Hypothalamic neuroendocrine integration of reproduction and metabolism in mammals.

Reproduction in mammals is an extremely energy-intensive process and is therefore tightly controlled by the body's energy status. Changes in the nutritional status of the body cause fluctuations in the levels of peripheral metabolic hormone signals, such as leptin, insulin, and ghrelin, which provide feedback to the hypothalamus and integrate to coordinate metabolism and fertility. Therefore, to link energy and reproduction, energetic information must be centrally transmitted to gonadotropin-releasing hormone (GnRH) neurons that act as reproductive gating. However, GnRH neurons themselves are rarely directly involved in energy information perception. First, as key factors in the control of GnRH neurons, we describe the direct role of Kisspeptin and Arg-Phe amide-related peptide-3 (RFRP-3) neurons in mediating metabolic signaling. Second, we focused on summarizing the roles of metabolic hormone-sensitive neurons in mediating peripheral energy hormone signaling. Some of these hormone-sensitive neurons can directly transmit energy information to GnRH neurons, such as Orexin neurons, while others act indirectly through other neurons such as Kisspeptin, RFRP-3 neuron, and (pituitary adenylate cyclase-activating polypeptide) PACAP neurons. In addition, as another important aspect of the integration of metabolism and reproduction, the impact of reproductive signaling itself on metabolic function was also considered, as exemplified by our examination of the role of Kisspeptin and RFRP-3 in feeding control. This review summarizes the latest research progress in related fields, in order to more fully understand the central neuropeptide network that integrates energy metabolism and reproduction.

Mutation of the ETS1 3'UTR interacts with miR-216a-3p to regulate granulosa cell apoptosis in sheep.

ETS1, an important member of the ETS transcription factor family, is involved in a variety of physiological processes in living organisms, such as cell development, differentiation, proliferation and apoptosis, and is thought to be associated with embryonic development and reproduction. However, the polymorphism of ETS1 has been rarely studied, and its potential impact on the formation of reproductive traits in sheep remains unclear. Here, we first analyzed polymorphisms of ETS1 in a population of 382 small-tailed Han sheep with a lambing number record using the Kompetitive Allele Specific PCR (KASP) technique. The results showed the presence of a SNP locus rs161611767 (T > C) in the 3'UTR of ETS1. The association analysis showed the lambing number of first, second and third parity in the individuals with the CC genotype (2.51 ± 0.108, 2.51 ± 0.179, 1.27 ± 0.196) was higher than that of individuals with the TT genotype (1.79 ± 0.086, 1.56 ± 0.102, 0.56 ± 0.100) (P < 0.05). Then, molecular biotechnologies were used to investigate the effects of the EST1 rs161611767 mutant locus on host gene expression in sheep and the underlying mechanism of its effect on sheep reproduction. The RT‒qPCR results showed that the expression of ETS1 was higher in individuals with the CC genotype than in those with the TT genotype (P < 0.05). The dual luciferase reporter assay showed that the luciferase activity of ETS1 in sheep with the TT genotype was decreased compared to CC genotype (P < 0.05), confirming the existence of EST1 rs161611767 in the 3'UTR as a functional SNP. Given that the 3'UTR is an important regulatory region of gene transcription and translation, we performed bioinformatics prediction and confirmed that the SNP rs161611767 of ETS1 was a direct functional target of miR-216a-3p using dual luciferase activity assay, and the binding capacity of allele T was stronger than that of allele C. Subsequently, the cell transfection results showed that miR-216a-3p suppressed the endogenous expression of ETS1 in sheep primary granulosa cells (GCs). Finally, CCK-8, EdU, WB detection of marker proteins and flow cytometry were used to detect the effects of miR-216a-3p on GCs viability and proliferation/apoptosis, respectively. The results showed that miR-216a-3p inhibited the proliferation of GCs while promoting apoptosis of GCs. In conclusion, these results demonstrate that the SNP rs161611767 of ETS1 is associated with lambing number in small-tailed Han sheep, and miR-216a-3p can act as a regulatory element binding to the T mutation in rs161611767 to regulate ETS1 expression and affect GCs development, which may indirectly affect the number of lambs in sheep. These studies provide evidence for the involvement of ETS1 polymorphisms in sheep reproduction and are expected to provide new insights to elucidate the molecular genetic mechanisms of lambing traits in sheep.

A unified GCNN model for predicting CYP450 inhibitors by using graph convolutional neural networks with attention mechanism.

