The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13-20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47-52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.
An alternative superovulator to replace clomiphene citrate (CC) is needed as it is unsuitable for women with polycystic ovarian syndrome and is associated with low pregnancy rates. Anastrozole is an effective superovulator, but has not been well researched. The aim of this study was to determine the optimal dose of anastrozole as a superovulator and ascertain its effects on implantation and uterine ultrastructure during early pregnancy in Wistar rats using scanning electron microscopy. The uterine morphological characteristics which were studied in day 1 and 6 pregnant rats include microvilli density, length, surface "beads", surface glycocalyx, cell borders and apices, uterine surface fording and large surface protrusions. A significant increase in implantation sites is seen in the 15 mg/kg anastrozole group, compared to control. Day 1 and 6 anastrozole groups have similar morphology to the control and different to the CC group. At day 6, large surface protrusions are mostly noted but not limited to anastrozole-treated rats; anastrozole also appears to retain glycocalyx to some extent. The increased number of implantation sites in the 15 mg/kg anastrozole group suggests that this dose superovulates and favors implantation. Anastrozole is probably dose-/species-specific and additionally the surface uterine morphology suggests that anastrozole is implantation friendly.
Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.
Kisspeptin is a hypothalamic neuropeptide, which acts directly on gonadotropin-releasing hormone (GnRH)-secreting neurons via its cognate receptor (GPR54 or Kiss-R) to stimulate GnRH secretion in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and Kiss-R types. Recent gene knockout studies have demonstrated that fish kisspeptin systems are not essential in the regulation of reproduction. Studying the detailed distribution of kisspeptin receptor in the brain and pituitary is important for understanding the multiple action sites and potential functions of the kisspeptin system. In the present study, we generated a specific antibody against zebrafish Kiss2-R (=Kiss1Ra/GPR54-1/Kiss-R2/KissR3) and examined its distribution in the brain and pituitary. Kiss2-R-immunoreactive cell bodies are widely distributed in the brain including in the dorsal telencephalon, preoptic area, hypothalamus, optic tectum, and in the hindbrain regions. Double-labeling showed that not all but a subset of preoptic GnRH3 neurons expresses Kiss2-R, while Kiss2-R is expressed in most of the olfactory GnRH3 neurons. In the posterior preoptic region, Kiss2-R immunoreactivity was seen in vasotocin cells. In the pituitary, Kiss2-R immunoreactivity was seen in corticotropes, but not in gonadotropes. The results in this study suggest that Kiss2 and Kiss2-R signaling directly serve non-reproductive functions and indirectly subserve reproductive functions in teleosts.
The ultrastructure of the pinealocytes of the Malaysian rat (Rattus sabanus), a mammal inhabiting a zone near the equator where the annual variations of daylength are inconspicuous, was examined and compared with that of pinealocytes of other mammals. On the basis of the presence of granular vesicles, only one population of pinealocytes was found. A large number of granular vesicles and vesicle-crowned rodlets is characteristic of the pinealocytes of this equatorial species. Vesicle-crowned rodlets are especially numerous in the endings of the pinealocyte processes and; they most often found in direct topographical connection with the perivascular spaces. The physiological significance of the presence of such large amounts of vesicle-crowned rodlets and of the secretory process characterized by the formation of granular vesicles is discussed.
Strategies based on mesenchymal stem cell (MSC) therapy for restoring injured articular cartilage are not effective enough in osteoarthritis (OA). Due to the enhanced inflammation and oxidative stress in OA microenvironment, differentiation of MSCs into chondrocytes would be impaired. This study aims to explore the effects of diallyl disulfide (DADS) on IL-1β-mediated inflammation and oxidative stress in human adipose derived mesenchymal stem cells (hADSCs) during chondrogenesis. MTT assay was employed to examine the effects of various concentrations of DADS on the viability of hADSCs at different time scales to obtain non-cytotoxic concentration range of DADS. The effects of DADS on IL-1β-induced intracellular ROS generation and lipid peroxidation were evaluated in hADSCs. Western blotting was used to analyze the protein expression levels of IκBα (np), IκBα (p), NF-κB (np) and NF-κB (p). Furthermore, the gene expression levels of antioxidant enzymes in hADSCs and chondrogenic markers at days 7, 14 and 21 of differentiation were measured using qRT-PCR. The results showed that addition of DADS significantly enhanced the mRNA expression levels of antioxidant enzymes as well as reduced ROS elevation, lipid peroxidation, IκBα activation and NF-κB nuclear translocation in hADSCs treated with IL-1β. In addition, DADS could significantly increase the expression levels of IL-1β-induced impaired chondrogenic marker genes in differentiated hADSCs. Treatment with DADS may provide an effective approach to prevent the pro-inflammatory cytokines and oxidative stress as catabolic causes of chondrocyte cell death and enhance the protective anabolic effects by promoting chondrogenesis associated gene expressions in hADSCs exposed to OA condition.
