Immunolocalisation of the uterine secretory proteins uterocalin, uteroferrin and uteroglobin in the mare's uterus and placenta throughout pregnancy

Previous studies have shown that the equine uterus produces many progesterone-dependent proteins throughout gestation. In particular, uterocalin and uteroferrin are detectable using electrophoresis or blot analyses but information regarding the immunohistochemical placental distribution of these two proteins is rare and information regarding uteroglobin is still lacking. The aim of the present study was to co-immunolocalise these three secretory proteins in the mare's uterus throughout gestation in an effort to understand their functional role in the maintenance of pregnancy. Therefore, endometrial biopsy samples were obtained from 20 pregnant mares between 16 and 309 days of gestation and labelled immunohistochemically for uteroglobin, uteroferrin and uterocalin. Uteroferrin remained detectable in almost every endometrial gland at all stages but with an increase in staining intensity as gestation advanced. The most progesterone-dependent protein, uterocalin, showed variable staining throughout gestation with the most intense labelling in early pregnancy and during the period of endometrial cup reaction. Uteroglobin secretion was only detectable in traces and only in individual glands throughout gestation. The results indicate that uterocalin and uteroferrin, but not uteroglobin, may play important roles in supplying nutrients for the conceptus, thereby contributing to the maintenance of pregnancy. However, further investigations are necessary to understand the role of uteroglobin during gestation.     

Developmental stages and chemical composition in embryos of the cockroach, Diploptera punctata, with observations on the effect of diet

A developmental time-table has been established for the embryos of viviparous Diploptera punctata. The percentage of gestation time occupied by pre-dorsal closure stages is only 19 per cent compared to 40 or 50 per cent in non-viviparous species. The increase in wet weight of the embryos begins several days before dorsal closure and continues throughout gestation. The increase in dry weight, protein, carbohydrate, and uric acid does not begin until shortly after dorsal closure but thereafter parallels the increase in wet weight.The increase in oöcyte protein during vitellogenesis is tenfold, less than in oviparous or ovoviviparous species. But the increase in embryo protein during gestation is sixtyfold; this characterizes viviparity in D. punctata.Although total lipid increases during gestation, lipid as a percentage of wet weight decreases most rapidly before dorsal closure and less rapidly there-after. It is suggested that before dorsal closure lipid is the major energy source for development. After dorsal closure the embryos are able to drink through their mouths the fluid nutrient provided by the mother.The ultimate source of nutrients required by developing embryos is the maternal diet during gestation. Females starved from ecdysis do not produce young; females fed only sugar from ecdysis produce viable larvae but suffer loss in body weight. Embryos from such females, although normal in length, are deficient in protein.     

Proteins synthesized and secreted during rat pancreatic development

The synthesis and secretion of proteins during development of the pancreas was analyzed using two-dimensional gel electrophoresis. The pattern of synthesis of the total proteins of the pancreas was found to change very little from 14 to 18 d gestation. In addition, the protein synthetic pattern of the embryonic pancreas was very similar to the protein patterns of several other embryonic tissues (gut, lung, and mesenchyme). Between 18 d gestation and the adult stage, the synthesis of the majority of protein species fades as the synthesis of the secretory (pro)enzymes becomes dominant. Thus, the terminal differentiation of the pancreas appears to involve the dominant expression of a limited set of genes (coding, in part, for the digestive [pro]enzymes) while the pattern of expression of the remaining domain remains relatively unchanged. Many of the secretory (pro)enzymes were identified and their synthesis during development was monitored. The synthesis of several secretory proteins was detected between 15 and 18 d gestation (e.g., amylase and chymotrypsinogen), whereas the synthesis of others was not detected until after 18 d gestation (i.e., trypsinogen, ribonuclease, proelastase, and lipase). Between 18 d gestation and the adult stage, the synthesis of the digestive (pro)enzymes increases to > 90% of pancreatic protein synthesis. The secretion of digestive (pro)enzymes was detected as early as 15 d gestation. The selective release of a second set of proteins was detected in the early embryo. These proteins are not detected in the adult pancreas or in zymogen granules but are also released by several other embryonic tissues. The function of this set of proteins is unknown.     

PGF2α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo–maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit–i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F₂α and PGE₂ concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF₂α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE₂. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo–maternal interactions in vivo in monotocous species.     