Undesirable drug-drug interactions (DDIs) may lead to serious adverse side effects when more than two drugs are administered to a patient simultaneously. One of the most common DDIs is caused by unexpected inhibition of a specific human cytochrome P450 (CYP450), which plays a dominant role in the metabolism of the co-administered drugs. Therefore, a unified and reliable method for predicting the potential inhibitors of CYP450 family is extremely important in drug development. In this work, graph convolutional neural network (GCN) with attention mechanism and 1-D convolutional neural network (CNN) were used to extract the features of CYP ligands and the binding sites of CYP450 respectively, which were then combined to establish a unified GCN-CNN (GCNN) model for predicting the inhibitors of 5 dominant CYP isoforms, i.e., 1A2, 2C9, 2C19, 2D6, and 3A4. Overall, the established GCNN model showed good performances on the test samples and achieved better performances than the recently proposed iCYP-MFE model by using the same datasets. Based on the heat-map analysis of the resulting molecular graphs, the key structural determinants of the CYP inhibitors were further explored.

The neuroendocrine pathways and mechanisms for the control of the reproduction in female pigs.

Within the hypothalamic-pituitary-gonad (HPG) axis, the major hierarchical component is gonadotropin-releasing hormone (GnRH) neurons, which directly or indirectly receive regulatory inputs from a wide array of regulatory signals and pathways, involving numerous circulating hormones, neuropeptides, and neurotransmitters, and which operate as a final output for the brain control of reproduction. In recent years, there has been an increasing interest in neuropeptides that have the potential to stimulate or inhibit GnRH in the hypothalamus of pigs. Among them, Kisspeptin is a key component in the precise regulation of GnRH neuron secretion activity. Besides, other neuropeptides, including neurokinin B (NKB), neuromedin B (NMB), neuromedin S (NMS), α-melanocyte-stimulating hormone (α-MSH), Phoenixin (PNX), show potential for having a stimulating effect on GnRH neurons. On the contrary, RFamide-related peptide-3 (RFRP-3), endogenous opioid peptides (EOP), neuropeptide Y (NPY), and Galanin (GAL) may play an inhibitory role in the regulation of porcine reproductive nerves and may directly or indirectly regulate GnRH neurons. By combining data from suitable model species and pigs, we aim to provide a comprehensive summary of our current understanding of the neuropeptides acting on GnRH neurons, with a particular focus on their central regulatory pathways and underlying molecular basis.

Genome-Wide Identification of microRNAs in Response to Salt/Alkali Stress in Medicago truncatula through High-Throughput Sequencing.

Saline-alkaline stress is a universal abiotic stress that adversely affects plant growth and productivity. Saline-alkaline conditions results in plant abnormal transcriptome expression finally manifesting as defective phenotypes. Considerable research has revealed the active role of microRNA in various stress conditions. This study was aimed to identify novel miRNAs and the miRNA expression patterns in the leguminous model plant R108 (Medicago truncatula). The miRNA contained in the total RNA extracted from Medicago truncatula seedlings (72 h) that had been treated with solutions mimicking saline and alkaline soils was subjected to miRNA deep sequencing. The Illumina HiSeq sequencing platform was used to analyze nine small RNA libraries of three treatment groups: distilled water, 20 mM NaCl + Na2SO4 and 5 mM Na2CO3 + NaHCO3. Sequencing revealed that 876 miRNAs including 664 known miRNAs and 212 potential novel miRNAs were present in all the libraries. The miR159 family, miR156 family, miR2086-3p, miR396, miR166, miR319, miR167, miR5213-5p, miR1510 and miR2643 were among the most expressed miRNAs in all libraries. The results of miRNAs expression under treatments were validated by reverse-transcription quantitative PCR (RT-qPCR). Target gene prediction through computational analysis and pathway annotation analysis revealed that the primary pathways affected by stress were related to plant development, including metabolic processes, single-organism processes and response to the stimulus. Our results provide valuable information towards elucidating the molecular mechanisms of salt/alkali tolerance in Medicago truncatula and provide insight into the putative role of miRNAs in plant stress resistance.

Identification of new progestogen-associated networks in mammalian ovulation using bioinformatics.

