Biochemical aspects of conceptus--endometrial interactions

Mammalian conceptuses must provide a chemical signal to the maternal system to insure maintenance of corpus luteum (CL) function and of progesterone production and continuation of uterine endometrial secretory activity. These events insure that the developing conceptus is provided with appropriate nutrients, regulatory enzymes and endocrine state to allow successful establishment and maintenance of pregnancy. Pig blastocysts begin to produce estrogens by Day 11 of pregnancy, which prevents secretion of the uterine luteolytic factor (PGF2 alpha) in an endocrine direction, but allows secretion in an exocrine direction, i.e., into the uterine lumen. Therefore, CL are "protected." Blastocyst estrogens also trigger secretion of a group of proteins, including uteroferrin, an iron transport protein, and a family of protease inhibitors whose biosynthesis within the uterine glandular epithelium is under the control of progesterone. Estrogen also appears to promote accumulation of glucose and fructose within the uterine lumen. A complex in vivo "culture medium" is thereby established to promote conceptus development. Pig blastocysts do not undergo invasive implantation within the uterine lumen although they produce the protease, plasminogen activator. Invasion may be prevented by endometrial secretion of progesterone-induced protease inhibitors which are produced in large amounts. In addition to estrogens of conceptus origin, calcium and prostaglandins PGF2 alpha and E2 may affect the uterine vasculature, water and electrolyte transport, capillary permeability, conceptus steroid production, and related events during pregnancy. The blastocysts of the large domestic animals also secrete proteins which include a large glycoprotein (Mr approximately 600,000) and a small acidic protein (Mr approximately 17,000). The latter has been purified from sheep and named ovine trophoblast protein I. These proteins may play unique roles in early pregnancy with respect to establishment and maintenance of pregnancy in the ewe, sow, mare, and cow.     

Effect of glutathione reductase knockdown in response to UVB-induced oxidative stress in human lung adenocarcinoma

Background

Glutathione reductase (GR) plays a critical role in the maintenance of physiological redox status in cells. However, the comprehensive investigations of GR-modulated oxidative stress have not been reported.

Methods

In the present study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate their differential responses to UVB-irradiation.

Results

We identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins involving protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression.

Conclusions

In summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression in response to UVB-irradiation. To our knowledge, this is the first global proteomic analysis to investigate the role of GR under UVB-irradiation in mammalian cell model.     

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

Effect of prenatal exposure to ethanol on hepatic elongation factor-2 and proteome in 21 d old rats: protective effect of folic acid

In this article, we study the effects of ethanol intake during pregnancy and lactation on hepatic and pancreatic elongation factor-2 (EF-2) of 21 d old progeny. At the same time, the effect of ethanol on the level of other relevant hepatic proteins was determined using proteomic analysis. The results show that ethanol not only produces a general increase of protein oxidation, but also produces an important depletion of EF-2 and several other proteins. Among the hepatic proteins affected by ethanol, the concomitant supplementation with folic acid to alcoholic mother rats prevented EF-2, RhoGDI-1, ER-60 protease, and gelsolin depletion. This protective effect of folic acid may be related to its antioxidant properties and suggests that this vitamin may be useful in minimizing the effect of ethanol in the uterus and lactation exposure of the progeny.     

The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico "matrisome" defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.     

Serum progesterone and corpus luteum function in pregnant pigtailed monkeys (Macaca nemestrina)

Corpus luteum (CL) function and control during pregnancy and early lactation in the pigtailed macaque was investigated. Peripheral concentrations of progesterone (P) on day 10 of pregnancy were 12.98 ± 2.21 ng/ml and decreased progressively to 7.96 ± 1.27 ng/ml by day 21 of pregnancy. The concentration of P increased around day 27 of gestation and reached peak levels of 18.48 ± 2.45 ng/ml on day 37, there-after gradually decreasing to a nadir at about midgestation. Ten days before parturition P concentrations increased again (P < 0.05). Concentrations of P decreased from 6.62 ± 1.48 ng/ml on the day of delivery to 2.16 ± 0.43 ng/ml on day 2 of lactation and remained low thereafter. Ovariectomy on day 35 did not affect the normal course of gestation or the patterns of P secretion during pregnancy. However, in these ovariectomized animals, in spite of suckling, P was not detectable after parturition. In intact monkeys, serum concentrations of P in the uteroovarian vein at days 80 and 159 of pregnancy were higher relative to the uterine vein. Incubation studies utilizing ³H-cholesterol as a substrate revealed that the CL were capable of synthesizing P on days 35 and 159 of gestation. Histologically, the CL contained active luteal cells at late pregnancy.Low serum concentrations of chorionic gonadotropin were detected on day 10 of gestation; concentrations of this hormone reached high levels between days 18 and 24 and the titers were nondetectable after day 40 of pregnancy. Luteinizing hormone was present in constant amounts in the circulation during pregnancy and lactation.These data suggest that the CL of pregnancy in the pigtailed monkey is functional or capable of functioning during various stages of pregnancy. However, the fetoplacental unit is the primary source of P during the latter 4.5 months of gestation. As in other primates, a functional CL is not required for maintenance of pregnancy after implantation nor for lactation. Thus, the physiological significance of CL function during pregnancy is unclear.     

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up-regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.     

Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients

Background

Strawberries (Fragaria ananassa) reproduce asexually through stolons, which have strong tendencies to form adventitious roots at their second node. Understanding how the development of the proximal (I-1) and distal (I-2) internodes of stolons differ should facilitate nursery cultivation of strawberries.

