VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Effects of a gel culture system made of polysaccharides (xanthan gum and locust bean gum) on in vitro bovine oocyte development and gene expression of the granulosa cells.

Xanthan gum (XG) and locust bean gum (LBG) are nontoxic polysaccharides that produce culture substrates. The present study examined the effect of XG-LBG gel on in vitro bovine oocyte growth and gene expression in granulosa cells. Oocytes and granulosa cell complexes (OGCs) were cultured in vitro on plastic culture plate (Plate) or XG-LBG gel for 16 days. OGCs formed a dome-like cavity surrounding the oocytes on plate but formed a spherical follicle structure on XG-LBG gel. The total granulosa cell numbers of the OGCs and their survival rate was greater for OGCs cultured on XG-LBG gel than for those cultured on plate. Oocytes grown on XG-LBG gels had higher lipid and mitochondrial content, as well as a larger diameter, than their plate counterparts. When oocytes grown in vitro were subjected to in vitro maturation and fertilization, the normal fertilization rate was significantly higher for oocytes developed on XG-LBG gel than that of oocytes cultured on the plate counterpart. RNAseq of the granulosa cells revealed that genes associated with focal adhesion, phosphatidylinositol 3'-kinase-Akt and Hippo signaling, and regulation of actin cytoskeleton were upregulated in granulosa cells of OGCs cultured on XG-LBG gel compared with those cultured on plate.

Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs.

BACKGROUND: Glaesserella parasuis, the causative agent of Glӓsser's disease, is widespread in swine globally resulting in significant economic losses to the swine industry. Prevention of Glӓsser's disease in pigs has been plagued with an inability to design broadly protective vaccines, as many bacterin based platforms generate serovar or strain specific immunity. Subunit vaccines are of interest to provide protective immunity to multiple strains of G. parasuis. Selected proteins for subunit vaccination should be widespread, highly conserved, and surface exposed. RESULTS: Two candidate proteins for subunit vaccination (RlpB and VacJ) against G. parasuis were identified using random mutagenesis and an in vitro organ culture system. Pigs were vaccinated with recombinant RlpB and VacJ, outer membrane proteins with important contributions to cellular function and viability. Though high antibody titers to the recombinant proteins and increased interferon-γ producing cells were found in subunit vaccinated animals, the pigs were not protected from developing systemic disease. CONCLUSIONS: It appears there may be insufficient RlpB and VacJ exposed on the bacterial surface for antibody to bind, preventing high RlpB and VacJ specific antibody titers from protecting animals from G. parasuis. Additionally, this work confirms the importance of utilizing the natural host species when assessing the efficacy of vaccine candidates.

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue.

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.

Evaluation of allogeneic freeze-dried platelet lysate in cartilage exposed to interleukin 1-ß in vitro.

BACKGROUND: Platelet-rich plasma (PRP) as well as other platelet-derived products have been used as a potential disease-modifying treatment for musculoskeletal diseases, such as osteoarthritis (OA). The restorative properties of such products rely mainly on the high concentrations of growth factors, demonstrating encouraging results experimentally and clinically. Yet, the autologous blood-derived nature of the PRP product lead to limitations that precludes it's widespread use. The main limitations for PRP use are; product variability, the need for minimum laboratory settings in most cases, and the need for storage at low temperatures to preserve its properties. Based on these limitations, the objective of this study was to investigate an allogeneic off-the-shelf platelet lysate (PL) in cartilage exposed to interleukin 1ß (IL-1ß). For this purpose, blood and cartilage were harvested from eight skeletally mature and healthy horses. Blood was processed into PL aliquots and divided into three groups (Frozen, Freeze-dried and Filtered freeze-dried), used in autologous and allogeneic conditions and in three different concentrations (1.5, 3 and 6-fold). Different PL preparations were then applied in cartilage culture with interleukin-1 beta and cultured for 10 days. Cartilage and media samples were collected and analyzed for total GAG and 35SO4-labeled GAG content. RESULTS: No significant differences between the controls and PL groups in cartilage and media were demonstrated. The effects of PL on cartilage matrix were concentration dependent and intermediate concentrations (3-fold) in PL showed increased 35SO4-labelled GAG in cartilage. CONCLUSION: In conclusion, the allogeneic freeze-dried PL presented equivalent effects compared to frozen autologous PL. Intermediate platelet concentration on average demonstrated improved results, demonstrating less GAG loss compared to other concentrations.

Effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex.

Oxygen concentration has been shown to influence in vitro viability and growth of ovarian follicles. The present study examined the effect of oxygen tension on in vitro development of dog follicles enclosed within the ovarian cortex. Ovaries were obtained from domestic dogs (age, 8 months to 2 years), and cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 4-well culture plate containing Eagle Minimum Essential Medium (MEM). Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability and diameter immediately after collection (Control) or after 2 or 5 days of in vitro incubation. Apoptotic cells were assessed using TUNEL assay. Comparisons of follicular viability and diameter were performed using analysis of variance followed by Tukey's test (p < 0.05). Comparisons of follicle density and apoptosis among treatments were conducted using Non-parametric Kruskal-Wallis test followed by Friedman's test (p < 0.05). No difference (p > 0.05) in follicle density was observed among groups at Day 2 of in vitro culture. However, the density of follicles within cortices cultured in 20% oxygen for 5 days significantly reduced compared to the Control and those incubated in 5% concentration. The viability of cultured follicles in all treatments decreased (p < 0.05) compared to the Control after 2 days incubation, and this value further reduced (p < 0.05) in 20% oxygen group at Day 5. There were no differences in the percentages of apoptotic follicles between the two treatment groups (p > 0.05). Nevertheless, after 5 days of culture, the percentage of TUNEL-positive follicles increased significantly (p < 0.05) in cortices incubated in 20% oxygen environment. In conclusion, our findings demonstrated that 5% oxygen level was superior to 20% concentration in sustaining in vitro viability of dog follicles enclosed within the ovarian cortex.

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue.

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α-MEM+ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non-cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.

Evaluation of in vitro culture systems for goat preantral follicles using reused ovaries from reproductive biotechniques: An alternative to maximize the potential of reproduction.

This study aimed to examine the in vitro culture of secondary preantral follicles, using reused ovaries, to compare both the 2D and 3D methods of in vitro culture of preantral follicles, and the system of medium replacement. Twenty-five pairs of ovaries from mixed-breed goats were used for the experiment. Follicular puncture of antral follicles was performed for in vitro production. After this procedure, the secondary preantral follicles were submitted to a microdissection procedure. The isolated preantral follicles were randomly divided into three treatments: (a) Two-dimensional culture with partial replacement of medium during culture (2D PR), (b) Three-dimensional culture with addition of medium during culture (3D AD) and (c) Three-dimensional culture with partial replacement of medium (3D PR). The culture period was 18 days. All treatments at the end of the in vitro culture period (18 days) presented a follicular survival rate which ranged from 59% to 70%, demonstrating that it was possible to perform an experiment with preantral follicles using ovaries that had previously been used in another reproductive biotechnique. The 3D AD treatment showed a survival percentage and follicular diameter higher than the 2D PR treatment, however, it did not differ from the 3D PR treatment. In conclusion, experiments employing the use of preantral follicles can be performed with success after the ovaries have been used for experiments with antral follicles. Moreover, the three-dimensional system with the addition of medium is recommended for in vitro culture of preantral follicles, since this system is more practical and financially feasible.

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification.

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.

Growth differentiation factor-9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles.

This study analysed the effect of growth differentiation factor-9 (GDF-9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α-MEM+ -control medium) or α-MEM+ supplemented with 1, 10, 50 or 100 ng/ml GDF-9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF-9 than other treatments, except for 10 ng/ml of GDF-9 (p > 0.05). Treatment containing 100 ng/ml GDF-9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF-9 showed more oocytes in MI than α-MEM+ , 1 or 50 ng/ml GDF-9 (p < 0.05). In conclusion, 100 ng/ml GDF-9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.

Age-related changes in the bovine corpus luteum function and progesterone secretion.

Decreased fertility associated with maternal ageing is a well-known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age-associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age-dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8 months), middle (80.1 months) and aged (188.9 months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6 months) and aged (183.1 months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3ß-HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation- and senescence-related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age-dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems.

BACKGROUND: A suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system. METHODS: Ovarian cortical tissues (1-2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined. RESULTS: In the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001). CONCLUSIONS: The CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.

Interactions between different media and follicle-stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758).