BACKGROUND: Progesterone plays an essential role in mammalian ovulation. Although much is known about this process, the gene networks involved in ovulation have yet to be established. When analyze the mechanisms of ovulation, we often need to determine key genes or pathways to investigate the reproduction features. However, traditional experimental methods have a number of limitations. RESULTS: Data, in this study, were acquired from GSE41836 and GSE54584 which provided different samples. They were analyzed with the GEO2R and 546 differentially expressed genes were obtained from two data sets using bioinformatics (absolute log2 FC > 1, P < 0.05). This study identified four genes (PGR, RELN, PDE10A and PLA2G4A) by protein-protein interaction networks and pathway analysis, and their functional enrichments were associated with ovulation. Then, the top 25 statistical pathway enrichments related to hCG treatment were analyzed. Furthermore, gene network analysis identified certain interconnected genes and pathways involved in progestogenic mechanisms, including progesterone-mediated oocyte maturation, the MAPK signaling pathway, the GnRH signaling pathway and focal adhesion, etc. Moreover, we explored the four target gene pathways. q-PCR analysis following hCG and RU486 treatments confirmed the certain novel progestogenic-associated genes (GNAI1, PRKCA, CAV1, EGFR, RHOA, ZYX, VCL, GRB2 and RAP1A). CONCLUSIONS: The results suggested four key genes, nine predicted genes and eight pathways to be involved in progestogenic networks. These networks provide important regulatory genes and signaling pathways which are involved in ovulation. This study provides a fundamental basis for subsequent functional studies to investigate the regulation of mammalian ovulation.

Reproductive role of miRNA in the hypothalamic-pituitary axis.

The hypothalamic-pituitary-gonadal (HPG) axis plays a critical role in regulating reproductive function. Gonadotropin-releasing hormone (GnRH), which is secreted by the hypothalamus, acts on pituitary gonadotrophs to stimulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion, ultimately affecting the animal's fertility. MicroRNAs are small, non-coding RNAs that are widely expressed throughout the brain and can fine-tune gene expression post-transcriptionally. Recently, growing evidence has unveiled the central position of miRNAs within a key regulatory process involving GnRH secretion and subsequent activation in the pituitary. Although transcriptional regulation of reproduction has been well studied, the post-transcriptional processes are less well understood. In this review, we elaborate comprehensively on the critical role of miRNAs in the reproductive process, including both temporal and spatial aspects. A better understanding of how miRNAs impact the neuroendocrine system may improve our knowledge of reproduction and provide novel targets for therapeutic development.

Investigation of hypoxia networks in ovarian cancer via bioinformatics analysis.

BACKGROUND: Ovarian cancer is a leading cause of the death from gynecologic malignancies. Hypoxia is closely related to the malignant growth of cells. However, the molecular mechanism of hypoxia-regulated ovarian cancer cells remains unclear. Thus, this study was conducted to identify the key genes and pathways implicated in the regulation of hypoxia by bioinformatics analysis. METHODS: Using the datasets of GSE53012 downloaded from the Gene Expression Omnibus (GEO), the differentially expressed genes (DEGs) were screened by comparing the RNA expression from cycling hypoxia group, chronic hypoxia group, and control group. Subsequently, cluster analysis was performed followed by the construction of the protein-protein interaction (PPI) network of the overlapping DEGs between the cycling hypoxia and chronic hypoxia using ClusterONE. In addition, gene ontology (GO) functional and pathway enrichment analyses of the DEGs in the most remarkable module were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Ultimately, the signaling pathways associated with hypoxia were verified by RT-PCR, WB, and MTT assays. RESULTS: A total of 931 overlapping DEGs were identified. Nine hub genes and seven node genes were screened by analyzing the PPI and pathway integration networks, including ESR1, MMP2, ErbB2, MYC, VIM, CYBB, EDN1, SERPINE1, and PDK. Additionally, 11 key pathways closely associated with hypoxia were identified, including focal adhesion, ErbB signaling, and proteoglycans in cancer, among which the ErbB signaling pathway was verified by RT-PCR, WB, and MTT assays. Furthermore, functional enrichment analysis revealed that these genes were mainly involved in the proliferation of ovarian cancer cells, such as regulation of cell proliferation, cell adhesion, positive regulation of cell migration, focal adhesion, and extracellular matrix binding. CONCLUSION: The results show that hypoxia can promote the proliferation of ovarian cancer cells by affecting the invasion and adhesion functions through the dysregulation of ErbB signaling, which may be governed by the HIF-1α-TGFA-EGFR-ErbB2-MYC axis. These findings will contribute to the identification of new targets for the diagnosis and treatment of ovarian cancer.

