[Clinical characteristics of 272 437 patients with different histopathological subtypes of primary esophageal malignant tumors].

Objective: To characterize the histopathological subtypes and their clinicopathological parameters of gender and onset age by common, rare and sparse primary esophageal malignant tumors (PEMT). Methods: A total of 272 437 patients with PEMT were enrolled in this study, and all of the patients were received radical surgery. The clinicopathological information of the patients was obtained from the database established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment from September 1973 to December 2020, which included the clinical treatment, pathological diagnosis and follow-up information of esophagus and gastric cardia cancers. All patients were diagnosed and classified by the criteria of esophageal tumor histopathological diagnosis and classification (2019) of the World Health Organization (WHO). The esophageal tumors, which were not included in the WHO classification, were analyzed separately according to the postoperative pathological diagnosis. The χ2 test was performed by the SPSS 25.0 software on count data, and the test standard α=0.05. Results: A total of 32 histopathological types were identified in the enrolled PEMT patients, of which 10 subtypes were not included in the WHO classification. According to the frequency, PEMT were divided into common (esophageal squamous cell carcinoma, ESCC, accounting for 97.1%), rare (esophageal adenocarcinoma, EAC, accounting for 2.3%) and sparse (mainly esophageal small cell carcinoma, malignant melanoma, etc., accounting for 0.6%). All the common, rare, and sparse types occurred predominantly in male patients, and the gender difference of rare type was most significant (EAC, male∶ female, 2.67∶1), followed with common type (ESCC, male∶ female, 1.78∶1) and sparse type (male∶ female, 1.71∶1). The common type (ESCC) mainly occurred in the middle thoracic segment (65.2%), while the rare type (EAC) mainly occurred in the lower thoracic segment (56.8%). Among the sparse type, malignant melanoma and malignant fibrous histiocytoma were both predominantly located in the lower thoracic segment (51.7%, 66.7%), and the others were mainly in the middle thoracic segment. Conclusion: ESCC is the most common type among the 32 histopathological types of PEMT, followed by EAC as the rare type, and esophageal small cell carcinoma and malignant melanoma as the major sparse type, and all of which are mainly occur in male patients. The common type of ESCC mainly occur in the middle thoracic segment, while the rare type of EAC mainly in the lower thoracic segment. The mainly sparse type of malignant melanoma and malignant fibrous histiocytoma predominately occur in the lower thoracic segment, and the remaining sparse types mainly occur in the middle thoracic segment.

Fatty acid composition differences between adipose depot sites in dairy and beef steer breeds.

The objective of the study was to compare fatty acid composition of longissimus dorsi (LD) and kidney fat (KF) in Holstein steers (HS), Simmental steers (SS) and Chinese LongDong Yellow Cattle steers (CLD). All steers received the same nutrition and management but in different locations. Cattle were harvested at approximately 550 kg and fatty acid composition of longissimus dorsi and kidney fat was analyzed in samples taken after 3 days of aging. There was evidence (P < 0.05) that C18:3n6 was greater in KF than LD in CLD cattle but not in HS or SS cattle. Percentage C18:1n9, C18:2n6, C18:3n3, and n6 fatty acids were greater in LD than KF for all breeds (P < 0.05), but the difference between fat sources for n6 in CLD cattle was smaller than the other two breeds. The LD had greater percentage of polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), and a greater ratio of n6:n3 PUFAs compared to the KF in each breed (P < 0.05). The △(9)-desaturase catalytic activity index was greater in LD than in KF in each breed group (P < 0.05). Percentage cis-9, trans-11 CLA was greater in KF than LD in HS (P < 0.05) but not SS or CLD cattle. These results indicate fatty acid percentages generally differed between longissimus dorsi fat and kidney fat. Further, there was some indication that some of these differences between fatty acid deposition sites were not consistent across breed group.

The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice.

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5alpha-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

The past, present and future of nongonadal LH/hCG actions in reproductive biology and medicine.

The past and present published studies reaffirm that nongonadal LH and hCG actions are real and here to stay. These actions have led to a better understanding of the biology of the hormones and more importantly begin to pave the way for novel therapies in reproductive medicine and in other areas.

