Single Germ Cell Research Articles

New Insights on the Male and Female Reproductive Organs of Centrorhynchus globocaudatus (Acanthocephala), Intestinal Parasite of Birds of Prey.

Acanthocephalans are dioecious parasites that gain sexual maturity in the alimentary canal of their definitive hosts (gnathostome vertebrates). This initial survey by light and transmission electron microscopy was conducted on the functional organization of the ovarian balls and uterine bell in mature females and on Saefftigen's pouch and the copulatory bursa in males. We studied these structures via the example of Centrorhynchus globocaudatus (Palaeacanthocephala) in Falco tinnunculus and Buteo buteo, from the Province of Ferrara (Northern Italy). Our study confirms that the ovarian balls have surface microvilli and consist of a multinucleate supporting syncytium and a cellular region with oogonial syncytium, single germ cells, zygotes, and shelled eggs. Germ cells are embedded in the supporting syncytium. The ultrastructural features of these components and data on fertilization, shell formation, and release from the ovarian ball, alongside insights into the likely egg sorting function of the uterine bell, are provided. We also present light and electron microscopy observations of Saefftigen's pouch and a suggestion regarding its hydrostatic functioning in the eversion of the copulatory bursa.

THU199 Using Primary Human Ovarian Stromal Cells To Elucidate Stromal Cell Influences On Egg Quality Across Pubertal Development

Abstract Disclosure: E. Tsui: None. M.M. Laronda: None. Follicles, the functional unit of the ovary with the potential to develop into mature eggs, are composed of a single germ cell and somatic support cells (i.e., granulosa and theca cells). While animal studies have demonstrated a positive effect of ovarian stromal cells to follicle development, the ovarian stromal compartment remains largely undescribed. Moreover, egg quality is maximized after the pubertal transition, but the impact of puberty on ovarian stromal cells and their influence on egg quality is not well understood. To elucidate the relationship between puberty, ovarian stromal cells, and follicle development, we utilized a follicle growth assay in which murine ovarian secondary follicles were cultured in three groups: alone (control), with pre-pubertal derived primary human ovarian stromal cells, or with post-pubertal derived primary human ovarian stromal cells. Follicles (n = 40 per group, N = 4 independent experiments) were monitored over 8 days by brightfield microscopy and evaluated for diameter and survival. No difference in follicle diameter or survival was detected between follicles grown with either pre-pubertal or post-pubertal derived stromal cells (diameter: p = 0.93 for days 2-8; survival: p > 0.9999 at day 8), however, each group of follicles co-cultured with stromal cells grew to larger diameters than follicles grown alone at days 2 (pre-pubertal and post-pubertal, p = 0.005), 4 (pre-pubertal, p = 0.0006; post-pubertal, p = 0.0002), 6 (pre-pubertal, p = 0.03; post-pubertal, p = 0.02), and 8 (pre-pubertal, p = 0.001; post-pubertal, p = 0.004). Survival between follicles grown alone or co-cultured with stromal cells trended towards significance (p = 0.0526 for both pre-pubertal and post-pubertal stroma groups), and all groups demonstrated average survival at day 8 above 70%. Follicle rupture was more commonly observed in follicles cultured with stromal cells (control, 10%; pre-pubertal, 40%; post-pubertal, 80%), with additional maturation studies in progress. Concurrently, the cellular landscape of ovarian stromal cells across puberty was evaluated with single cell RNA sequencing (n = 11, age range = 0.34 to 16.09 years, Tanner stages 1 and 3-5). Analyses revealed 12 sub-clusters representing five major cell types: endothelial cells, theca cells, vascular smooth muscle cells, granulosa cells, and immune cells. This data is screened through existing human secretome databases to identify candidate secreted factors that will be investigated in future follicle growth assays. These results demonstrate a benefit of human ovarian stromal cells in our follicle growth assay and, in conjunction with ongoing maturation and sequencing experiments, validate the utility of this assay to further understand how puberty affects the stromal microenvironment to impact egg quality, insight that can be used to develop and improve future reproductive technologies. Presentation: Thursday, June 15, 2023

The study of features in sperm and spermatogenesis from males of the genus Ovis with different genotypes