The potential regulatory mechanisms of the gonadotropin-releasing hormone in gonadotropin transcriptions identified with bioinformatics analyses.

BACKGROUND: The regulation of gonadotropin synthesis and release by gonadotropin-releasing hormone (GnRH) plays an essential role in the neuroendocrine control of reproduction. However, the mechanisms underlying gonadotropin regulation by GnRH pulse frequency and amplitude are still ambiguous. This study aimed to explore the molecular mechanisms and biological pathways associated with gonadotropin synthesis by GnRH pulse frequencies and amplitudes. METHODS: Using GSE63251 datasets downloaded from the Gene Expression Omnibus (GEO), differentially expressed genes (DEGs) were screened by comparing the RNA expression from the GnRH pulse group, the GnRH tonic group and the control group. Pathway enrichment analyses of DEGs was performed, followed by protein-protein interaction (PPI) network construction. Furthermore, sub-network modules were constructed by ClusterONE and GO function and pathways analysed by DAVID. In addition, the relationship between the metabolic pathways and the GnRH pathway was verified in vitro. RESULTS: In total, 531 common DEGs were identified in GnRH groups, including 290 up-regulated and 241 down-regulated genes. DEGs predominantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including 11 up-regulated pathways (signallingsignallingmetabolic pathways, signallingand GnRH signalling pathway) and 5 down-regulated pathways (type II diabetes mellitus). Moreover, FBJ osteosarcoma oncogene (FOS) and jun proto-oncogene (JUN) had higher connectivity degrees in the PPI network. Three modules in the PPI were identified with ClusterONE. The genes in module 1 were significantly enriched in five pathways, including signallingthe insulin resistance and GnRH signalling pathway. The genes in modules 2 and 3 were mainly enriched in metabolic pathways and steroid hormone biosynthesis, respectively. Finally, knockdown leptin receptor (LEPR) and insulin receptor (INSR) reversed the GnRH-modulated metabolic related-gene expression. CONCLUSIONS: The present study revealed the involvement of GnRH in the regulation of gonadotropin biosynthesis and metabolism in the maintenance of reproduction, achieved by bioinformatics analyses. This, indicates that the GnRH signalling pathway played a central linkings role in reproductive function and metabolic balance. In addition, the present study identified the difference response between GnRH pulse and GnRH tone, indicated that abnormal GnRH pulse and amplitude may cause disease, which may provide an improved understanding of the GnRH pathway and a new insight for disease diagnosis and treatment.

MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development.

Ovaries, which provide a place for follicular development and oocyte maturation, are important organs in female mammals. Follicular development is complicated physiological progress mediated by various regulatory factors including microRNAs (miRNAs). To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing. Furthermore, via bioinformatic analyses, we dissected the associated functional networks of the observed significant miRNAs, in terms of interacting with signal pathways and transcription factors. During the growth and selection of dominant follicles, 15 dysregulated miRNAs and 139 associated pathways were screened out. In comparison of different styles of follicles, 7 commonly abundant miRNAs and 195 pathways, as well as 10 differentially expressed miRNAs and 117 pathways in dominant follicles in comparison with subordinate follicles, were collected. Furthermore, SMAD2 was identified as a hub factor in regulating follicular development. The regulation of miR-26a/b on smad2 messenger RNA has been further testified by real time PCR. In conclusion, we established functional networks which play critical roles in follicular development including pivotal miRNAs, pathways, and transcription factors, which contributed to the further investigation about miRNAs associated with mammalian follicular development.

Application of epigenetic markers in molecular breeding of the swine.

Livestock phenotypes are determined by the interaction of a variety of factors, including the genome, the epigenome and the environment. Epigenetics refers to gene expression changes without DNA sequence alterations. Epigenetic markers mainly include DNA methylation, histone modifications, non-coding RNAs, and imprinting genes. More and more researches show that epigenetic markers play an important role in the traits of pigs by modulating phenotype changes via gene expression. However, the role of epigenetic markers has caught little attention in swine breeding. The mechanism that influences important traits of swine has not been analyzed in detail, and it still lacks adequate scientific basis for practical applications. From the aspects of nutrition, diseases, important economic traits and trans-generational inheritance, we summarize the research, application prospects and challenges in the field of utilizing epigenetic markers in molecular breeding of pigs, thus providing a more comprehensive theoretical basis to promote more rapid research development in this field.

[A mechanistic review of how hypoxic mircroenvironment regulates mammalian ovulation].