Ghrelin, a gut-brain peptide hormone, is implicated in a multiplicity of biological functions, including energy homeostasis and reproduction. Neuronal systems that are involved in energy homeostasis as well as reproduction traverse the hypothalamus; however, the mechanism by which they control energy homeostasis is not fully understood. The present study analyzes the anatomical relationship of neurons expressing gonadotropin-releasing hormone (GnRH), neuropeptide Y (NPY) and growth hormone-releasing hormone (GHRH) in a cichlid, tilapia (Oreochromis niloticus). Additionally, we examine in vivo effects of ghrelin on these hypothalamic neurons and plasma growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. Double-immunofluorescence showed neuronal fiber associations between GnRH, NPY and GHRH in the brain and pituitary. Intracerebroventricular injection of ghrelin had no effect on numbers, soma size, or optical density of GnRH and NPY neurons, whereas the number of GHRH neurons was significantly decreased in the animals injected with ghrelin when compared to controls, which may indicate administered ghrelin promoted GHRH release. Plasma GH and pituitary GH mRNA levels were significantly increased in the animals injected with ghrelin. These results suggest that central administration of ghrelin primarily act on hypothalamic GHRH neurons to stimulate GH release from the pituitary in the tilapia.
Articular cartilage defect remains the most challenging joint disease due to limited intrinsic healing capacity of the cartilage that most often progresses to osteoarthritis. In recent years, stem cell therapy has evolved as therapeutic strategies for articular cartilage regeneration. However, a number of studies have shown that therapeutic efficacy of stem cell transplantation is attributed to multiple secreted factors that modulate the surrounding milieu to evoke reparative processes. This systematic review and meta-analysis aim to evaluate and compare the therapeutic efficacy of stem cell and secretome in articular cartilage regeneration in animal models. We systematically searched the PubMed, CINAHL, Cochrane Library, Ovid Medline and Scopus databases until August 2017 using search terms related to stem cells, cartilage regeneration and animals. A random effect meta-analysis of the included studies was performed to assess the treatment effects on new cartilage formation on an absolute score of 0-100% scale. Subgroup analyses were also performed by sorting studies independently based on similar characteristics. The pooled analysis of 59 studies that utilized stem cells significantly improved new cartilage formation by 25.99% as compared with control. Similarly, the secretome also significantly increased cartilage regeneration by 26.08% in comparison to the control. Subgroup analyses revealed no significant difference in the effect of stem cells in new cartilage formation. However, there was a significant decline in the effect of stem cells in articular cartilage regeneration during long-term follow-up, suggesting that the duration of follow-up is a predictor of new cartilage formation. Secretome has shown a similar effect to stem cells in new cartilage formation. The risk of bias assessment showed poor reporting for most studies thereby limiting the actual risk of bias assessment. The present study suggests that both stem cells and secretome interventions improve cartilage regeneration in animal trials. Graphical abstract ᅟ.
Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25 mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.
Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P
Rhogocyte is a unique molluscan cell that synthesises a supramolecular respiratory protein known as hemocyanin. Its ability to synthesise the protein has eluded the scientists despite hemocyanin's importance as a carrier protein and complex molecule with anti-viral activity. Although a hypothetical model of hemocyanin release from the rhogocytes lacunae was proposed based on colloid-osmotic pressure mechanism, lack of in vitro studies limits further validation of this model. In this study, we aim to investigate the impact of cell culture conditions and nature of hemocyanin biosynthesis of rhogocyte cells dissociated from Haliotis laevigata mantle tissue. Population of cells with different hemocyanin expression levels was profiled using flow cytometry, while hemocyanin concentrations in the media were elucidated by ELISA assay. We demonstrated that addition of lipoprotein supplement into the media resulted in a burst secretion of hemocyanin into the culture media. Over 7 days of culture, the population of cells tagged with hemocyanin antibody increased steadily while hemocyanin release in the media decreased significantly. Variation of culture medium, temperature, growth supplement type and concentration also impacted the cell growth and hemocyanin biosynthesis. These results indicated the possibility of an active process triggered by the addition of supplement to synthesise the protein at the highest amount during the first hour. The current study provides a glimpse of the hemocyanin biosynthesis by rhogocyte that may be significant to understand the cell ability to synthesise supramolecular protein and secretion through lacunae.