Relevance. Interspecific hybridization of domestic animals with wild related species is considered as one of the promising directions in animal husbandry in the framework of increasing the genetic biodiversity of the gene pool of agricultural animals. The article presents the results of studies of the reproductive characteristics in animals of the genus Ovis with different genotypes.Methods. The objects of research were purebred sheep of the Romanov breed, mouflon and interspecific hybrids from sheep of the Romanov breed with mouflon. The qualitative and quantitative indicators of the sperm at the age of 9, 12 and 18 months were studied. An assessment of the morphometric parameters of spermatozoa from interspecific hybrids is given in comparison with the original parental species. The testes histological studies of purebred and hybrid animals at the age of 12 months were carried out.Results. Differences in several indicators of sperm production and spermatogenesis in purebred and hybrid animals depending on the genotype were revealed. A decrease in the volume of ejaculate and concentration of spermatozoa in hybrid animals relative to purebred males at the age of 12 and 18 months was established in 3.5, 2.6 times and in 1.6, 2.1 times, respectively (р < 0,01). An increase in the proportion of spermatozoa with abnormal morphology in hybrid animals relative to purebred males in 2.9–3.3 times was revealed (р < 0,01). The obtained data are confirmed by histological studies. There is a decrease in the area and volume of seminiferous tubules in hybrid animals by 9.7% and 37.1%, respectively, compared with similar indicators of purebred males of the Romanov breed (р < 0,01). It was shown that in purebred animals in the lumen of the seminiferous tubule have many mature germ cells — sperm, while in hybrid males the presence of single germ cells was established, which indicates a later puberty of hybrid animals compared to the original maternal form — sheep of the Romanov breed.

Diagnostic importance of hepatocyte nuclear factor 1 beta (HNF1β) in testicular tumors and its sensitivity for the detection of yolk sac tumors: an immunohistochemical analysis

Objectives: Testicular tumors are common solid malignancies in young fertile men, and most are germ cell tumors. In general, they originate from a single germ cell and transform into different tumor types or present with the coexistence of different morphological patterns. Due to the heterogeneity of these tumors, immunohistochemical markers are frequently used in their differential diagnosis. In recent years, some studies have indicated hepatocyte nuclear factor 1 beta (HNF1β) can be used in the differential diagnosis of testicular tumors, especially yolk sac tumors (YSTs). In this study, we aimed to investigate the general expression status of HNF1β in all testicular tumors and determine its importance in YST detection. Methods: A total of 144 testicular tumors treated with orchiectomy between 2011 and 2020 were included in our study. The pathological diagnosis reports of these cases were retrospectively reviewed and their general prognostic features were determined. HNF1β immunohistochemical staining was applied to the characteristic paraffin blocks representing the lesions. Staining was evaluated in terms of severity and prevalence. Results: Most cases (38.2%) were seminomas, followed by mixed germ cell tumors (34.0%, 49/144), embryonic carcinomas (7.6%), pure YSTs (4.9%), and others (Leydig cell tumors, mesenchymal tumors, lymphomas, etc.). No HNF1β immunostaining was observed in any of the seminomatous lesions. A high level of staining was present in almost all the pure YSTs and tumor areas with the YST component. HNF1β had a specificity of 95.1% and sensitivity of 87.1% in the detection of YSTs. Conclusions: HNF1β has high specificity and sensitivity in detecting YSTs among testicular tumors, and therefore we consider that it can be routinely used to detect the presence of YSTs, especially in patients with mixed germ cell tumors.

Single-cell RNA-Seq reveals a highly coordinated transcriptional program in mouse germ cells during primordial follicle formation.