Mammalian ovulation is a complicated process that includes development of follicles, ovulation, formation of corpus luteum and luteolysis. The three different stages of the ovulation activity are affected by hypoxic microenvironment and hypoxia-induced factors (HIF), which play a crucial role in physiologyical processes, such as angiogenesis and inflammation. Although the process of ovulation has been well elucidated, the molecular mechanism regulated by hypoxia needs an in depth study. In this review, we summarize how hypoxic and HIF regulate gene expression during mammalian ovulation in order to provide a better understanding of ovulation mechanism, which may lay a theoretical basis for prevention and therapy of various ovarian diseases.

[The mechanism of miRNA-mediated PGR signaling pathway in regulating female reproduction].

MicroRNAs (miRNAs) are involved in several physiological processes as important post-transcriptional regulators. Progesterone (P4), an important steroid hormone, produces physiological effect through binding specific receptor progesterone receptors (PGR) which regulates functions of both reproductive and non-reproductive tissues as a member of the nuclear receptor superfamily. P4/PGR and miRNAs could regulate female reproduction independently, however, it is still unclear how miRNAs and P4/PGR interaction regulates female reproductive activities such as ovulation in female reproduction. In this review, we summarize the possible ways in which miRNAs regulate P4 production and PGR gene expression as well as P4/PGR regulate miRNAs expression, which provide a theoretical basis for further studying the role of miRNAs and P4/PGR in female reproduction.

The Inhibitory Effects of RFamide-Related Peptide 3 on Luteinizing Hormone Release Involves an Estradiol-Dependent Manner in Prepubertal but Not in Adult Female Mice.

The mammalian gonadotropin-inhibitory hormone (GnIH) ortholog, RFamide-related peptide (RFRP), is considered to act on gonadotropin-releasing hormone (GnRH) neurons and the pituitary to inhibit gonadotropin synthesis and release. However, there is little evidence documenting whether RFamide-related peptide 3 (RFRP-3) plays a primary role in inhibition of the hypothalamo-pituitary-gonadal (HPG) axis prior to the onset of puberty. The present study aimed to understand the functional significance of the neuropeptide on pubertal development. The developmental changes in reproductive-related gene expression at the mRNA level were investigated in the hypothalamus of female mice. The results indicated that RFRP-3 may be an endogenous inhibitory factor for the activation of the HPG axis prior to the onset of puberty. In addition, centrally administered RFRP-3 significantly suppressed plasma luteinizing hormone (LH) levels in prepubertal female mice. Surprisingly, centrally administered RFRP-3 had no effects on plasma LH levels in ovariectomized (OVX) prepubescent female mice. In contrast, RFRP-3 also inhibited plasma LH levels in OVX prepubescent female mice that were treated with 17beta-estradiol replacement. Our study also examined the effects of RFRP-3 on plasma LH release in adult female mice that were ovariectomized at dioestrus, with or without estradiol (E2). Our results showed that the inhibitory effects of RFRP-3 were independent of E2 status. Quantitative real-time PCR and immunohistochemistry analyses showed that RFRP-3 inhibited GnRH expression at both the mRNA and protein levels in the hypothalamus. These data demonstrated that RFRP-3 could effectively suppress pituitary LH release, via the inhibition of GnRH transcription and translation in prepubescent female mice, which is associated with estrogen signaling pathway and developmental stages.

[Progesterone receptor-mediated molecular mechanisms on mammalian female reproduction].

The steroid hormone, progesterone, plays a critical role in regulation of mammalian female reproductive activities. Besides the non-genomic activity of progesterone on target cells, its main physiological effect is caused through genomic action by the ligand-dependent nuclear progesterone receptor. The genomic and non-genomic effects of progesterone collectively mediate various female reproductive functions, including ovulation, embryo implantation, maintenance of pregnancy, initiation of parturition, and development of mammary gland. Although a large number of candidate genes regulated by progesterone have been identified by gene chip technology, the traditional progesterone response elements located in the promoter region of downstream target genes havenot been detected. Accordingly, it was suggested thatthe mechanism of nuclear progesterone receptors regulating transcription may be different from other nuclear steroid receptors. In this review, we summarized the mechanisms of progesterone receptors mediating the physiological effects in various female re-productive activities.

[Effects of RFRP-3 on reproductive function and energy balance in mammals].