Pregnancy Loss Following Coxsackievirus B3 Infection in Mice during Early Gestation Due toHigh Expression of Coxsackievirus-Adenovirus Receptor (CAR) in Uterus and Embryo

Coxsackieviruses are important pathogens in children and the outcomes of neonatal infection can be serious or fatal. However, the outcomes of coxsackievirus infection during early gestation are not well defined. In this study, we examined the possibility of vertical transmission of coxsackievirus B3 (CVB3) and the effects of CVB3 infection on early pregnancy of ICR mice. We found that the coxsackievirus and adenovirus receptor (CAR) was highly expressed not only in embryos but also in the uterus of ICR mice. CVB3 replicated in the uterus 1 to 7 days post-infection (dpi), with the highest titer at 3 dpi. The pregnancy loss rate in mice infected with CVB3 during early gestation was 38.3%, compared to 4.7% and 2.7% in mock-infected and UV-inactivated-CVB3 infected pregnant mice, respectively. These data suggest that the uterus and embryo, which express abundant CAR, are important targets of CVB3 and that the vertical transmission of CVB3 during early gestation induces pregnancy loss.     

The in vitro effect of progesterone on the orexin system in porcine uterine tissues during early pregnancy

Background

Orexin A (OXA) and orexin B (OXB) are hypothalamic-derived peptides that participate in the regulation of energy metabolism, food intake and reproductive function by influencing the hypothalamic-pituitary-ovarian axis. Orexins are also produced in the endometrium, myometrium and placenta, which suggests that they could act as a link between energy metabolism and the reproductive system. Changes in the expression of orexin and the orexin receptor genes and proteins during the oestrous cycle and early gestation in pigs imply that orexin activity may be regulated by local factors within the uterus. The aim of this study was to investigate the influence of progesterone (P₄) on the expression of orexin system genes, and proteins in the porcine uterus during early gestation. Gene expression was analyzed by real-time PCR. Adiponectin secretion was determined by ELISA, and the receptors proteins content was defined using western blot analysis.

Results

In the endometrium, P₄ enhanced OXA secretion on days 10 to 11 of gestation and OXB secretion on days 12 to 13. In the myometrium, P₄ inhibited the secretion of both orexins on days 15 to 16 and OXB secretion also on days 12 to 13. In the endometrium, P₄ inhibited the expression of orexin receptor 1 (OX1R) protein at nearly all times analyzed, whereas the expression of orexin receptor 2 (OX2R) protein was inhibited only on days 15 to 16 of gestation. In the myometrium, P₄ stimulated OX1R protein expression on days 12 to 13 and 15 to 16 of gestation and inhibited OX1R protein expression on days 27 to 28. The expression of OX2R protein in the myometrium increased on days 12 to 13 and decreased on days 10 to 11 and 15 to 16.

Conclusions

The results indicate that P₄ could regulate the expression of the orexin system in the porcine uterus during early pregnancy, which suggests the presence of a local feedback loop that could play an important role in the regulation of maternal metabolism during pregnancy. The findings may contribute to the existing knowledge of the mechanisms linking maternal energy metabolism with the regulation of the reproductive system during pregnancy.

     

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi.     

Changes in the mitochondrial proteome of developing maize seed embryos

Mitochondria are required for seed development, but little information is available about their function and role during this process. We isolated the mitochondria from developing maize (Zea mays L. cv. Nongda 108) embryos and investigated the mitochondrial membrane integrity and respiration as well as the mitochondrial proteome using two proteomic methods, the two‐dimensional gel electrophoresis (2‐DE) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH). Mitochondrial membrane integrity and respiration were maintained at a high level up to 21 days after pollination (DAP) and decreased thereafter, while total mitochondrial number, cytochrome c oxidase activity and respiration per embryo exhibited a bell‐shaped change with peaks at 35–45 DAP. A total of 286 mitochondrial proteins changed in abundance during embryo development. During early stages of seed development (up to 21 DAP), proteins involved in energy production, basic metabolism, protein import and folding as well as removal of reactive oxygen species dominated, while during mid or late stages (35–70 DAP), some stress‐ and detoxification‐related proteins increased in abundance. Our study, for the first time, depicted a relatively comprehensive map of energy production by mitochondria during embryo development. The results revealed that mitochondria were very active during the early stages of maize embryo development, while at the late stages of development, the mitochondria became more quiescent, but well‐protected, presumably to ensure that the embryo passes through maturation, drying and long‐term storage. These results advance our understanding of seed development at the organelle level.     

Uterine and oviducal protein secretion during early pregnancy in the mouse

Changes in the protein composition of the embryo's environment during early development were studied by analysis of proteins synthesized and secreted by oviducal and uterine explants on Days 1-6 of pregnancy. Although secretions from ampullar and isthmic oviduct and uterus contained many proteins in common, each area also produced its own characteristic proteins. In the uterus, changes in the secretion pattern were found during the peri-implantation period, including both increases and decreases in particular proteins which appear to be dependent on the presence of embryos. Embryo-induced effects on uterine secretion began between 09:00 h of Day 4 and 09:00 h of Day 5. Oviducal secretions exhibited many of the embryo-dependent proteins found in the uterus, but the expression of these proteins did not appear to be influenced by the presence of embryos on Day 1 or Day 3. The characteristic pattern of secreted protein expression by each portion of the reproductive tract may reflect the specialization of each area for certain developmental events.     

