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Spermatogonial stem cells are self-renewal and differentiate into sperm in post-pubertal mammals. There exists a balance between the self-renewal and differentiation in the testes. Spermatogonial stem cells make up only 0.03% of testicular cells in adult mice. These cells maintain sperm production by differentiating after puberty. Therefore, analyzing the expression of genes associated with spermatogenesis is critical for understanding differentiation. The present study aimed to establish the postnatal period of cells in relation to spermatogenesis. To study the expression of differentiated and undifferentiated marker genes in enriched spermatogonial stem cells, in vitro culture was performed and cells from pup (6–8-day-old) and adult (4-months-old) testicular tissues were isolated. As a result, undifferentiated genes, Pax7, Plzf, GFRa1, Etv5 and Bcl6b , were highly increased in cultured spermaotogonial stem cells compared with pup and adult testicular cells. On the other hands, differentiated gene, c-kit was highly increased in adult testicular cells, Also Stra8 gene was highly increased in pup and adult testicular cells. This study provides a better understanding of spermatogenesis-associated gene expression during postnatal periods.

Research question Can specimen types (cells versus tissues) and additive cryoprotectant agents contribute to efficient cryopreservation of primate spermatogonial stem cells (SSC)? Design Testicular tissues or cells from four prepubertal monkeys were used in this study. The freezing efficacy of testicular tissue was compared with cell suspensions using conventional freezing media (1.4 mol/l dimethyl sulfoxide [DMSO]) and the efficacy of cryoprotectant additives (1.4 mol/l DMSO combined with trehalose 200 mmol/l, hypotaurine 14 mmol/l, necrostatin-1 50 µmol/l or melatonin 100 µmol/l) was evaluated in testicular tissue freezing. Results The survival rate (46.0 ± 4.8% versus 33.7 ± 6.0%; P = 0.0286) and number of recovered cells (5.0 ± 1.5 × 106 cells/g versus 0.7 ± 0.8 × 106 cells/g; P = 0.0286) were significantly higher in frozen tissues than in frozen cell suspensions. After tissue freezing, a higher number of recovered PGP9.5+ cells were observed with 200 mmol/l trehalose treatment than in DMSO controls (2.4 ± 0.6 × 106 cells/g versus 1.1 ± 0.3 × 106 cells/g; P = 0.0164). Normal establishment of donor-derived colony was observed in SSC after tissue freezing with 200 mmol/l trehalose. Conclusions Testicular tissue freezing is more effective than single cell suspension freezing for higher recovery of undifferentiated spermatogonia. Moreover, it was verified that slow freezing using 200 mmol/l trehalose, 1.4 mol/l DMSO and 10% KnockOut™ Serum Replacement in Dulbecco's phosphate-buffered saline is an effective cryopreservation protocol for primate testicular tissue.

Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene (

BACKGROUND Cryopreservation can expand the usefulness of spermatogonial stem cells (SSCs) in various fields. However, previous investigations that have attempted to modulate cryoinjury-induced mechanisms to increase cryoprotective efficiency have mainly focused on apoptosis and necrosis. OBJECTIVES This study aimed to establish an effective molecular-based cryoprotectant for SSC cryopreservation via autophagy modulation. MATERIALS AND METHODS To determine the efficacy of autophagy modulation, we assessed the recovery rate and relative proliferation rate and performed western blotting for the determination of autophagy flux, immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization, and spermatogonial transplantation for in vivo SSC functional activity. RESULTS The results showed that a basal level of autophagy caused a higher relative proliferation rate (pifithrin-μ 0.01 μM, 184.2 ± 11.2%; 3-methyladenine 0.01 μM, 175.3 ± 10.3%; pifithrin-μ 0.01 μM + 3-methyladenine 0.01 μM, P3, 224.6 ± 22.3%) than the DMSO control (100 ± 6.2%). All treatment groups exhibited normal characteristics, suggesting that these modulators could be used as effective cryoprotectants without changing the properties of the undifferentiated germ cells. According to the results of the in vivo spermatogonial transplantation assay, the colonies per total number of cultured SSCs was significantly higher in the pifithrin-μ 0.01 μM (1596.7 ± 172.5 colonies), 3-methyladenine 0.01 μM (1522.1 ± 179.2 colonies), and P3 (1727.5 ± 196.5 colonies) treatment groups than in the DMSO control (842.8 ± 110.08 colonies), which was comparable to that of the fresh control (1882.1 ± 132.1 colonies). DISCUSSION A basal level of autophagy is more essential for resilience in frozen SSCs after thawing, rather than the excessive activation or inhibition of autophagy. CONCLUSION A basal level of autophagy plays a critical role in the pro-survival response of frozen SSCs after thawing; herein, a new approach by which SSC cryoprotective efficiency can be improved was identified.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B&ndash, Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

