Establishment of bovine expanded potential stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Modulatory effect of heat stress on viability of primary cultured chicken satellite cells and expression of heat shock proteins ex vivo.

Satellite cells promote muscle repairing and muscle growth. Thereby the intention of the present study was to investigate the beneficial effects of heat stress at different time intervals on chicken satellite cells' viability. Satellite cells were isolated from 1-day-old chicks and treated at two different temperatures (37 °C and 41 °C) for various time periods (6 h, 12 h, 24 h, 48 h, and 72 h). Both temperatures significantly increased cell viability after 24 h and 48 h. After 12 h, cell viability was significantly increased at 41 °C compared to 37 °C. However, more apoptotic cells were observed at end of the experiment of 41 °C compared to 37 °C. In addition, more live cells were found at early of experimental period at 41 °C than 37 °C. Additionally, protein and mRNA expression of HSP70, HP60 and HSP47 were significantly upregulated throughout the experimental period at temperature of 41 °C compared to those at 37 °C. These results indicate that cell viability and expression of heat stress related proteins/genes are induced by high temperature of 41 °C via heat stress pathway whereas activation of heat stress related proteins/genes are lower at 37 °C. Thus, 41 °C can trigger satellite cells' viability essential for better cell survival than 37 °C at early incubation time.

Primary cell culture of canine corneal endothelial cells.

OBJECTIVE: To establish a primary cell culture and clarify the characteristics of canine corneal endothelial cells in vitro. PROCEDURES: The eyes were enucleated from dogs that were euthanized for reasons unrelated to this study. Enucleated canine eyes were dissected, and the intact corneas were isolated from the globes. Using enzymes, the corneal endothelial cells were dispersed from the cornea. The obtained canine corneal endothelial cells were cultured in a cell culture dish. Cultured corneal endothelial cells were morphologically evaluated using phase-contrast microscopy. Immunohistochemical analysis of the cultured cells, particularly of the corneal endothelial cell marker, zonula occludens-1 (ZO-1), Na+ /K+ -ATPase, and vimentin, was performed to clarify whether the cultured cells were actually corneal endothelial cells. Furthermore, the post-passage morphology of cultured cells was evaluated. RESULTS: Canine primary cultured corneal endothelial cells showed morphologically small, cobblestone-like structures. The isolated cells had proliferative ability in vitro and demonstrated positive expression of the corneal endothelial cell markers, ZO-1, Na+ /K+ -ATPase, and vimentin. However, repeated passages resulted in larger cell sizes as assessed by phase-contrast microscopy. Repeated passages also resulted in lower cell density. CONCLUSIONS: This study demonstrated the successful culture of canine corneal endothelial cells. This might enhance the understanding of corneal endothelial cell characteristics in dogs.

Interleukin-6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing.

PURPOSE: To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. METHODS: Fourteen equine corneas were randomly assigned to one of two groups: wounded (n = 8) or unwounded (n = 6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6 mm filter paper disk soaked in 1N-NaOH for 60 s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope in situ hybridization for interleukin-6 (IL-6) and alpha-smooth muscle actin (αSMA) expression at T24, T48, and T72 h after wounding. Media of the cultured corneas were evaluated for the presence of lactate dehydrogenase (LDH) by a colorimetric assay. RESULTS: The ulcerated area of the wounded corneas decreased over time and all corneas healed within 72 h. Histologically, normal corneal architecture was observed including healthy epithelium (in areas other than the ulcerated ones), minimal stromal edema, intact endothelium, and Descemet's membrane. IL-6 expression was increased in wounded corneas compared with unwounded controls. LDH expression was elevated for both wounded and unwounded corneas at T24 but decreased substantially and was not detected at T48 in media from wounded and unwounded corneas, respectively. No αSMA expression was detected from either wounded or unwounded corneas. CONCLUSIONS: The equine air-liquid interface, ex vivo, corneal epithelial wound healing model is effective and physiologically relevant. This model can be used in future studies evaluating various corneal therapies.

Characterization of rodent Sertoli cell primary cultures.

