Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life. The objective of this research was to xenograft testis tissue from multiple ages of bull calves for 24 or 36 wk in order to gain a better understanding of early bovine testis development. Testis tissue from 1-, 2-, 4-, and 8-wk-old calves was grafted onto the backs of castrated immunodeficient mice. Testis tissue from all donor ages grew, differentiated, and produced testosterone and elongating spermatids. Testis tissue grafts from 1- and 8-wk-old calves had elongating spermatids in greater than 5.5% of seminiferous tubule cross sections at the time of graft removal regardless of grafting period. Four-week-old donor tissue never had more than 5.2% of seminiferous tubule cross sections with elongating spermatids. Extending the grafting period from 24 to 36 wk resulted in an increase in the percentage of seminiferous tubule cross sections with elongating spermatids from 2% to 10% in 2-wk donor tissue. These data demonstrate that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting.

Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 μg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.

Endometriosis, the presence of a functional endometrium outside of the uterine cavity, is associated with infertility. In our simulated model of pregnancy in baboons with experimental endometriosis, hCG infusion fails to induce expression of the immunoregulatory protein glycodelin. To test the hypothesis that the development of endometriosis is associated with an aberrant endometrial immunological environment, we examined the expression of a series of immunoregulatory genes in endometrium from baboons with and without endometriosis. Six months following intraperitoneal inoculation with menstrual endometrium, eutopic endometrium was surgically collected between Days 9 and 11 postovulation. Control endometrium was similarly collected from disease-free animals. Total RNA was extracted, and biotinylated cDNA probes were hybridized to the SuperArray GEArray Q series Th1/Th2/Th3 cDNA array, representing 96 genes. Gene expression levels were determined using ScanAlyze and GEArray Analyzer software. Seven genes were upregulated, including JUND, FOS, CCL11, NFKB1 and others, in the endometrium from baboons with endometriosis compared with the endometrium from disease-free animals; one gene, IL1R1, was downregulated. Quantitative RT-PCR confirmed upregulation of FOS and CCL11 in endometriotic eutopic endometrium. Immunohistochemical analysis revealed altered levels and distribution of FOS protein in the eutopic endometrium of baboons with induced endometriosis. These data suggest that in an induced model of endometriosis an aberrant eutopic immunological environment results in a decreased apoptotic potential and in rapid alterations in endometrial gene expression. We propose that the reduced fecundity associated with endometriosis has a multifold etiology in spontaneous and induced disease.

Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 ± 0.42 fmol of immunoreactive ADM per milligram of protein and 84 ± 8 fg of ADM mRNA per picogram of Actb (β-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22–52) and by human calcitonin gene-related peptide (8–37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22–52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis.

A-kinase anchor proteins (AKAPs) spatially restrict cAMP-dependent protein kinase by tethering it to various cellular structures. In the polarized sperm cell, various compartmentalized functions, such as motility generated by the flagellum, are modulated by cAMP-dependent protein kinase. This important regulatory enzyme is associated with AKAP4, the principal component of the fibrous sheath; AKAP4 is synthesized as a precursor, pro-AKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. To define the domains responsible for the intracellular distribution and assembly of AKAP4 into a macromolecular complex, various AKAP4-green fluorescent protein (GFP) constructs were introduced into somatic cell lines. The presence of the pro domain, either alone or as part of pro-AKAP4, resulted in a diffuse cytoplasmic localization of the GFP fusion protein, suggesting that, the pro domain keeps the AKAP4 precursor unassembled in vivo until it is transported to the developing tail structure and incorporated into the fibrous sheath. When the mature AKAP4-GFP fusion protein was expressed, it localized in a punctate cytoplasmic pattern. Two domains critical for this punctate localization, T_2a and T_2b, are homologous to the T₂-tethering domain of rat AKAP5 that is important for binding to the actin cytoskeleton in transfected HEK293 cells. In contrast to AKAP5, the distribution of AKAP4 was dependent on the microtubular cytoskeleton. The interaction of AKAP4 with the microtubular network provides evidence that the longitudinal columns of the fibrous sheath, which contain AKAP4, may interact directly with the outer microtubular doublets of the sperm axoneme.

Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.