The aim was to verify the effect of follicle-stimulating hormone (FSH) supplementation to α-MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non-cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag-NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7-day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag-NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7-day culturing conditions.

Sequential medium with GH and IGF-1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue.

The growth hormone (GH) and growth insulin-like factor-1 (IGF-1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF-1 in the long-duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1-7), second half (days 7-14) or during 14 culture days. Treatments were identified as: αMEM+; GH â†’ IGF-1; IGF-1 â†’ GH and GH + IGF-1. The culture was designed in 24-well plates, in an incubator at 37°C and 5% CO2 . The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH â†’ IGF-1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF-1 â†’ GH and GH + IGF-1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH â†’ IGF-1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.

Resveratrol promotes in vitro activation of ovine primordial follicles by reducing DNA damage and enhancing granulosa cell proliferation via phosphatidylinositol 3-kinase pathway.

We aimed to study the effects of resveratrol on the morphology, DNA fragmentation, follicular activation and cell proliferation after in vitro culture of ovine ovarian tissue, and to verify if PI3K pathway is involved in resveratrol action in the sheep ovary. Ovaries were collected and divided into fragments. One fragment was fixed for histology (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+ ) alone or with resveratrol (2, 10 or 30 µM). After culture, ovarian tissue was destined to morphological analysis. TUNEL and proliferating cell nuclear antigen (PCNA) analyses were performed in the fresh control, α-MEM+ and 2 µM resveratrol. Inhibition of PI3K activity was performed through pre-treatment with LY294002. The percentage of normal follicles was similar between α-MEM+ and 2 µM resveratrol, and higher than those in other resveratrol treatments. An increase in follicular activation was observed in all treatments compared to fresh control. DNA fragmentation decreased in tissues cultured in 2 µM resveratrol compared to α-MEM+ . Moreover, PCNA-positive cells were higher in 2 µM resveratrol than in α-MEM+ . LY294002 inhibited follicular activation stimulated by α-MEM+ and 2 µM resveratrol. In conclusion, 2 µM resveratrol promotes primordial follicle activation compared to the fresh control by reducing DNA fragmentation and stimulating granulosa cell proliferation through activation of the PI3K pathway.

Effects of tumour necrosis factor-alpha and interleukin-1 beta on in vitro development of bovine secondary follicles.

The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.

Effects of dissolved carbon dioxide on the integrity of the rumen epithelium: An agent in the development of ruminal acidosis.

The carbon dioxide released and dissolved in rumen fluid may easily permeate across the epithelial cell membrane. Thus, we hypothesized that CO2 may act as proton carrier and induce epithelial damage under acidotic conditions. Ovine ruminal epithelia were mounted in Ussing chambers under short-circuit conditions. The serosal buffer solution had a constant pH of 7.4 and was gassed either with 100% oxygen or with carbogen (95% O2 /5% CO2 ). The mucosal solution was gassed with either 100% oxygen or 100% carbon dioxide. The mucosal pH was lowered stepwise from 6.6 to 5.0 in the presence or absence of short-chain fatty acids (SCFA). The transepithelial conductance (Gt ) as an indicator of epithelial integrity and the short-circuit current (Isc ) as an indicator of active electrogenic ion transfer were continuously monitored. At an initial mucosal pH of 6.6, there was no significant difference in Gt between the treatment groups. In the absence of both SCFA and CO2 , Gt remained constant when the mucosal solution was acidified to pH 5.0. In the presence of SCFA, mucosal acidification induced a significant rise in Gt when the solutions were gassed with oxygen. A small increase in Gt was observed in the mucosal presence of CO2 . However, no difference in final Gt was observed between SCFA-containing and SCFA-free conditions under carbon dioxide gassing during stepwise mucosal acidification. The SCFA+proton-induced increase in Gt could also be minimized by serosal gassing with carbogen. Because of the SCFA+proton-induced changes in Gt and their attenuation by CO2 , a protective role for mucosally available carbon dioxide may be assumed. We suggest that this effect may be due to the intraepithelial conversion of carbon dioxide to bicarbonate. However, the serosal presence of CO2 at a physiological concentration may be sufficient to protect the epithelia from SCFA+proton-induced damage for a certain period of time.

In vitro meat: A future animal-free harvest.