Sertoli cells play a vital role in spermatogenesis by offering physical and nutritional support to the differentiating male germ cells. They form the blood-testis barrier and secrete growth factors essential for germ cell differentiation. Sertoli cell primary cultures are critical for understanding the regulation of spermatogenesis; however, obtaining pure cultures has been a challenge. Rodent Sertoli cell isolation protocols do not rule out contamination by the interstitial or connective tissue cells. Sertoli cell-specific markers could be helpful, but there is no consensus. Vimentin, the most commonly used marker, is not specific for Sertoli cells since its expression has been reported in peritubular myoid cells, mesenchymal stem cells, fibroblasts, macrophages, and endothelial cells, which contaminate Sertoli cell preparations. Markers based on transcription and growth factors also have limitations. Thus, the impediment to obtaining pure Sertoli cell cultures pertains to both the method of isolation and marker usage. The aim of this review is to discuss improvements to current methods of rodent Sertoli cell primary cultures, assess the properties of prepubertal versus mature Sertoli cell cultures, and propose steps to improve cellular characterization. Potential benefits of using contemporary approaches, including lineage tracing, specific cell ablation, and RNA-seq for obtaining Sertoli-specific transcript markers are discussed. Evaluating the specificity and applicability of these markers at the protein level to characterize Sertoli cells in culture would be critical. This review is expected to positively impact future work using primary cultures of rodent Sertoli cells.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Vector.

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

Development of primary cell culture from spleen of giant gourami Osphronemus goramy for propagation of giant gourami iridovirus (GGIV).

The severe mortality of fish due to the infection of megalocytivirus caused significant economic losses. Since 2011, megalocytivirus (giant gourami iridovirus (GGIV)) has become the main pathogen in giant gourami (Osphronemus goramy), particularly in West Java, Central Java and Bali. This study aimed to develop primary cell culture from spleen as the target organ for propagating megalocytivirus in vitro, which was developed by explant method with enzymatic dissociation. Optimization was carried out at incubation temperature, medium and serum concentrations. The origin of the primary cell, cell susceptibility and GGIV pathogenicity were observed. The results showed that the primary cell (GP cells) can grow well in 10% foetal bovine serum L-15 medium at 27°C, which was sufficient for cell growth. PCR and BLAST analyses showed the primary cell was originated from giant gourami. In infected GP cells, cell enlargement and cell rounding were observed. Virus propagated in GP cells was highly virulent when injecting giant gourami in an artificial infection experiment. Intraperitoneal injection of diluted virus supernatant showed 100% mortality in 7-11 days post-injection and 97% mortality in 21 days post-cohabitation, with abnormalities observed in spleen and kidney. In conclusion, GP cell was successfully subcultured for more than 30 passages and susceptible to GGIV.

In-vitro differentiation of early pig spermatogenic cells to haploid germ cells.

Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1) were upregulated in the presence of RA. The addition of an RA receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility.

Cryopreservation of Canine Primary Dorsal Root Ganglion Neurons and Its Impact upon Susceptibility to Paramyxovirus Infection.

Canine dorsal root ganglion (DRG) neurons, isolated post mortem from adult dogs, could provide a promising tool to study neuropathogenesis of neurotropic virus infections with a non-rodent host spectrum. However, access to canine DRG is limited due to lack of donor tissue and the cryopreservation of DRG neurons would greatly facilitate experiments. The present study aimed (i) to establish canine DRG neurons as an in vitro model for canine distemper virus (CDV) infection; and (ii) to determine whether DRG neurons are cryopreservable and remain infectable with CDV. Neurons were characterized morphologically and phenotypically by light microscopy, immunofluorescence, and functionally, by studying their neurite outgrowth and infectability with CDV. Cryopreserved canine DRG neurons remained in culture for at least 12 days. Furthermore, both non-cryopreserved and cryopreserved DRG neurons were susceptible to infection with two different strains of CDV, albeit only one of the two strains (CDV R252) provided sufficient absolute numbers of infected neurons. However, cryopreserved DRG neurons showed reduced cell yield, neurite outgrowth, neurite branching, and soma size and reduced susceptibility to CDV infection. In conclusion, canine primary DRG neurons represent a suitable tool for investigations upon the pathogenesis of neuronal CDV infection. Moreover, despite certain limitations, cryopreserved canine DRG neurons generally provide a useful and practicable alternative to address questions regarding virus tropism and neuropathogenesis.

Betanodavirus infection in primary neuron cultures from sole.

Nervous necrosis virus (NNV), G. Betanodavirus, is the causative agent of viral encephalopathy and retinopathy, a disease that causes mass mortalities in a wide range of fish species. Betanodaviruses are neurotropic viruses and their replication in the susceptible fish species seems to be almost entirely restricted to nerve tissue. However, none of the cell lines used for NNV propagation has a nervous origin. In this study, first we established a protocol for the primary culture of neurons from Senegalese sole, which made it possible to further study virus-host cell interactions. Then, we compared the replication of three NNV strains with different genotypes (SJNNV, RGNNV and a RGNNV/SJNNV reassortant strain) in sole neuron primary cultures and E-11 cells. In addition, to study how two amino acid substitutions at the c-terminal of the capsid protein (positions 247 and 270) affect the binding to cell receptors, a recombinant strain was also tested. The results show that sole neural cells enabled replication of all the tested NNV strains. However, the recombinant strain shows a clearly delayed replication when compared with the wt strain. This delay was not observed in virus replicating in E-11 cells, suggesting a viral interaction with different cell receptors. The establishment of a sole primary neuronal culture protocol provides an important tool for research into betanodavirus infection in sole.