Human endometrium is a dynamic tissue under the influence of numerous hormones, growth factors, and cytokines interacting to maintain a balance of cellular growth, differentiation, and apoptosis. We have previously demonstrated that several factors including interleukin-8, extracellular matrix, and steroid hormones modulate FASLG, one of the apoptotic molecules, in human endometrium. Chemokine ligand 2 (CCL2), a monocyte chemoattractant and activating factor, is a cytokine involved in endometrial function. CCL2 is elevated in the peritoneal fluid of women with endometriosis. We hypothesize that increased levels of CCL2 in the endometriotic environment may upregulate FASLG expression in human endometrial stromal cells and induce a local immunotolerance in endometriosis. To test our hypothesis, we studied the in vitro regulation of FASLG expression and apoptosis by CCL2 in endometrial stromal cells. Western blot analysis revealed that CCL2 upregulated FASLG protein expression in cultured endometrial stromal cells. Based on semiquantitative RT-PCR analysis, CCL2 did not alter either FAS or FASLG mRNA expression in endometrial stromal cells. Immunocytochemistry results from the same cells treated with CCL2 demonstrated upregulation of FASLG protein expression. CCL2 did not change rate of apoptosis in endometrial stromal cells as evaluated by TUNEL assay. However, an increased apoptotic rate was detected in Jurkat (T lymphocytes) cells cocultured with endometrial stromal cells previously treated with CCL2. We speculate that increased FASLG expression by CCL2 may induce apoptosis of T lymphocytes and thus produce an immunotolerant environment for the development of ectopic implants.

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.

Steroidal regulation of gene expression in follicular cells is not completely defined. Granulosa cells from 5 mm bovine follicles were cultured and treated and steady-state mRNA levels determined for FSHR (follicle-stimulating hormone receptor) and CYP19A1 (aromatase). Cells were treated for 5 days with (0.1–300 ng/ml) 17beta-estradiol (E₂), testosterone (T), or 5alpha-dihydrotestosterone (DHT). FSHR mRNA was increased by T and DHT but not E₂. In contrast, CYP19A1 mRNA was induced by all doses of E₂ but only high doses of T and DHT. Similarly, varying treatment duration (1–5 days) showed that FSHR was increased by T and DHT and CYP19A1 mRNA increased by E₂ and T at all times. Synergism between steroid hormones and FSH or forskolin was also evaluated. FSH or E₂ did not alter FSHR mRNA and did not enhance DHT stimulation of FSHR mRNA. In contrast, DHT alone had no effect on CYP19A1 mRNA but synergized with FSH plus E₂ to increase CYP19A1 mRNA, probably due to induction of FSHR by DHT. Effects of E₂ and T on CYP19A1 were blocked by ICI 182,780, indicating mediation by estrogen receptors. However, the specific androgen receptor antagonist bicalutamide did not block E₂ or T effects on CYP19A1 but did block T and DHT stimulation of FSHR. Thus, FSHR is specifically regulated through androgen receptor, whereas CYP19A1 is regulated by multiple pathways, including estrogen receptors and cAMP/protein kinase A induced by FSHR activation in granulosa cells. These inter- and intracellular regulatory mechanisms may be critical for normal follicle growth and dominant follicle selection.

Ithas been notoriously difficult to successfully cryopreserve swine embryos, a task that has been even more difficult for in vitro-produced embryos. The first reproducible method of cryopreserving in vivo-produced swine embryos was after centrifugation and removal of the lipids. Here we report the adaptation of a similar process that permits the cryopreservation of in vitro-produced somatic cell nuclear transfer (SCNT) swine embryos. These embryos develop to the blastocyst stage and survive cryopreservation. Transfer of 163 cryopreserved SCNT embryos to two surrogates produced 10 piglets. Application of this technique may permit national and international movement of cloned transgenic swine embryos, storage until a suitable surrogate is available, or the long-term frozen storage of valuable genetics.