Development of cerebrospinal fluid and the blood-cerebrospinal fluid barrier in rabbits.

Blood plasma and cerebrospinal fluid (CSF) samples were collected from adult female rabbits (New Zealand White), newborn, and embryos at 18, 20, 24, and 28 days of gestation. Samples were analyzed for total protein using the Folin phenol reagent. During development, mean total protein of blood plasma rose sharply from 12.45 to 12.51 mg/ml at 18 to 20 days to 37.56 mg/ml at 28 days. Levels further increased to 54.06 mg/ml in the newborn and to 66.18 mg/ml in the adult. The protein concentration of cerebrospinal fluid was constant at 5.20 to 5.29 mg/ml between 18 and 20 days of gestation, but steadily decreased to 3.53 mg/ml at 28 days. By birth, the CSF protein concentration was further reduced to 2.08 mg/ml, and this level differed only slightly (P < 0.05) from CSF protein values determined for adults. These data indicate that the blood-cerebrospinal fluid barrier to proteins begins to function by 18 to 20 days of gestation, and the protein concentration of cerebrospinal fluid approaches the normal adult value soon after birth.     

The role of the pituitary in modifications of the uterine secretion of spayed, oestradiol-primed rats

Tests performed on spayed, adult female estradiol-primed Ivanovas rats, with ligated uteri and normal pituitary function have shown that treatment with sexual steroids, including progesterone and testosterone, modifies uterine secretion. One half of the animals were hypophysectomized. In estradiol primed hypophysectomized controls, growth was retarded about 28%, the weight of the empty uterus reduced, and the quantity of uterine secretion diminished in comparison with the values for the nonhypophysectomized controls. In test rats treated with estradiol, gain in body weight was virtually arrested in the nonhypophysectomized rats and a reduction in weight was observed in both groups treated with the highest dose of estradiol tested (300 mcg/kg daily). In rats treated with progesterone, no significant differences were found between the two groups. In treated groups, a dose-related reduction in the weight of the empty uterus was found. Treatment caused a marked reduction in the quantity of the uterine secretion, the effect appearing greater in nonhypophysectomized rats. Increasing doses of progesterone produced a rapid rise in the viscosity of the uterine fluid, as well as a decrease in the pH of the uterine lumen. In both hypophysectomized and nonhypophysectomized rats, testosterone induced a dose-related increase in body weight, statistically significant only in animals with intact pituitaries treated with 100 mg/kg daily. The weight of the empty uterus also increased. The quantity of uterine fluid was reduced by testosterone only when it was given in massive doses to nonhypophysectomized rats. Doses of 100-300 mg/kg daily were needed to produce the same response as a dose of about 10 mg/kg daily of progesterone. In response to large doses, viscosity of secretion rose slightly and the pH of uterine lumen and secretion decreased. It may be concluded that the progestative modifications induced by progesterone in the uterus of spayed, estradiol-primed rats, including particularly changes in uterine secretion, are the effects of a peripheral mechanism not involving the pituitary. Testosterone appears to be an exception as far as the quantity and viscosity of uterine secretion are concerned, since modifications in these parameters are only observed in the presence of a functional pituitary body.     

Protein production by sheep embryos during the period of maternal recognition of pregnancy

An embryo must be present in the uterus 12-13 days after estrus to prevent regression of the ovine corpus luteum. The present experiments were designed to determine if embryo-specific secretory proteins could be detected in the maternal blood at the time of maternal recognition of pregnancy. In two experiments, 92 embryos were flushed from 47 ewes at 14-15 days after estrus. Embryos were incubated in vitro for 24 h and the proteins in the media were harvested. Antisera to proteins in both flushing and incubation medium were produced in rabbits. In experiment 1, crude fractions were used for antibody production and radioimmunoassays were established for protein peaks separated on a 1.1 X 75 cm G-100 Sephadex column. Two low molecular weight fractions (EPiv and EPv) appeared to be embryo specific but were not detectable in jugular vein sera of 14- to 15-day pregnant animals. In experiment 2, proteins derived from uterine flushes and from embryo incubations were chromatographed on a 2.5 X 85 cm column of G-100 Sephadex. The protein peaks were measured, pooled, lyophilized, and used for immunization of rabbits. As in experiment 1, antisera were generated, some of which seemed to be directed against embryo-specific proteins. However, we could not detect these fractions in the uterine vein blood of pregnant animals. Thus, embryo-specific proteins are either confined to the uterus or they appear in the blood in quantities that are undetectable with our assay system.     