Isolation and biological characteristics of multipotent mesenchymal stromal cells derived from chick embryo intestine.

1. Over the past decade, rapid advancement in isolation methods for identifying markers of the once elusive intestinal stem cell (ISC) populations has laid the foundation for unravelling their complex interrelationships during homeostasis. Study on ISC in avian intestinal tissue might play a pivotal foundation for further studies on the epithelial-to-mesenchymal transition (EMT) in gastrointestinal disease and cell-based therapy as well as intestinal tissue engineering. 2. The following experiment isolated a population of fibroblast-like, plastic adhering cells derived from chick embryo intestine, showing a strong self-renewing and proliferative ability, which was maintained in vitro up to passage 25. The findings included growth characteristics, detected expression of cell surface markers and characterised the capability of these cells to differentiate towards the osteogenic, adipogenic, and chondrogenic cell lineages. 3. RT-PCR analysis showed that these cells from chick embryos expressed mesenchymal stromal cell markers CD44, CD90 and VIMENTIN as well as ISC-specific genes LGR5, MI1, SMOC2, BMI1, and HOPX. Immunofluorescence and flow cytometry confirmed this biology characterisation further. 4. In conclusion, cells were isolated from the intestine of 18-day-old chicken embryos that exhibited the biological characteristics of mesenchymal stromal cells as well as markers of intestinal stem cells. Our findings may provide a novel insight for in vitro cell culture and characteristics of ISCs in avian species, which may also indicate a benefit for obtaining cell source for intestinal tissue engineering as well as cell-based investigation for gastrointestinal disease and treatment.

Separation of somatic and germ cells is required to establish primate spermatogonial cultures.

STUDY QUESTION: Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions? SUMMARY ANSWER: We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired. WHAT IS KNOWN ALREADY: Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells. STUDY DESIGN, SIZE, DURATION: Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells. MAIN RESULTS AND THE ROLE OF CHANCE: This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth of more rapidly expanding somatic cells to be a major problem when establishing spermatogonial cultures. Initiating germ cell cultures from the supernatant and maintaining germ cells in suspension cultures minimized the somatic cell contamination and provided enriched germ cell fractions which displayed after 11 days of culture a significantly higher expression of germ cell markers genes (DDX-4, MAGE A-4; P < 0.05) compared with separately cultured attached cells. Additionally, germ cell transplantation experiments demonstrated a significantly higher absolute number of cells with colonization ability (P < 0.001) in supernatant cells after 11 days of separate culture. LIMITATIONS, REASONS FOR CAUTION: This study presents a relevant aspect for the successful setup of spermatogonial cultures but provides limited data regarding the question of whether the long-term maintenance of spermatogonia can be achieved. Transfer of these preclinical data to man may require modifications of the protocol. WIDER IMPLICATIONS OF THE FINDINGS: Spermatogonial cultures from rodents have become important and innovative tools for basic and applied research in reproductive biology and veterinary medicine. It is expected that spermatogonia-based strategies will be transformed into clinical applications for the treatment of male infertility. Our data in the marmoset monkey may be highly relevant to establish spermatogonial cultures of human testes. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by the DFG-Research Unit FOR 1041 Germ Cell Potential (SCHL394/11-2) and by the Graduate Program Cell Dynamics and Disease (CEDAD) together with the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Isolation of peste des petits ruminants virus using primary goat kidney cell culture from kidneys obtained at slaughter.

BACKGROUND: Traditionally isolation of peste des petits ruminant virus (PPRV) is performed in Vero cells that takes several blind passages before observing typical cytopathic effects (CPEs). As an alternate, researchers have been using lamb kidney (LK) cells but day-old lambs are difficult to obtain and requires animal sacrifice. OBJECTIVE: We established a primary goat kidney (GK) cell culture from the kidneys obtained at slaughter. METHODS: The kidney of Black Bengal goats were collected from slaughter house and processed to make single cell suspension. The cells were resuspended in appropriate culture medium and maintained under optimum culture condition. RESULTS: The 80% confluent monolayer of GK cells was obtained after 15-20 days post seeding. Upon infection with a field isolate of PPRV, the well-developed CPEs characterized by cell rounding, vacuolation in the cytoplasm and fusion of cells were observed after 48 hr post infection. Virus quantification in the culture supernatant revealed more viral RNA in GK cells than LK cells. The multicycle growth analysis of PPRV showed a steady increase in the virus loads in the culture supernatant of infected GK cells, suggesting an adaptation of the PPRV in GK cells. CONCLUSIONS: The findings suggest that primary GK cells can be successfully prepared from the mature kidney cortical tissues and can be used for the isolation of PPRV. This system could reduce the unnecessary sacrifice of lambs or kids. Since kidneys of slaughtered goats are available throughout the year, using this protocol primary cell culture from mature goat kidney can provide primary cells to the laboratory throughout the year.