Selective deletion of Pten in theca-interstitial cells leads to androgen excess and ovarian dysfunction in mice.

Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis.

Targeted disruption of LH receptor gene revealed the importance of uterine LH signaling.

Numerous previous studies have demonstrated that LH and hCG can directly regulate several uterine functions. We investigated in the present study, whether uterine phenotype in LH receptor knockout animals resulted also from the absence of direct actions of LH in the uterus. The phenotype consisted of marked growth reduction of uterus, decreased thickness of endometrial and myometrial layers, number of endometrial glands, height of luminal epithelium and vascular space. Analysis of uterine gene expression by mouse genome U74Av2 Affymetrix genechips revealed a differential expression of 155 genes by more than 3-fold (range 3-53-fold) between null and wild-type animals. Of these, 89 genes decreased and 66 increased in uterus of null animals. Semi-quantitative RT-PCR confirmed the differential expression of several selected genes. The decreased genes can be clustered into 18 functional families and the increased into 15 functional families. Semi-quantitative RT-PCR, Western blotting and immunocytochemistry demonstrated a decreased expression of ERbeta, PR-A, PR-B and AR in uterus of null animals as compared with wild-type siblings. Twenty-one-day estradiol and progesterone replacement therapy did not normalize the decrease in the number of endometrial glands and several genes that either decreased or increased in expression. The partial success of therapy suggests that direct LH actions could be required to completely normalize the uterus. In summary, findings on the knockout model reaffirm that LH and hCG control uterine functions directly as well as indirectly through increasing ovarian synthesis of steroid hormones and both actions are required for normal uterine biology.

Novel presence of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and the down-regulating action of hCG on gonadotropin-releasing hormone gene expression in immortalized hypothalamic GT1-7 neurons.

We recently demonstrated that rat preoptic area and anterior hypothalamus, sites of GnRH neurons, contain receptors for LH/hCG. We investigated in the present study whether LH/hCG receptor and GnRH genes are coexpressed in the same neurons and whether LH/hCG can directly regulate GnRH gene expression in immortalized hypothalamic GT1-7 neurons. The immunostaining for both LH/hCG receptors and GnRH are present in the same neurons in rat preoptic area and the GT1-7 neurons. The reverse transcription-nested polymerase chain reaction generated an expected 255-basepair LH/hCG receptor fragment in GT1-7 neurons. Northern blotting showed the presence of a major 1.8-kilobase and minor 2.6- and 4.3-kilobase receptor transcripts. Immunoblotting detected an 80-kilodalton receptor protein. Covalent receptor cross-linking studies showed that [125I]hCG binds to an 80-kilodalton protein with a specificity expected of LH/hCG receptors. Scatchard plot analysis demonstrated that GT1-7 neurons contain a single class of high affinity (Kd = 3.8 x 10(-11) M) and low capacity (5000 sites/neuron) LH/hCG receptors. Culturing GT1-7 neurons with highly purified hCG resulted in a dose- and time-dependent decrease in steady state GnRH, but not glyceraldehyde-3-phosphate dehydrogenase, messenger RNA (mRNA) levels. Human and rat LH, but not hCG alpha or -beta, FSH, or TSH, mimicked the down-regulating action of hCG on GnRH mRNA levels. Pretreatment of GT1-7 neurons with LH/hCG receptor antisense, but not sense, phosphorothioate oligodeoxynucleotides for 48 h resulted in decreases in [125I]hCG binding and the GnRH mRNA response to exogenous hCG. The half-life of GnRH mRNA transcripts, as determined by blocking transcription by actinomycin-D, was 32.5 +/- 2.5 h. This half-life was virtually unchanged by treatment with 100 ng/ml hCG (30.5 +/- 3.5 h). Treatment of GT1-7 neurons with 100 ng/ml hCG resulted in a dramatic decrease in nuclear run-on transcription of GnRH, but not beta-actin, gene compared to that in the controls. The same hCG concentrations and time points that decreased steady state GnRH mRNA levels also decreased cellular GnRH protein levels. Paradoxically, hCG stimulated the secretion of preexisting GnRH until the levels were depleted. In summary, GnRH neurons in the rat preoptic area and GT1-7 neurons coexpress LH/hCG receptor gene. Treatment of GT1-7 neurons with LH/hCG results in a decrease in steady state GnRH mRNA levels. This decrease is dose and time dependent and hormone specific, and requires the presence of cellular LH/hCG receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.