ACE2 expression and activity are enhanced during pregnancy

In the current study, we investigated the expression and activity of ACE2 during pregnancy in normotensive and hypertensive rats, focusing on the relative contribution of the uterus and the placentas, the kidney serving as a reference. We used the Sabra rat model of salt-sensitive hypertension. We confirmed a systemic vasodilatory state during the third trimester of pregnancy, as evidenced by a reduction in blood pressure, both in normotensive and hypertensive rats. At the time that blood pressure was reduced, ACE2 was expressed abundantly in the reproductive organs. The relative levels of ACE2 mRNA in the pregnant animal were placenta > kidneys > or = uterus and of ACE2 activity kidney > placenta > uterus. In the uterus and the placenta, ACE2 expression was unaffected by strain, salt-loading, or the level of blood pressure. ACE2 activity in the uterus of the nonpregnant rat was not affected by any of these variables either, but during pregnancy increased in salt-loaded animals. When estimating the total contribution of the uterus to ACE2 mRNA and activity during pregnancy, we found that the amount of ACE2 mRNA increased in both strains irrespective of diet, but that ACE2 activity increased only in salt-loaded animals. We further estimated the relative total contribution of the uterus, placentas, and kidneys to ACE2 expression and activity during pregnancy by adjusting for mass and number of organs and found that the placentas were the major contributors, followed by the kidney and the uterus. We conclude that during pregnancy, the placentas, in particular, but also the uterus, constitute important sources of ACE2, in addition to its normal production in the kidney, leading to an estimated twofold increase in total ACE2 activity. These data are consistent the hypothesis that transient ACE2 overexpression and increased activity during pregnancy may be important in modulating systemic, as well as local hemodynamics in the uteroplacental unit.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Electrophoretic characterization of proteins in oviduct fluid of cows during the estrous cycle

This study was undertaken to examine the in vivo protein composition of the bovine oviduct during the estrous cycle. Oviduct fluid was collected daily from four oviduct-cannulated dairy cows for a total of four complete estrous cycles. Fluid secretion followed a definite cyclic pattern, with maximum secretion occurring at estrus in all cycles. Protein concentration fluctuated during the cycle and varied among animals. In general, protein concentration was lower at the time of estrus. Total protein in oviduct fluid, however, was higher around estrus, indicating increased transudation or secretion by the oviduct. One-dimensional SDS-PAGE separation revealed the protein pattern of oviduct fluid to be generally similar to that of blood serum and follicular fluid. Two proteins appeared to be oviduct-specific. The first, a protein of approximately 47 kDa, was evident in oviduct fluid throughout the estrous cycle. The second protein, evident as a broad diffuse staining band above albumin, appeared for only 3-4 days at or near ovulation. This protein had a molecular weight of 80-95 kDa and stained positive for carbohydrate with periodic acid-Schiff reagent. These studies indicate that the in vivo protein composition of oviduct fluid varies with the estrous cycle, and that around estrus, an oviduct-specific glycoprotein is present.     

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.     

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.     

Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans

Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging.     

Interaction between Lysophosphatidic Acid,Prostaglandins and the Endocannabinoid System during the Window of Implantation in the Rat Uterus

Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids’ receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins’ synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids’ levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.     

Analysis of proteins secreted by mouse embryos developing in vivo and in vitro

Proteins secreted by mouse blastocysts developing in vitro were compared to these from blastocysts developing in utero to determine if a simple medium supporting blastocyst development also supports secreted protein expression. In-vivo embryos were collected on days 3, 4, or 5 of pregnancy and incubated in 35S-methionine to produce conditioned medium containing released, labeled proteins. Embryos for culture were collected on day 3 and after 48 or 72 h labeled conditioned medium was produced. Labeled proteins were separated by two-dimensional electrophoresis and compared using a digital image analysis system. Day 3 embryos did not release proteins in detectable amounts, although synthesis of intracellular proteins was substantial. Day-4 and -5 blastocysts released proteins in increasing amount and complexity, consistent with previous results. When day-3 embryos were cultured in medium containing 4 mg/ml BSA for 48 h, secreted protein patterns were similar but not identical to those of day-5 uterine blastocysts. Although most of the proteins produced by uterine blastocysts were secreted by cultured embryos, differences were found in the relative quantities of certain proteins. Neither crystallized BSA nor polyvinyl alcohol at 4 mg/ml supported development of protein secretion as well as the crude fraction-V BSA. Blastocysts restricted to the oviduct also exhibited quantitative differences in protein secretion patterns compared to uterine blastocysts. Thus, although blastocyst development and the expression of many secreted proteins are supported outside the uterus, the full pattern of secretion characteristic of the peri-implantation embryo may be dependent on specific uterine influences.