A lumpy skin disease virus which underwent a recombination event demonstrates more aggressive growth in primary cells and cattle than the classical field isolate.

Genomic changes by recombination have been recently observed in lumpy skin disease viruses circulating in Russia. The first characterized naturally occurring recombinant lumpy skin disease virus Saratov/2017 occurred through recombination between a live attenuated virus vaccine and the Southern African lumpy skin disease virus. Understanding if recombination can increase or decrease virulence of viruses through changes in different gene regions is required to improve the understanding of capripoxvirus biology. In this study, the in vitro and in vivo growth of the recombinant Saratov/2017 and the classical field isolate Dagestan/2015 was compared. Primary lamb kidney and lamb testis cells as well as the goat ovarian cell line were used to assess virus replication. In the goat ovarian cell line, Saratov/2017 and Dagestan/2015 induced comparable cytopathic activity and virus titres. In contrast, in primary lamb kidney and lamb testis cells, Saratov/2017 grew more aggressively causing more massive rounding up of cells, detachment and agglomeration compared to Dagestan/20152015. Growth curves of Saratov/2017 and Dagestan/2015 were assessed in primary lamb testis cells using different multiplicities of infection (MOI), with Saratov/2017 demonstrating faster replication at the different MOI and time points evaluated post-infection. In cattle, Saratov/2017 demonstrated more pronounced skin reactions when titrated by skin inoculation of serially diluted virus. In both primary cells and cattle, the titre of Saratov/2017 was significantly higher compared to Dagestan/2015 (p ≤ .05). These results demonstrate recombinant Saratov/2017 exhibits more aggressive replication properties.

Intercellular interactions between mast cells and stromal fibroblasts obtained from canine cutaneous mast cell tumours.

Mast cell tumours (MCTs) are the most frequent malignant skin neoplasm in dogs. Due to the difficulty in purifying large numbers of canine neoplastic mast cells, relatively little is known about their properties. A reproducible in vitro model is needed to increase the understanding about the phenotype and functional properties of neoplastic mast cells. In the present study, we describe the establishment of primary cocultures of neoplastic mast cells from canine cutaneous MCTs and cancer-associated fibroblasts. We confirmed the inability of canine neoplastic mast cells to remain viable for long periods in vitro without the addition of growth factors or in vivo passages in mice. Using a transwell system, we observed that mast cell viability was significantly higher when there is cell-to-cell contact in comparison to non-physical contact conditions and that mast cell viability was significantly higher in high-grade than in low-grade derived primary cultures. Moreover, the use of conditioned medium from co-cultured cells led to a significantly higher tumoral mast cell viability when in monoculture. Signalling mechanisms involved in these interactions might be attractive therapeutic targets to block canine MCT progression and deserve more in-depth investigations.

Establishment of immortalised cell lines derived from female Shiba goat KNDy and GnRH neurones.

Kisspeptin plays a critical role in governing gonadotrophin-releasing hormone (GnRH)/gonadotrophin secretion and subsequent reproductive function in mammals. The hypothalamic arcuate nucleus (ARC) kisspeptin neurones, which co-express neurokinin B (NKB) and dynorphin A (Dyn) and are referred to as KNDy neurones, are considered to be involved in GnRH generation. The present study aimed to establish cell lines derived from goat KNDy and GnRH neurones. Primary-cultured cells of female Shiba goat foetal hypothalamic ARC and preoptic area (POA) tissues were immortalised with the infection of lentivirus containing the simian virus 40 large T-antigen gene. Clones of the immortalised cells were selected by the gene expression of a neuronal marker, and then the neurone-derived cell clones were further selected by the gene expression of KNDy or GnRH neurone markers. As a result, we obtained a KNDy neurone cell line (GA28) from the ARC, as well as two GnRH neurone cell lines (GP11 and GP31) from the POA. Immunocytochemistry revealed the expression of kisspeptin, NKB and Dyn in GA28 cells, as well as GnRH in GP11 and GP31 cells. GnRH secretion from GP11 and GP31 cells into the media was confirmed by an enzyme immunoassay. Moreover, kisspeptin challenge increased intracellular Ca2+ levels in subsets of both GP11 and GP31 cells. Kisspeptin mRNA expression in GA28 cells, which expressed the oestrogen receptor alpha gene, was significantly reduced by 17ß-oestradiol treatment. Furthermore, the transcriptional core promoter and repressive regions of the goat NKB gene were detected using GA28 cells. In conclusion, we have established goat KNDy and GnRH neurone cell lines that could be used to analyse molecular and cellular mechanisms regulating KNDy and GnRH neurones in vitro, facilitating the clarification of reproductive neuroendocrine mechanisms in ruminants.