The expression of 15-lipoxygenase gene and the presence of functional enzyme in cytoplasm and nuclei of pregnancy human myometria.

The expression of the 15-lipoxygenase (15-LO) gene in pregnancy human myometria, from messenger RNA (mRNA) to the product of the enzyme reaction, was investigated. In situ hybridization with antisense riboprobe synthesized from human reticulocyte 15-LO complementary DNA has revealed the presence of mRNA in myometrial smooth muscle as well as in myometrial blood vessels. Immunoblot analysis with a polyclonal antibody to recombinant human 15-LO enzyme showed a single 110-kilodalton immunoreactive protein in myometria. Light microscope immunocytochemistry using polyclonal antibodies has demonstrated the presence of immunoreactive 15-LO protein and 15-hydroxyeicosatetraenoic acid (15-HETE), the primary product of the 15-LO pathway. While myometrial blood vessels did not show any obvious change, myometrial smooth muscle showed lower 15-LO mRNA, 15-LO immunoreactive protein, and 15-HETE at term pregnancy and during labor. Immunogold electron microscopy showed the presence of immunoreactive 15-LO in rough endoplasmic reticulum and 15-HETE in myofilaments. Quite unexpectedly, both are also present in nuclear chromatin. In summary, the present study demonstrates for the first time the expression of 15-LO gene in pregnancy human myometria and the mRNA and catalytically active enzyme are lower at term pregnancy and during labor. Quite unexpectedly, catalytically active 15-LO is also present in nuclear chromatin. These findings suggest that 15-LO/15-HETE may have genomic as well as nongenomic actions, both of which may either initiate and/or facilitate the progression of labor in women.

Expression of epidermal growth factor (EGF) receptor and its ligands, EGF and transforming growth factor-alpha, in human fallopian tubes.

Although human uterus is known to contain epidermal growth factor (EGF) and its receptors, it is virtually unknown whether human fallopian tubes, which are an anatomical continuation of the uterus, also contain them. Therefore, the present studies investigated whether EGF and its structural and functional homolog, i.e. transforming growth factor-alpha (TGF-alpha), and their common receptor are expressed in human fallopian tubes. Human fallopian tubes contain major 10.5-kilobase (kb) and minor 6.0-kb receptor messenger RNA (mRNA) transcripts, a single 5.0-kb EGF mRNA transcript, and a single 170-kilodalton receptor protein. The transcripts, along with their corresponding proteins and TGF-alpha protein, are present in ciliated and nonciliated epithelial cells, tubal smooth muscle, vascular smooth muscle, and endothelium. The cellular distribution and reproductive state dependency of these three regulatory molecules varied. For all of them, however, ampullary segments contained more than isthmus; proliferative phase and/or postpartum specimens contained more than secretory phase; and postmenopausal specimens contained the lowest amounts. The cell periphery and nuclear/perinuclear area of the cells contained EGF, TGF-alpha, and their receptors. Immunogold electron microscopy showed the receptors to be present in cell membranes, cilia, basal bodies which control ciliary activity, endoplasmic reticulum, nuclear membranes, and chromatin. In summary, human fallopian tubes contain EGF, EGF/TGF-alpha receptor mRNA and protein, and TGF-alpha protein. The expression of all these regulatory molecules was dependent on anatomical region, cell type, and reproductive state of the fallopian tubes. These findings suggest that EGF and TGF-alpha may regulate numerous tubal functions, thus potentially influencing fertility in women.

Human chorionic gonadotropin down-regulates the expression of gonadotropin-releasing hormone receptor gene in GT1-7 neurons.