In vitro meat production is a novel idea of producing meat without involving animals with the help of tissue engineering techniques. This biofabrication of complex living products by using various bioengineering techniques is a potential solution to reduce the ill effects of current meat production systems and can dramatically transform traditional animal-based agriculture by inventing "animal-free" meat and meat products. Nutrition-related diseases, food-borne illnesses, resource use and pollution, and use of farm animals are some serious consequences associated with conventional meat production methods. This new way of animal-free meat production may offer health and environmental advantages by reducing environmental pollution and resource use associated with current meat production systems and will also ensure sustainable production of designer, chemically safe, and disease-free meat as the conditions in an in vitro meat production system are controllable and manipulatable. Theoretically, this system is believed to be efficient enough to supply the global demand for meat; however, establishment of a sustainable in vitro meat production would face considerably greater technical challenges and a great deal of research is still needed to establish this animal-free meat culturing system on an industrial scale.

Dual estrogenic regulation of the nuclear progestin receptor and spermatogonial renewal during gilthead seabream (Sparus aurata) spermatogenesis.

Studies in teleosts suggest that progestins have crucial functions during early spermatogenesis. However, the role of the different progestin receptors in these mechanisms is poorly understood. In this work, we investigated the expression pattern and hormonal regulation of the classical nuclear progestin receptor (Pgr) in the gilthead seabream at three different stages of spermatogenesis: the resting (postspawning) phase, onset of spermatogenesis, and spermiation. Immunolocalization experiments using a seabream specific Pgr antibody revealed that the receptor was expressed in Sertoli and Leydig cells, and also in a subset of spermatogonia type A, throughout spermatogenesis. Short-term treatment of testis explants with 17ß-estradiol (E2) increased pgr mRNA expression at all stages, while the progestin 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) had the opposite effect. At the resting stage, Sertoli cell Pgr expression was positively correlated with the occurrence of proliferating spermatogonia type A in the tubules, and both processes were incremented in vitro by E2 likely through the estrogen receptor alpha (Era) expressed in Sertoli and Leydig cells. In contrast, treatment with 17,20ßP downregulated Pgr expression in somatic cells. The androgen 11-ketotestosterone (11-KT) upregulated pgr expression in Leydig cells and promoted the proliferation of mostly spermatogonia type B, but only during spermiation. No relationship between the changes in the cell type-specific expression of the Pgr with the entry into meiosis of germ cells was found. These data suggest a differential steroid regulation of Pgr expression during seabream spermatogenesis and the potential interplay of the E2/Era and 17,20ßP/Pgr pathways for the maintenance of spermatogonial renewal rather than entry into meiosis.

Glucose, amino acids and fatty acids directly regulate ghrelin and NUCB2/nesfatin-1 in the intestine and hepatopancreas of goldfish (Carassius auratus) in vitro.

Ghrelin and nesfatin-1 are two peptidyl hormones primarily involved in food intake regulation. We previously reported that the amount of dietary carbohydrates, protein and lipids modulates the expression of these peptides in goldfish in vivo. In the present work, we aimed to characterize the effects of single nutrients on ghrelin and nesfatin-1 in the intestine and hepatopancreas. First, immunolocalization of ghrelin and NUCB2/nesfatin-1 in goldfish hepatopancreas cells was studied by immunohistochemistry. Second, the effects of 2 and 4hour-long exposures of cultured intestine and hepatopancreas sections to glucose, l-tryptophan, oleic acid, linolenic acid (LNA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on ghrelin and nesfatin-1 gene and protein expression were studied. Co-localization of ghrelin and NUCB2/nesfatin-1 in the cytoplasm of goldfish hepatocytes was found. Exposure to glucose led to an upregulation of preproghrelin and a downregulation of nucb2/nesfatin-1 in the intestine. l-Tryptophan mainly decreased the expression of both peptides in the intestine and hepatopancreas. Fatty acids, in general, downregulated NUCB2/nesfatin-1 in the intestine, but only the longer and highly unsaturated fatty acids inhibited preproghrelin. EPA exposure led to a decrease in preproghrelin, and an increase in nucb2/nesfatin-1 expression in hepatopancreas after 2h. These results show that macronutrients exert a dose- and time-dependent, direct regulation of ghrelin and nesfatin-1 in the intestine and hepatopancreas, and suggest a role for these hormones in the digestive process and nutrient metabolism.