The assembly of primordial follicles in mammals represents one of the most critical processes in ovarian biology. It directly affects the number of oocytes available to a female throughout her reproductive life. Premature depletion of primordial follicles contributes to the ovarian pathology primary ovarian insufficiency (POI). To delineate the developmental trajectory and regulatory mechanisms of oocytes during the process, we performed RNA‐seq on single germ cells from newborn (P0.5) ovaries. Three cell clusters were classified which corresponded to three cell states (germ cell cyst, cyst breakdown, and follicle) in the newborn ovary. By Monocle analysis, a uniform trajectory of oocyte development was built with a series of genes showed dynamic changes along the pseudo‐timeline. Gene Ontology term enrichment revealed a significant decrease in meiosis‐related genes and a dramatic increase in oocyte‐specific genes which marked the transition from a germ cell to a functional oocyte. We then established a network of regulons by using single‐cell regulatory network inference and clustering (SCENIC) algorithm and identified possible candidate transcription factors that may maintain transcription programs during follicle formation. Following functional studies further revealed the differential regulation of the identified regulon Id2 and its family member Id1, on the establishment of primordial follicle pool by using siRNA knockdown and genetic modified mouse models. In summary, our study systematically reconstructed molecular cascades in oocytes and identified a series of genes and molecular pathways in follicle formation and development.

Three distinct hematological malignancies from a single germ cell tumor: a case report

BackgroundThe association between non seminomatous germ cell tumors (GCTs) and hematological malignancies of rare lineage has been described in the literature. In some of these cases there is evidence that the leukemia derives from a pluripotent primitive clone present in the original germ cell tumor.Case presentationWe present a highly unusual case of a 23-year-old man of South Asian origin with a history of Klinefelter’s syndrome who initially developed mediastinal non seminomatous GCT. Following treatment with surgery and standard chemotherapy he went on to develop three different hematological malignancies of distinct lineages in sequential fashion over a short time period. Despite treatment with multiple intensive chemotherapy regimens and a matched unrelated donor allogeneic stem cell transplant, he died 41 months after initial diagnosis of his GCT and 10 months after the first diagnosis of hematological malignancy.ConclusionsThis is an extreme case that highlights the pluripotency and aggressiveness of these GCT-derived hematological malignancies, and the need for novel therapeutic approaches.

Single-cell RNA sequencing reveals the landscape of early female germ cell development.

Meiosis initiation is a crucial step for the production of haploid gametes, which occurs from anterior to posterior in fetal ovaries. The asynchrony of the transition from mitosis to meiosis results in heterogeneity in the female germ cell populations, which limits the studies of meiosis initiation and progression at a higher resolution level. To dissect the process of meiosis initiation, we investigated the transcriptional profiles of 19363 single germ cells collected from E12.5, E14.5, and E16.5 mouse fetal ovaries. Clustering analysis identified seven groups and defined dozens of corresponding transcription factors, providing a global view of cellular differentiation from primordial germ cells toward meiocytes. Furthermore, we explored the dynamics of gene expression within the developmental trajectory with special focus on the critical state of meiosis. We found that meiosis initiation occurs as early as E12.5 and the cluster of oogonia_4 is the critical state between mitosis and meiosis. Our data provide key insights into the transcriptome features of peri-meiotic female germ cells, which offers new information not only on meiosis initiation and progression but also on screening pathogenic mutations in meiosis-associated diseases.

A novel pattern of germ cell divisions in the production of hymenopteran insect eggs.

Egg development is a defining process of reproduction in higher eukaryotes. In the fruit fly, Drosophila melanogaster, this process begins with four mitotic divisions starting from a single germ cell, producing a cyst of 16 cystocytes; one of these cells will become the oocyte and the others supporting nurse cells. These mitotic divisions are exceptional because cytokinesis is incomplete, resulting in the formation of cytoplasmic bridges known as ring canals that interconnect the cystocytes. This organization allows all cystocytes to divide synchronously during each mitotic round, resulting in a final, power-of-2 number of germ cells. Given that numerous insects obey this power-of-2 rule, we investigated if strict cell doubling is a universal, underlying cause. Using confocal microscopy, we found striking departures from this paradigm in three different power-of-2 insects belonging to the Apocrita suborder (ants, bees and wasps). In these insects, the earliest-formed cystocytes cease to divide during the latter mitotic cycles while their descendants undergo further division, thereby producing a 'radial' direction of division activity. Such cystocyte division patterns that depart from strict cell doubling may be 'fine-tuned' in order to maintain a final, power-of-2 germ cell number.