Genomic imprinting in domestic animals contributes to the variance of performance traits. However, research remains to be done on large-scale detection of epigenetic landscape of porcine imprinted loci including the GNAS complex locus. The purpose of this study was to generate porcine parthenogenetic fetuses and comprehensively identify imprinting patterns of the GNAS locus in transcript levels. To this end, both normally fertilized and bimaternal (uniparental) parthenogenetic porcine fetuses were generated, and whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) were performed to construct methylome and transcriptome, respectively. Differentially methylated regions (DMRs) between the fetuses were identified through methylome analysis, and parental-origin-specific expression patterns of transcripts were examined with transcriptome. As a result, three major DMRs were identified: paternally methylated Nesp DMR, maternally methylated Nespas-Gnasxl DMR, and maternally methylated Exon1B–Exon1A DMR. Parental-origin-specific expressions of those five DMR-affected transcripts were found, including a novel imprinted transcript, Exon1B, in pigs. In conclusion, using parthenotes, parental-origin-specific imprinting patterns in the porcine GNAS locus was comprehensively identified, and our approach paves the way for the discovery of novel imprinted genes and loci in a genomic context across species.

Cryopreservation of spermatogonial stem cells (SSCs) is important to preserve the lineages of valuable livestock and produce transgenic animals. Although interest in molecular-based cryopreservation methods have been increasing to improve their efficiency, the issue of necroptosis has not yet been considered. Therefore, the purpose of this study was to understand the role of necroptosis using necrostatin-1 (Nec-1), necroptosis inhibitor, in SSC cryopreservation, and to investigate the potential application of Nec-1 as a cryoprotectant. To determine the cryopreservation efficiency of Nec-1, we assessed recovery rate, proliferation potential, cellular membrane damage, RIP1 protein expression, apoptosis, and its mechanism. Stable characterization and functional activity of SSCs was determined via immunofluorescence, RT-qPCR, and in vivo transplantation of SSCs. Our results showed a higher proliferation potential in 50 μM Nec-1 (146.5 ± 16.8%) than in DMSO controls (100.0 ± 3.4%). Furthermore, the cryoprotective effects of Nec-1 were verified by a decrease in RIP1 expression (3.1 ± 0.2-fold vs. 1.3 ± 0.3-fold) and in early apoptosis (4.3 ± 0.8% vs. 2.6 ± 0.1%) compared to DMSO controls. Normal functional activity was observed in the SSCs after cryopreservation with 50 μM Nec-1. In conclusion, necroptosis could be a cause of cryoinjury, and their inhibitor may serve as potential effective cryoprotectant. This study will contribute to establish a molecular-based cryopreservation method, and thereby expanding the use of SSCs into the domestic livestock industry as well as for clinical applications.