Results

Herein, we compared the proteomic profiles of the strawberry stolon I-1 and I-2 internodes. Proteins extracted from the internodes were separated by two-dimensional gel electrophoresis, and 164 I-1 protein spots and 200 I-2 protein spots were examined further. Using mass spectrometry and database searches, 38 I-1 and 52 I-2 proteins were identified and categorized (8 and 10 groups, respectively) according to their cellular compartmentalization and functionality. Many of the identified proteins are enzymes necessary for carbohydrate metabolism and photosynthesis. Furthermore, identification of proteins that interact revealed that many of the I-2 proteins form a dynamic network during development. Finally, given our results, we present a mechanistic scheme for adventitious root formation of new clonal plants at the second node.

Conclusions

Comparative proteomic analysis of I-1 and I-2 proteins revealed that the ubiquitin-proteasome pathway and sugar-hormone pathways might be important during adventitious root formation at the second node of new clonal plants.     

Androstenedione interferes in luteal regression by inhibiting apoptosis and stimulating progesterone production

Androgens, in concert with lactogenic hormones, contribute to the maintenance of function of the corpus luteum (CL) in pregnant rats. Whereas some of the androgenic actions in the CL are clearly mediated by intracrine conversion to estrogen, pure androgenic effects are also implicated in the regulation of this transient endocrine gland. In this report, we have established, to our knowledge for the first time, the expression of androgen receptor (AR) mRNA and protein throughout gestation in the rat CL. We have found that the AR remains expressed in the CL of gestation on Day 4 postpartum and becomes expressed in the newly formed CL after postpartum ovulation. An AR immunoreactive protein was identified in the CL of pregnancy as well as in prostate and epididymis, which were used as positive controls. The luteal AR protein had mainly nuclear localization, yet some diffuse cytoplasmic staining was also observed. Moreover, we have established that androstenedione, the main circulating androgen in pregnant rats, significantly reduces the decline in luteal weight observed during postpartum structural regression. This effect was correlated with a decrease in the number of cells undergoing apoptosis and with enhanced levels of circulating progesterone. In addition, in vivo administration of androstenedione delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that the interference with apoptosis in vitro elicited by androstenedione is accompanied by an increased capacity of the CL to secrete progesterone. In summary, the results of this study have established that the rat CL expresses AR throughout pregnancy and after parturition, and they have defined a potential role for androstenedione in opposing postpartum luteal regression through inhibition of apoptosis and stimulation of progesterone production.     

iTRAQ-based analysis of sperm proteome from normozoospermic men achieving the rescue-ICSI pregnancy after the IVF failure

Background

In the assisted reproduction, the infertile molecules of spermatozoa from normozoospermic men who underwent the unexplained failure of in vitro fertilization (IVF) due to the lack of sperm binding to the normal zona pellucida, and then achieved pregnancy with the rescue intracytoplasmic sperm injection (R-ICSI) remain unclear. More works are still necessary to explore this male infertile mechanism.

Methods

Normozoospermicmen with the IVF pregnancy and normozoospermic men with the R-ICSI pregnancy after the conventional IVF failure were collected. iTRAQ-based proteomic approach were performed to reveal the new infertile causes between the IVF pregnancy men and the R-ICSI pregnancy men. To validate the confidence of proteome data, the individual samples were analyzed by western blot and immunofluorescence. Further, the spontaneous acrosome reactions were measured to evaluate the sperm quality.

Results

Compared with IVF pregnancy group, 56 sperm proteins were differentially expressed in the R-ICSI pregnancy group. Bioinformatic analyses (PANTHER, DAVID, PubMed and STRING) indicated these altered sperm proteins were involved in various molecular functions: reproduction, chromosome organization, and sperm-oocyte interaction. Moreover, the confidence of proteome data was confirmed by western blot and immunofluorescence using the individual samples, which were consistent with our proteomic data. Additionally, an increased rate of the spontaneous acrosome reaction rate was found in the R-ICSI pregnancy group.

Conclusions

The sealtered sperm proteins and the increased spontaneous acrosome reaction rate might account for this unexplained male infertility in the R-ICSI pregnancy patients. The present proteomic results will throw light on the better understanding of the unexplained infertile mechanisms underlying these normozoospermic man who achieved R-ICSI pregnancy after IVF failure.

     

Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.     

Expression of the whey acidic protein in transgenic pigs impairs mammary development

The whey acidic protein has been found in milk of mice, rats, rabbits and camels, and its gene is expressed specifically in mammary tissue at late pregnancy and throughout lactation. A characteristic of whey acidic protein is the ‘four-disulfide-core’ signature which is also present in proteins involved in organ development. We have generated six lines of transgenic pigs which carry a mouse whey acidic protein transgene and express it at high levels in their mammary glands. Transgenic sows from three lines could not produce sufficient quantities of milk to support normal development of healthy offspring. This phenotype appears to be similar, if not identical, to themilchlos phenotype exhibited by mice expressing whey acidic protein transgenes. Mammary tissue from post-partummilchlos sows had an immature histological appearance, which was distinct from that observed during normal development or involution. Expression of the whey acidic protein transgene was found in mammary tissue from sexually immature pigs frommilchlos lines, but not in sows from lines that appeared to lactate normally. We suggest that precocious synthesis of whey acidic protein impairs mammary development and function. Impaired mammary development due to inappropriate timing of whey acidic protein expression is consistent with the notion that proteins with the ‘four-disulfide-core’ signature participate in tissue formation.