The hypothalamo-pituitary-gonadal (HPG) axis integrates internal and external cues via a balance of stimulatory and inhibitory neurochemical systems to regulate reproductive function in mammals. However, RFRP-3 is a unique inhibitor of HPG axis at the hypothalamuic level in mammals to date. A large number of previous studies have confirmed that RFamide-related peptide (RFRP-3) suppresses gonadotropin-releasing hormone (GnRH) system and luteinizing hormone (LH) secretion, thereby affecting the reproduction. However, whether the inhibition of LH secretion by RFRP-3 occurs at the pituitary level or the hypothalamus level is not clear. It is interesting that RFRP-3 is also related to signal pathway of melatonin modulating mammal seasonal reproduction, but little is known about the effects of melatonin on the RFRP-3 neuron up to now. In addition, RFRP-3 also plays an important role in the regulation of energy balance and behavior. The regulatory mechanism of RFRP-3 in HPG axis and role of RFRP-3 in modulating mammalian energy balance, as well as behavior, are systematically elaborated and the remaining unsolved problems are also discussed in this paper.

[The neuroendocrine regulatory mechanisms of mammalian seasonal reproduction].

The seasonal reproduction of mammal means the reproduction experiences an annual period from quiescence to renaissance. Studies have shown that kisspeptin and RFRP play an important role in the reproductive seasonality. The non-breeding season is characterized by an increase in the negative feedback effect of estrogen on GnRH, and this effect is transmitted by kisspeptin neurons, which may be an important factor affecting the reproduction activities. The expression of RFRP depends on melatonin secretion, and shows an apparent inhibition on reproduction in non-breeding season. In addition, thyroid hormones influence termination of the breeding season. Dopaminergic neuron A14/A15 also contributes to the seasonal changes in estrogen negative feedback. These neural systems may synergistically modulate the seasonal changes of reproductive function with the photoperiod. This review makes a systematic expatiation on the relationship between seasonal reproduction and these neural systems.

Molecular cloning and characterization of a novel gene encoding zinc finger protein from Medicago sativa L.

A suppression subtraction hybridization (SSH) cDNA library had been constructed to identify differentially expressed genes. Based on the sequence of an expressed sequence tag (EST) homologous to Pisum sativum zinc finger protein mRNA (Accession number: AF160911), the full-length cDNA of 1,676 nucleotides was cloned from alfalfa by rapid amplification of cDNA ends (RACE). It was designated as MsZFN, encoding a protein of 418 amino acids. The amino acid sequence compared by blast revealed high homology with zinc finger protein of other plants. Sequence comparison showed that there were five conserved typical zinc finger motifs, and one sugar transfer protein signature. The calculated molecular weight of the MsZFN protein was 45.8 k Da, and theoretical isoelectric point was 8.13. The MsZFN localized in nucleus. Under normal growth conditions, differential expression of MsZFN exhibited that the expression was the highest in leaf and the lowest in root. MsZFN was quickly and transiently induced by NaCl treatment and reached its maximum at 30 min.

[Advances on progesterone receptor gene].

The steroid hormone, progesterone, plays a key role in diverse events associated with female reproduction. In humans and other vertebrates, the biological activity of progesterone is mediated by modulation of the transcriptional activity of two progesterone receptors, PGR-A and PGR-B. This review introduced the structure, expression regulation and polymorphism of progesterone receptor gene. The relationship between progesterone receptor gene and reproductive function was also discussed in mammals.

Cloning and expression analysis of a vacuolar Na+/H+ antiporter gene from alfalfa.

A full-length cDNA, named MsNHX1, encoding a vacuolar Na+/H+ antiporter was cloned from alfalfa (Medicago sativa), using degenerate primers, followed by 3' and 5' RACE. The cDNA sequence was 2225 bp long and included an open reading frame encoding a deduced protein of 541-amino-acid polypeptide. The deduced amino acid sequence showed high similarity (more than 73%) to those of the previously cloned Na+/H+ antiporters form Arabidopsis thaliana, Qryza sativa, Atriplex gemlinin, Beta vulgaris and Hordeum vulgare. Southern hybridization showed that MsNHX1 has only one copy in alfalfa genome. Semiquantitative RT-PCR analysis indicated that the mRNA level of MsNHX1 was moderate without stress and markedly up-regulated after treatment by NaCl and ABA (abscisic acid). Those results suggest that the MsNHX1 product play an important role in salt tolerance of the alfalfa, and its transcript expression is possibly partially regulated through ABA-dependent signaling pathway.