Previous studies have shown that human CG (hCG) can down-regulate the expression of GnRH gene in GT1-7 neurons and that these neurons contain GnRH receptors and a self-stimulatory mechanism in the synthesis and release of GnRH. These findings have led us to hypothesize that hCG may down-regulate GnRH receptors to disrupt the self-stimulatory mechanism. To test this hypothesis, we cultured GT1-7 neurons in the presence or absence of an optimal concentration of highly purified hCG (100 ng/ml) and then measured steady-state levels of GnRH receptor messenger RNA (mRNA) transcripts and protein. Northern blotting demonstrated that GT1-7 neurons contain a major 5.5-kb and minor 2.4-kb and 1.6-kb GnRH receptor mRNA transcripts. Ligand blotting showed that GT1-7 neurons also contain 53-kDa and 43-kDa GnRH receptor proteins. Culturing with hCG resulted in a significant decrease in steady-state levels of GnRH receptor mRNA transcripts by 12 h and GnRH receptor proteins by 6 and 12 h, followed by a return to the controls by 24 h. The treatment with hCG had no obvious effect on the transcription rate of GnRH receptor gene. The hCG treatment, however, significantly decreased the half-life of GnRH receptor mRNA transcripts from 27 h to 16 h. In summary, we conclude that treatment with hCG can down-regulate the expression of GnRH receptor gene by decreasing the stability of transcripts in GT1-7 neurons. By down-regulating GnRH receptors, hCG may disrupt the self-stimulatory mechanism in GnRH synthesis.

Transcriptional and posttranscriptional mechanisms in epidermal growth factor regulation of human chorionic gonadotropin (hCG) subunits and hCG receptor gene expression in human choriocarcinoma cells.

Epidermal growth factor (EGF) stimulates the secretion of hCG in choriocarcinoma cells. However, the molecular mechanisms involved in this EGF action have never previously been investigated. The present study investigated them as well as EGF regulation of the hCG/LH (LH) receptor gene in JEG-3 human choriocarcinoma cells. The JEG-3 cells contain multiple EGF receptor messenger RNA (mRNA) transcripts and a single 170-kilodalton immunoreactive receptor protein. The human EGF can bind to the receptor protein and stimulate the receptor autophosphorylation as well as the phosphorylation of four other membrane proteins. Culturing JEG-3 cells with recombinant human EGF resulted in a dose- and time-dependent increase in hCG secretion. The maximal effect was seen at 100 ng/ml EGF, with a time lag of about 5 h. Tyrosine kinase, but not protein kinase-C or protein kinase-A, signaling was involved in the EGF action to increase hCG secretion. The EGF-induced increase in hCG secretion was not due to an increase in cell number or differentiation into multinuclear syncytia. EGF treatment resulted in a dose- and time-dependent increase in steady state levels of hCG alpha and hCG beta mRNAs. This increase was due to the stabilization of subunit mRNA transcripts. The increase in subunit mRNAs preceded the increase in hCG secretion. The EGF treatment resulted in a dose- and time-dependent decrease in steady state levels of the hCG/LH receptor mRNA transcripts. The decrease was due to a transcriptional inhibition of receptor gene. EGF treatment paradoxically stabilized hCG/LH receptor protein. In summary, EGF treatment up-regulates hCG subunits gene expression and down-regulates hCG/LH receptor mRNAs involving transcriptional and posttranscriptional mechanisms in JEG-3 human choriocarcinoma cells.

The central role of human chorionic gonadotropin in the formation of human placental syncytium.

A number of trophoblast products, including human chorionic gonadotropin (hCG), can increase the formation of human placental syncytium through the differentiation of mononuclear cytotrophoblasts. The present study investigated the central role of hCG in this process by using antisense receptor phosphorothioate oligodeoxynucleotides (ODNs). Culturing cytotrophoblasts with the hCG/LH receptor antisense, but not sense, ODN resulted in a significant decrease in receptor protein levels and inhibited spontaneous as well as exogenous hCG induced increase in differentiation. The hCG/LH receptor antisense ODN also inhibited epidermal growth factor (EGF), TGF-alpha, leukemia inhibitory factor (LIF), but not 8-bromo-cAMP, induced increases in differentiation, suggesting that hCG is required for EGF, TGF-alpha and LIF, but not for the cAMP actions. Although antisense EGF receptor and LIF receptor ODNs inhibited EGF and LIF induced increase in differentiation, respectively, they were ineffective against hCG, suggesting that they use separate pathways, but they both converge on a common pathway requiring the hCG actions. Mechanism of action studies revealed that EGF treatment activates its receptors and MAPK, both of which are required for EGF to increase the differentiation, cAMP levels and activate protein kinase A. In summary, our results demonstrate that hCG is an autocrine and paracrine regulator that is required for EGF, TGF-alpha, and LIF, but not for cAMP to increase human placental syncytium formation. Direct activation of protein kinase A seems to bypass the hCG pathway, perhaps by targeting genes associated with the differentiation.