In the overnourished adolescent sheep, maternal tissue synthesis is promoted at the expense of placental growth and leads to a major decrease in lamb birth weight at term. Maternal growth hormone (GH) concentrations are attenuated in these pregnancies, and it was recently demonstrated that exogenous GH administration throughout the period of placental proliferation stimulates uteroplacental and fetal development by Day 81 of gestation. The present study aimed to determine whether these effects persist to term and to establish whether GH affects fetal growth and body composition by increasing placental size or by altering maternal metabolism. Adolescent recipient ewes were implanted with singleton embryos on Day 4 postestrus. Three groups of ewes offered a high dietary intake were injected twice daily with recombinant bovine GH from Days 35 to 65 of gestation (high intake plus early GH) or from Days 95 to 125 of gestation (high intake plus late GH) or remained untreated (high intake only). A fourth moderate-intake group acted as optimally nourished controls. Pregnancies were terminated at Day 130 of gestation (6 per group) or were allowed to progress to term (8–10 per group). GH administration elevated maternal plasma concentrations of GH, insulin, glucose, and nonesterified fatty acids during the defined treatment windows, while urea concentrations were decreased. At Day 130, GH treatment had reduced the maternal adiposity score, percentage of fat in the carcass, and internal fat depots and leptin concentrations, predominantly in the high-intake plus late GH group. Placental weight was lower in high-intake vs. control dams but independent of GH treatment. In contrast, fetal weight was elevated by late GH treatment, and these fetuses had higher relative carcass fat content, perirenal fat mass, and liver glycogen concentrations than all other groups. Expression of leptin mRNA in fetal perirenal fat and fetal plasma leptin concentrations were not significantly altered by maternal nutritional intake or GH. In pregnancies proceeding to term, the duration of gestation, fetal placental mass, and lamb birth weight were reduced in high-intake compared with control dams but were not significantly affected by GH treatment. In conclusion, exogenous GH has profound effects on maternal endocrinology, metabolism, and body composition when administered during early and late pregnancy. Treatment during late pregnancy has a modest effect on fetal growth independent of placental size and a profound effect on fetal adiposity, which may have implications beyond the fetal period.

We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.

In mammals, removal of one testis results in compensatory testicular hypertrophy (CTH) of the remaining gonad. Although CTH is ubiquitous among juveniles of many species, laboratory rats, laboratory mice, and humans unilaterally castrated in adulthood fail to display CTH. We documented CTH in pre- and postpubertally hemi-castrated Syrian and Siberian hamsters and tested whether day length affects CTH in juvenile and adult Siberian hamsters. Robust CTH was evident in long-day hemi-castrates of both species and was preceded by increased serum FSH concentrations in juvenile Siberian hamsters. In sharp contrast, CTH was undetectable in short-day hemi-castrated Siberian hamsters for several months and only made its appearance with the development of neuroendocrine refractoriness to short day lengths; serum FSH concentrations of juveniles also did not increase above sham-castrate values until the onset of refractoriness. Long-day hemi-castrated Siberian hamsters with hypertrophied testes underwent complete gonadal regression after transfer to short days, albeit at a reduced rate for the first 3 weeks of treatment. Blood testosterone concentrations of adult hamsters did not differ between long-day hemicastrates and sham-castrates 9–12 weeks after surgery. We conclude that CTH is suppressed by short day lengths in Siberian hamsters at all ages and stages of reproductive development; in short day lengths, but not long day lengths, the remaining testis produces sufficient negative feedback inhibition to restrain FSH hypersecretion and prevent CTH.

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

Peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth and development are primarily regulated by secretions from the uterus. This study investigated the effects of progesterone on preimplantation conceptus development and endometrial galectin 15 (LGALS15). Ewes received daily injections of either corn oil (CO) vehicle or 25 mg progesterone (P4) from 36 h postmating to hysterectomy. Treatment with P4 increased blastocyst diameter by 220% on Day 9 and advanced time of elongation of blastocysts to a filamentous conceptus on Day 12. Effects of P4 treatment on blastocyst development were blocked by administration of RU486, a progesterone receptor antagonist. Consistent with early elongation of blastocysts, interferon tau (IFNT) protein was about 50-fold greater in uterine flushes from Day 12 in ewes receiving P4 compared with those receiving CO. Expression of cathepsin L (CTSL) and radical S-adenosyl methionine domain containing 2 (RSAD2), both IFNT-stimulated genes, was increased in endometria of Day 12 P4-treated ewes. LGALS15 mRNA, expressed only in the endometrial luminal epithelium and superficial glands, was detected between Days 9 and 12 and was more abundant in ewes receiving P4 than in those receiving CO on both Days 9 and 12. RU486 treatment ablated P4 induction of LGALS15 mRNA in the endometrial epithelia. LGALS15 protein in uterine flushings was not different on Day 9 but tended to be greater in P4-treated ewes than in those receiving CO on Day 12. The advanced development of blastocysts in P4-treated ewes is hypothesized to involve early induction of specific genes in the endometrial epithelia, such as LGALS15, and undoubtedly components of uterine histotroph.

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1–4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor.

All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes.

Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.