First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells.

Porcine circovirus type 3 (PCV3) was first detected in aborted fetuses in 2015 when sows displaying clinical signs that looked like porcine dermatitis and nephropathy syndrome died suddenly. Primary porcine kidney cells were selected for both the isolation and propagation of PCV3 strain SNUVR181115 (GenBank accession number MK503331) as these cells were permissive to PCV3 infection. PCV3 did not produce cytopathic effect on infected monolayers, therefore PCV3 infection was confirmed by in situ hybridization with a PCV3 specific DNA probe. Electron microscopy was used to analyze cell culture for the presence of virus. The intracytoplasmic inclusion bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell. Virus replication peaked at 6th passage yielding titers close to 106 genomic copies of PCV3 per mL. PCV3 strain SNUVR181115 isolated from primary porcine kidney cells was highly conservative and was clustered with the Korean and Chinese strains. These results demonstrated that primary porcine kidney cells are useful for PCV3 isolation and replication.

Bioactivity of recombinant hFSH glycosylation variants in primary cultures of porcine granulosa cells.

Previous studies have reported hypo-glycosylated FSH and fully-glycosylated FSH to be naturally occurring in humans, and these glycoforms exist in changing ratios over a woman's lifespan. The precise cellular and molecular effects of recombinant human FSH (hFSH) glycoforms, FSH21 and FSH24, have not been documented in primary granulosa cells. Herein, biological responses to FSH21 and FSH24 were compared in primary porcine granulosa cells. Hypo-glycosylated hFSH21 was significantly more effective than fully-glycosylated hFSH24 at stimulating cAMP accumulation and protein kinase A (PKA) activity, leading to the higher phosphorylation of CREB and ß-Catenin. Compared to fully-glycosylated hFSH24, hypo-glycosylated hFSH21 also induced greater levels of transcripts for HSD3B, STAR and INHA, and higher progesterone production. Our results demonstrate that hypo-glycosylated hFSH21 exerts more robust activation of intracellular signals associated with steroidogenesis than fully-glycosylated hFSH24 in primary porcine granulosa cells, and furthers our understanding of the differing bioactivities of FSH glycoforms in the ovary.

Isolation, characterization and germline chimera preparation of primordial germ cells from the Chinese Meiling chicken.

Primordial germ cells (PGCs) are precursors of germline cells that can generate sperm and eggs in adults, making them promising tools for transgenic animal preparation and germplasm preservation, especially in avians. In this study, we purified the PGCs from circulating embryonic blood of Chinese Meiling chickens using Nycodenz density centrifugation, and characterized them by alkaline phosphatase (AKP) staining, periodic acid-Schiff (PAS) staining and stage-specific embryonic antigen-1 (SSEA-1) immunostaining and PGC-specific gene amplification. The purified PGCs were also labeled with PKH26 and transferred into donor chicken embryos at the Hamburger-Hamilton (HH) stage 14 to 16, and cells with red fluorescence were observed in the gonads of 8-d-old embryos. When using about 200 PGCs isolated from Chinese Meiling chickens, microinjection into the dorsal aortas of recipient chickens with white feathers at stage HH14 to 16 resulted in germline chimeras that hatched and attained sexual maturity. The frequency of donor-derived yellow-feathered offspring from germline chimeric chickens was 12.6 ± 2.6% after mating with the white-feathered chickens. These results demonstrate that we had successfully purified the PGCs from the Chinese Meiling chicken. These germline cells could be used to preserve Chinese Meiling chickens.

Bioencapsulation of Oocytes and Granulosa Cells.

A protocol for the encapsulation in sodium alginate of granulosa cells in primary culture and co-culture of oocyte-cumulus complexes is reported. Sodium alginate forms strong gels when jellified with barium ions, allowing the self-organization of cells into a 3D structure. This method of encapsulation is simple and cheap, allowing the culture of cells in a 3-dimensional fashion.