Exposure to bisphenol A (BPA) in the gestational period damages the reproductive health of offspring, detailed evidence regarding BPA-induced damage in testicular germ cells of offspring is still limited. In this study, pregnant mice (F0) were gavaged with three BPA doses (50 &mu, g, 5 mg, and 50 mg/kg body weight (bw)/day, tolerable daily intake (TDI), no-observed-adverse-effect-level (NOAEL), and lowest-observed-adverse-effect level (LOAEL), respectively) on embryonic days 7 to 14, followed by investigation of the transgenerational effects of such exposure in male offspring. We observed that the NOAEL- and LOAEL-exposed F1 offspring had abnormalities in anogenital distance, nipple retention, and pubertal onset (days), together with differences in seminiferous epithelial stages and testis morphology. These effects were eradicated in the next F2 and F3 generations. Moreover, there was an alteration in the ratio of germ cell population and the apoptosis rate in germ cells increased in F1 offspring at the LOAEL dose. However, the total number of spermatogonia remained unchanged. Finally, a reduction in the stemness properties of spermatogonial stem cells in F1 offspring was observed upon LOAEL exposure. Therefore, we provide evidence of BPA-induced disruption of physiology and functions in male germ cells during the gestational period. This may lead to several reproductive health issues and infertility in offspring.

In vivo cells express their characteristics in three-dimensional (3D) microenvironments via cell-cell interactions through autocrine, contact-dependent, paracrine, and synaptic signaling, often between heterologous cell types. Various in vitro 3D microwell-based culture methods have been proposed to further identify cellular characteristics by recreating cellular environments, typically in the form of spheroids and organoids, thereby realizing contact-based cell-cell interactions. However, in vivo cells generally exhibit multiple cellular interaction modes that have not been completely evaluated using existing microwell-based methods. This has led to a demand for more advanced and comprehensive methods. This study introduces a novel apparatus, the membrane-bottomed microwell (MBM) for non-contact co-cultures and 3D cell cultures. The MBM is a combination of a Transwell and a microwell array; these have previously been utilized to facilitate heterologous cell co-culturing and spheroid 3D cell culturing, respectively. In the Transwell insert, the lower part of the MBM is immersed in the culture media in which the cells are being two-dimensionally (2D) cultured, and the spheroids of the MBM are affected by the 2D cultured cells via the membrane at the bottom of the microwell. Here, we describe the methods for manufacturing the MBM in detail and elucidate the results of simulations of diffusion through the bottom of the membrane. We validate the proposed MBM for the spheroid culture of spermatogonial stem cells (SSCs), which had previously been 2D co-cultured with Sandos inbred mouse (SIM)-derived 6-thioguanine- and ouabain-resistant (STO; a mouse embryonic feeder cell line) feeder cells. The proposed system is shown to facilitate successful SSC spheroid culturing with paracrine signaling of STOs through an apparatus that simplifies both the loading and the evaluation processes; therefore, we believe that our findings will enable a more comprehensive understanding of SSCs and associated phenomena and that our system can be applied to various in vitro cell and tissue experiments.

Bisphenol-A (BPA) exposure in an adult male can affect the reproductive system, which may also adversely affect the next generation. However, there is a lack of comprehensive data on the BPA-induced disruption of the association and functional characteristics of the testicular germ cells, which the present study sought to investigate. Adult male mice were administered BPA doses by gavage for six consecutive weeks and allowed to breed, producing generations F1&ndash, F4. Testis samples from each generation were evaluated for several parameters, including abnormal structure, alterations in germ cell proportions, apoptosis, and loss of functional properties of spermatogonial stem cells (SSCs). We observed that at the lowest-observed-adverse-effect level (LOAEL) dose, the testicular abnormalities and alterations in seminiferous epithelium staging persisted in F0&ndash, F2 generations, although a reduced total spermatogonia count was found only in F0. However, abnormalities in the proportions of germ cells were observed until F2. Exposure of the male mice (F0) to BPA alters the morphology of the testis along with the association of germ cells and stemness properties of SSCs, with the effects persisting up to F2. Therefore, we conclude that BPA induces physiological and functional disruption in male germ cells, which may lead to reproductive health issues in the next generation.