Functional nuclear epidermal growth factor receptors in human choriocarcinoma JEG-3 cells and normal human placenta.

Immunocytochemistry with a monoclonal antiepidermal growth factor (anti-EGF) receptor antibody directed against the extracellular domain which can inhibit ligand binding to the receptors showed that nuclei of choriocarcinoma JEG-3 cells and normal placental trophoblasts were distinctly immunostained for EGF receptors. This finding led us to investigate the structure and function of nuclear EGF receptors. Western immunoblotting revealed that cell membranes, isolated intact pure nuclei, and nuclear membranes contain a 170-kilodalton EGF receptor protein. Covalent receptor cross-linking demonstrated that the 170-kilodalton receptor protein in nuclei and nuclear membranes can bind [125I]EGF just as in cell membranes, and that this binding is inhibited by excess unlabeled EGF. As in cell membranes, the addition of EGF resulted in an increased receptor autophosphorylation in the nuclei and nuclear membranes. In addition, the activated receptor kinase stimulated, and in some cases inhibited, tyrosine phosphorylation of a number of lower molecular size proteins, especially in nuclei and nuclear membranes. Although the identity of these proteins is not known, none of them could bind [125I]EGF. The addition of EGF to isolated nuclei resulted in a time-dependent specific transcriptional inhibition of hCG/LH receptor gene. In summary, our data demonstrating the presence of functional nuclear EGF receptors are novel, potentially important, and go against the traditional concepts of growth factors action. The nuclear receptors have the capacity to transduce signals from EGF and may mediate intracrine and paracrine actions of EGF in the regulation of trophoblast functions.

Novel role of human chorionic gonadotropin in differentiation of human cytotrophoblasts.

Our laboratory previously demonstrated that cytotrophoblasts and syncytiotrophoblasts in human placental tissue contain hCG/LH receptors. From this finding, we postulated that one role of hCG might be to promote the differentiation of cytotrophoblasts into syncytiotrophoblasts. To test this postulate, cytotrophoblasts were isolated from human term pregnancy placentas and cultured with and without increasing concentrations of highly purified hCG. The results showed that hCG had a biphasic effect on 1) the aggregation of cells without intervening plasma membranes; 2) the expression of cadherin, a cell adhesion receptor that facilitates cellular aggregation; 3) the expression of hCG/LH receptor gene; and 4) the expression of three different hormonal markers of differentiation. The hCG effects were time and dose dependent and hormone specific. The addition of excess polyclonal hCG antibody, but not normal rabbit serum or nonspecific antirabbit immunoglobulin G, decreased basal responses as well as those to exogenous hCG. The polyclonal hCG/LH receptor antibody increased differentiation and dramatically stimulated hCG secretion in the presence or absence of exogenous hCG. (Bu)2cAMP mimicked the actions of hCG. H-89, a protein kinase-A inhibitor, decreased basal as well as exogenous hCG responses. Calphostin, a protein kinase-C inhibitor and lavendustin, a tyrosine kinase inhibitor, on the other hand, had no effect. In summary, it is novel that hCG made in human placenta can regulate the differentiation of cytotrophoblasts, which make little hCG, into syncytiotrophoblasts, which make considerable amounts of hCG.

Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin in mucosal cells from human fallopian tubes.

