. This report is the first description of a differential regulation of the Goliath protein between germ cells and Leydig cells.

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.

The peptide hormone relaxin, which attains high circulating levels during pregnancy, has been shown to depress small-bowel motility through a nitric oxide (NO)-mediated mechanism. In the present study we investigated whether relaxin also influences gastric contractile responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 μg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. Evaluation of the expression of nitric oxide synthase (NOS) isoforms was performed by immunohistochemistry and Western blot. In control mice, neurally induced contractile responses elicited by electrical field stimulation (EFS) were reduced in amplitude by addition of relaxin to the organ bath medium. In the presence of the NO synthesis inhibitor l-NNA, relaxin was ineffective. Direct smooth muscle contractile responses were not influenced by relaxin or l-NNA. In strips from relaxin-pretreated mice, the amplitude of neurally induced contractile responses was also reduced in respect to the controls, while that of direct smooth muscle contractions was not. Further addition of relaxin to the bath medium did not influence EFS-induced responses, whereas l-NNA did. An increased expression of NOS I and NOS III was observed in gastric tissues from relaxin-pretreated mice. In conclusion, the peptide hormone relaxin depresses cholinergic contractile responses in the mouse gastric fundus by up-regulating NO biosynthesis at the neural level.

We tested the hypothesis that leptin could prevent fasting-mediated reductions in pulsatile secretion and modify GnRH-mediated release of LH in heifers approaching puberty. Thirteen crossbred, prepubertal heifers (13.5–16 mo; 280–350 kg) exhibiting frequencies of pulses of LH between 0.67 and 1 pulse/h, were assigned randomly to two groups: 1) control (n = 6), fasted for 72 h with s.c. injections of saline at 12-h intervals, and 2) leptin (n = 7), fasted for 72 h with s.c. injections of oleptin (19.2 μg/kg) at 12-h intervals. Blood samples were collected intensively for 6 h on Days 0 and 3. This was followed on Day 3 with sequential administration of physiological (0.0011 μg/kg, i.v.) and pharmacological (0.22 μg/kg, i.v.) doses of GnRH and additional blood sampling. Leptin treatment increased (P = 0.0003) plasma concentrations of leptin 5–6-fold compared to controls. Fasting caused a marked decline (P = 0.01) between Days 0 and 3 in the frequency of LH pulses in controls; however, this effect was prevented in the leptin group, with pulse frequency increasing (P < 0.008) from Day 0 to 3. Leptin treatment increased GnRH-induced release of LH at both low (P = 0.04) and high (P = 0.02) doses. Plasma insulin and insulin-like growth factor-1 were reduced by fasting and unaffected by leptin. Leptin increased mean concentrations of growth hormone. Results indicate, for the first time, that exogenous leptin can prevent fasting-mediated reductions in the frequency of LH pulses and modify GnRH-mediated release of LH in intact, prepubertal heifers.

LH consists of α- and β-subunits, and synthesis of the β-subunit has been reported to be the rate-limiting step in LH production. In this study, we found that activin A increased both the LHβ mRNA level and LH content in cells of the gonadotroph cell line, LβT2. We next examined the effects of activin A and GnRH on LHβ promoter activity by reporter gene assay and compared the signal transduction pathways. Activin A and GnRH activated the LHβ promoter, and the response to a combination of activin A and GnRH was higher than that to activin A or GnRH alone. The effects of activin A and GnRH were specifically inhibited by inhibin-like peptide and antide, a GnRH antagonist, respectively. The activation of the LHβ promoter by GnRH was inhibited by PD098059 and U0126, MAP kinase kinase (MEK) inhibitors. In contrast, these protein kinase inhibitors did not inhibit the activin A-induced activation. GnRH, but not activin A, activated MAP kinase in LβT2 cells. Overexpression of constitutively active MEK1 or MEK kinase activated both MAP kinase and the LHβ promoter. Furthermore, GnRH, but not activin A, strongly induced SRE-mediated transcription, a known target of the MAP kinase pathway. These results suggest that GnRH activates the LHβ promoter via the MAP kinase pathway and that activin A-induced activation of the LHβ promoter is independent of the MAP kinase pathway.

The factors regulating the dynamic expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the primate corpus luteum (CL) during the menstrual cycle are unknown. We hypothesized that LH or progesterone (P) regulate interstitial-collagenase (MMP-1), the gelatinases (MMP-2 and -9), TIMP-1, and TIMP-2 in the CL. Hormone ablation/replacement was performed in rhesus monkeys on Days 9–11 of the luteal phase in five treatment groups (n = 4/group): control (no treatment), antide (GnRH antagonist), antide LH; antide LH trilostane (TRL; 3β-hydroxysteroid dehydrogenase inhibitor), and antide LH TRL R5020 (nonmetabolizable progestin). On Day 12, the CL was removed and the RNA and protein isolated for real-time polymerase chain reaction and immunoassays, respectively. The MMP-1 mRNA increased 20-fold with antide, whereas LH replacement maintained MMP-1 mRNA at control levels. Likewise, TRL increased MMP-1 mRNA 54-fold, and R5020 prevented this effect. Immunodetectable MMP-1 protein also increased with antide or TRL; these increases were abated with LH or R5020. Gelatinase mRNA and/or protein levels increased with antide (e.g., 3-fold, MMP-2 mRNA), and LH replacement reduced protein levels (e.g., 11-fold, MMP-2). The TRL increased MMP-9, but not MMP-2, expression; however, R5020 replacement had no effect on mRNA or protein levels. The LH treatment increased TIMP-1 and -2 mRNA and TIMP-1 protein expression compared to controls and antide groups, whereas R5020 enhanced only immunodetectable TIMP-1. These data strongly suggest that LH suppresses MMP-1 in the primate CL via P and that it also suppresses gelatinases, either at the mRNA (MMP-2) or protein (MMP-2 and -9) levels, perhaps in part via steroids, including P. In contrast, LH promotes TIMP expression, perhaps via steroids, including P.

Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased (∼4-fold) with term labor in amnion (n = 15), and with preterm labor (∼30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r² = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1β and tumor necrosis factor α. Production of ENA-78 by choriodecidual explants was increased modestly after 2–4 h of exposure to lipopolysaccharide (5 μg/ml). An immunoreactive doublet (∼8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.