Data Availability Statement: Full-length MYH3 CDS sequence from KNP, KX538787; full-length MYH3 CDS sequence from Landrace, KX538788. Two-kb of 5’-UTR MYH3 genomic DNA sequence from KNP, KX549312; 0.5-kb of 3’-UTR MYH3 genomic DNA sequence from KNP, KX549313. Two-kb of 5’-UTR MYH3 genomic DNA sequence from Landrace, KX549311; 0.5-kb of 3’-UTR MYH3 genomic DNA sequence from Landrace, KX549314. Resequencing data access information is provided in S10 Table. All the raw data to produce figures and tables are available at https://datadryad.org/review?doi=doi:10.5061/dryad.dr32n87, Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5'-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs., This work was carried out with the support of >Cooperative Research Program for Agriculture Science & Technology Development (Project title: A research on performance improvement of ‘Nanchukmacdon’, a high quality pig breed developed using Korean native black pigs (KNP) in Jeju Island, Project No. PJ01012301 and PJ01262701)> Rural Development Administration (http://www.rda.go.kr), Republic of Korea. This work was also supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program, Republic of Korea (www.kribb.re.kr). M.P.E work was funded by AGL2016-78709-R (Ministerio de Economía y Competitividad, Spain; http://www.mineco.gob.es/portal/site/mineco/). T

The Kit gene is proto-oncogene, encoded stem cell factor and involved in melanocyte development. Also, Kit is expressed in epidermis and proliferation of melanoblasts which occur to pigmentation in skin, eyes and other tissues. Here we established porcine Kit genes overexpressed transgenic mice. The role of Kit gene was regulation in melanogensis remains unclear in porcine. Therefore, present study aims to ectopic expression porcine Kit gene in pigmentation of skin tissues. Our results showed that no effect on melanogenesis in ectopic Kit gene overexpressed transgenic mice. Further studies on Kit gene will provide insight into other specific roles in porcine and mice species.

Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the mouseOct4gene, which were demonstrated to controlOct4expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pigOct4and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.

Human embryonic stem cells (hESCs) are pluripotent, self-renewing cells derived from the inner cell mass of human blastocysts. During normal development, hESCs differentiate into 3 germ layers. Cellular lineages differentiated from hESCs express a set of genes that are exclusive to these specialized cells. Therefore, we hypothesized that endothelial cells derived from hESCs would express genes specific to endothelial cells. We previously isolated endothelial cells from human embryonic stem cells (hESC-ECs) using fluorescence-activated cell sorter (FACS). The aim of the current study was to identify genes associated with hESC-derived endothelial-like cells. Using suppression subtractive hybridization (SSH), we identified a set of genes specific to cells differentiated from hESC-ECs. We obtained 113 clones of expressed sequences that were more abundant in hESC-ECs compared with hESCs. Based on the NCBI GenBank database, 56 of these clones were known genes, 13 clones corresponded to nucleotides, 2 clones showed homology with chromosome sequences, and 42 clones showed no significant homology with any nucleotide sequences. These identified genes are related to cytoskeleton and cell adhesion, development, heat shock protein, metabolism, signal transduction, and transcription/nuclear-specific proteins. We anticipate that further study of these genes will provide crucial insights into their specific roles in the development of endothelial cells from hESCs.

Ribosomal protein L21 (RPL21) is a structural component of the 60S subunit of the eukaryotic ribosome. This protein has an important role in protein synthesis and the occurrence of hereditary diseases. Pig is a common laboratory model, however, to the best of our knowledge, its RPL21 gene has not been cloned to date. In this study, we cloned and identified the full-length sequence of the pig RPL21 gene for the first time. In addition, we examined its expression pattern and function by using overexpression or knockdown approaches. As a result, we obtained a 604 bp segment that contains a 483 bp open reading frame encoding 160 amino acids. The pig RPL21 gene is located in the “+” strand of chromosome 11, which spans 2167 bp from 4199792 to 4201958. Pig RPL21 protein has nine strands and two helices in its secondary structure. Pig RPL21 is predominantly expressed in ovary and lung, at lower levels in kidney, small intestine, and skin, and at the lowest levels in heart and liver. Furthermore, RPL21 expression is closely connected with cell proliferation and cell cycle arrest. The results are intended to provide useful information for the further study of pig RPL21.