The present study investigated the regulation of cyclooxygenase-2 (COX-2) gene by human CG (hCG) in mucosal cells from human fallopian tubes. The mucosal cells contained a major [4.3 kilobase (kb)] and several minor (3.6, 2.4, and 1. 8 kb) messenger RNA (mRNA) transcripts of LH/hCG receptors and also an 80-kDa receptor protein. The receptor protein can bind 125I-hCG. Culturing mucosal cells with increasing concentrations of highly purified hCG resulted in a dose- and time-dependent increase in steady-state levels of a 4.4-kb mRNA transcript and 72-kDa protein of COX-2. Whereas hLH and hCG could mimic each other in increasing COX-2 protein levels, FSH, TSH, PRL, and isolated alpha- and beta-subunits of hCG had no effect, suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. Culturing mucosal cells with increasing concentrations of hCG also resulted in a dose-dependent increase in media PGE2, levels, suggesting that the COX-2 protein increased by hCG is catalytically active. To determine the molecular mechanism of hCG action responsible for increasing the steady-state COX-2 mRNA levels, we measured the transcription rate of the COX-2 gene by nuclear run-on assay and the stability of its transcripts by an actinomycin D blocking method. The results showed that although hCG treatment had no effect on the transcription rate of COX-2 gene, it significantly increased the stability of COX-2 transcripts from 3.7 h in the control to 7.3 h after treatment. In summary, we conclude that tubal mucosal cells contain LH/hCG receptor transcripts and the receptor protein that can bind hCG. Culturing these cells with exogenous hCG and LH can up-regulate the expression of COX-2 gene by increasing the stability of transcripts. Through this up-regulation, LH and hCG may influence tubal functions that are important for early pregnancy in women.

cis-acting elements and trans-acting proteins in the transcription of chorionic gonadotropin/luteinizing hormone receptor gene in human choriocarcinoma cells and placenta.

We investigated the cis-acting elements and trans-acting proteins responsible for a higher basal rate of transcription of hCG/LH receptor gene in human choriocarcinoma JEG-3 cells compared with normal term pregnancy placenta. Sequential deletion of the 5'-flanking region of the gene revealed that there are three negative control regions (NCRs) designated NCR1 (-1457 to -1373 bp), NCR2 (-1051 to -835 bp), and NCR3 (-480 to -184 bp), and a promoter (-184 to -1 bp). NCR3 was more inhibitory than the other two; nearly 60-70% of the inhibitory activity resides in a sequence between -480 to -276 bp, and the rest resides in the sequence between -276 to -184 bp. Gel mobility shift assays showed that the nuclear extracts from JEG-3 cells contained proteins that form three complexes with NCR1, two with NCR2, and six with NCR3. Many of the proteins that form the complexes in NCR3 are shared with the other two NCRs. Most of the proteins that form these complexes are less abundant in nuclear extracts from JEG-3 cells than in those from placenta. The JEG-3 cell nuclear extracts also contained proteins that form three complexes with the proximal promoter of the hCG/LH receptor gene. These proteins were identified as Ap2, Ap2-like I, and Sp1 from the competition studies with synthetic excess unlabeled Ap2, Sp1, and CTF/NF1 consensus oligodeoxynucleotides and/or supershift in gel mobility assays with anti-Ap2 antibody. Although the JEG-3 cell nuclear extracts contained abundant Ap2-like protein I and low levels of Ap2 and Sp1 proteins, the placental nuclear extracts contained low levels of Ap2-like protein I and very low to nondetectable levels of Ap2 and Sp1 proteins. Deoxyribonuclease I footprinting revealed that the nuclear extracts from JEG-3 cells and placent protected the -116 to -93 bp and -65 to -45 bp regions in the proximal promoter of the hCG/LH receptor gene that contain Sp1 and Ap2 binding sites, respectively. However, the nuclear extracts from placenta only partially protected these regions, which is consistent with lower levels of proteins that bind to the proximal promoter of the gene. In summary, we conclude that the presence of low levels of proteins that bind to the NCRs and the high levels of proteins, especially Ap2-like I, that bind to the proximal promoter can potentially explain higher transcription of the hCG/LH receptor gene in JEG-3 cells compared with that in normal term pregnancy human placenta.

Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua.

Human endometrial stromal cells contain human CG (hCG)/LH receptors and in vitro, hCG/LH can promote stromal cells differentiation into decidua. In the present study, we tested the hypothesis that the treatment of stromal cells with exogenous hCG/LH to promote in up-regulation of cyclooxygenase-2 (COX-2) gene expression. The stromal cells from proliferative phase endometria were cultured for 10 days with 10 ng/ml estradiol and 100 ng/ml progesterone in the presence or absence of increasing concentrations of highly purified hCG. Northern blotting demonstrated that the cells contained a 4.4-kb COX-2 messenger RNA transcript whose levels significantly increased after treatment with hCG. Western blotting showed that the cells contained a 72-kDa COX-2 protein which also significantly increased after treatment with hCG. The effect of hCG on COX-2 messenger RNA and protein was seen at 10 ng/ml and higher concentrations sustained the increased levels. Although human LH mimicked hCG, human FSH, TSH, and isolated alpha- and beta-subunits of hCG had no effect on COX-2 protein levels suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. The effect of hCG on COX-2 protein paralleled the increase in media prostaglandin E2 levels indicating that the increased COX-2 gene and half-life of its transcripts to determine the molecular mechanism of hCG action. The results showed that hCG treatment had no significant effect on transcription rate of the COX-2 gene. On the other hand, treatment with hCG significantly increased the half-life of the COX-2 transcripts from 2.6 h in the control to 6.7 h after treatment. In summary, we conclude that treatment of human endometrial stromal cells with exogenous hCG to promote their differentiation into decidua results in an up-regulation of COX-2 gene expression by increasing the stability of the transcripts.

Novel self-regulation of human chorionic gonadotropin biosynthesis in term pregnancy human placenta.

Term pregnancy human placenta contains hCG/LH receptor mRNA transcripts and immunoreactive receptor protein. Both the receptor transcripts and receptor proteins are present only in trophoblasts. These findings led us to investigate whether hCG can regulate its own synthesis in term pregnancy human placenta. Treatment of placental tissue in static cultures or in a dynamic superfusion system with increasing concentrations of highly purified hCG provoked a biphasic effect on the steady state hCG subunit mRNA levels. Although low concentrations of hCG (< 200 mIU/ml) were not effective, moderate concentrations (200-1000 mIU/ml) increased, and high concentrations (> or = 5000 mIU/ml) either had no effect or actually decreased mRNA levels relative to the control values. This response was specific, because none of the hCG concentrations tested had any effect on glyceraldehyde-3-phosphate dehydrogenase or beta-actin mRNA levels. The effects of hCG on steady state hCG subunit mRNA levels were paralleled by corresponding changes in tissue hCG protein levels. Endogenous hCG appears to down-regulate alpha-subunit mRNA levels and hCG secretion. The hCG effect is probably receptor mediated, because a receptor antagonist, deglycosylated hCG, partially antagonized the hCG action. Treatment with exogenous hCG also down-regulated its own receptor mRNA and receptor protein levels. hCG regulation of its alpha-subunit and receptor levels involved both transcriptional as well as posttranscriptional mechanisms. In summary, this is the first demonstration of hCG regulating its own synthesis in term pregnancy human placenta. The findings of this study could offer a potential molecular explanation for the profile of hCG levels in normal pregnant women.

Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain.

LH from anterior pituitary and hCG from placenta bind to a common receptor in gonadal and nongonadal reproductive tissues. There have been numerous examples suggesting that the brain may also contain hCG/LH receptors, yet there has been no evidence for their existence so far. We now demonstrate by reverse transcription-nested polymerase chain reaction and northern blotting that the rat brain contains hCG/LH receptor mRNA. A major receptor transcript of 2.6 kilobases and minor transcripts of 1.8 and 4.4 kilobases were found. Western immunoblotting, ligand blotting, and covalent receptor cross-linking studies have shown that rat brain also contains an 80-kilodalton receptor protein that can bind hCG and hLH, but not hFSH. Rat testis used as a positive control showed a higher abundance of multiple transcripts and an 80-kilodalton receptor protein that can bind [125I]hCG. Rat liver used as a negative control did not contain any receptor transcripts or protein. In situ hybridization, dot blotting, immunocytochemistry, and topical autoradiography have revealed that hCG/LH receptors are present in rat hippocampus; dentate gyrus; hypothalamus; cerebellum; choroid plexus; ependymal cells of third, fourth, and lateral ventricles; cortex; brainstem; bovine hypothalamus; and human area postrema. These novel findings could potentially explain numerous previous observations and suggest new possibilities concerning the regulation of brain functions by hCG and LH.