Sacrococcygeal teratoma: a case report

Teratomas are germ cell tumours composed of multiple types of cells derived from more than a single germ cell layer. The most common site of an extra gonadal teratoma is the sacrococcygeal region. We report a case of a 10-day-old female child with a large swelling in the sacrococcygeal region extending laterally into the buttocks. X-ray showed a large mass projecting from the lower pelvic region and delineate the extent of the tumour and the involvement of tissues. Image findings and clinical presentation led to the diagnosis of sacrococcygeal teratoma. Tumour was surgically excised with prior pre-anaesthetic checkup, post-operative period was uneventful. She was discharged with advice to come in follow-up surgical outpatient clinic. There was no evidence of tumour recurrence till 1 year of age. She had normal growth curve for her age with normal developmental milestones.

Germ cell proliferation and cluster behavior in ovarioles of Sialis flavilatera (Megaloptera: Sialidae) during larval growth

Telotrophic meroistic insect ovaries are assigned to four different types. The Sialis type is found in Sialidae (Megaloptera), Raphidioptera and a coleopteran subgroup (Myxophaga: Hydroscaphidae). King and Büning (1985) proposed a hypothetical model for the development of this ovariole type; however, a detailed description of ovarian development in Sialis was missing so far. Using light and electron microscopy, we investigated developing ovaries of Sialis flavilatera starting in the 10th month of the biennial larval phase until adulthood. At least from the 10th month onwards, a Sialis ovariole anlage contains a single germ cell syncytium, whose growth is promoted by a mitotic cell population maintained in its anterior compartment. The stem-like, dividing germ cells form synchronous sub-clusters consisting of 2–16 cystocytes, which are spatially arranged in bigger rosettes that stay connected to each other via cytoplasmic tubes. Within individual rosettes, cells communicate by centrally gathering intercellular bridges. Following each round of cystocyte division and subsequent rosette formation, plasma membrane wrinkles sprout near newborn bridges, elongate, and interdigitate with the preexisting membrane tubes. In this way the membrane labyrinth emerges and grows. Germ cells leaving the proliferation zone posteriorly enter meiotic prophase. Hypotheses on the phylogenetic origin of this ovary type are discussed in the light of our results.

In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells.

229 LONG-TERM PROPAGATION AND CRYOPRESERVATION OF CAT SPERMATOGONIAL STEM CELLS

Spermatogonial stem cells (SSC) are unique adult stem cells that reside within the seminiferous tubules of the testis. As stem cells, SSC maintain the ability to self-replicate, providing a potentially unlimited supply of cells and an alternate source for preservation of the male genome. While self-renewing, long-term SSC culture has been achieved in mice, there is virtually no information regarding culture requirements of felid SSC. Therefore, the objectives of this study were to (1) evaluate the ability of 3 feeder cell lines to support germ cell colony establishment in domestic cats (Felis catus), and (2) assess long-term culture using the best feeder(s). Cells isolated enzymatically from peripubertal cat testes (n = 4) and enriched by differential plating were cultured on mouse embryonic fibroblasts (STO line), mouse-derived C166 endothelial cells, and primary cat fetal fibroblasts (cFF). Colony morphology was assessed every other day and immunocytochemistry (ICC) was performed to investigate expression of SSC markers. At 5 days in vitro (DIV), a cluster forming activity assay was used to estimate the number of SSC supported by each feeder cell line. Differences among treatments were compared using Tukey-Kramer adjustment for pair-wise mean comparisons. Data were expressed as mean cluster number ± SE per 105 cells input. When cultured on STO feeders, cat germ cells were distributed as individual cells. On both C166 cells and cFF feeders, germ cell clumps (morphologically consistent with SSC colonies in other species) were observed. Immunocytochemistry revealed that the single germ cells present on STO feeders were positive for UCHL1 and weakly expressed PLZF and OCT4. Cells within the germ cell clumps on C166 cells and cFF co-expressed all 3 SSC markers. The C166 cells supported a higher number of germ cell clusters (77.4 ± 13.8) compared with STO (3.5 ± 1.1, P = 0.0003) or cFF (22.7 ± 1.0, P = 0.0024). Therefore, subsequent subculture experiments were performed exclusively with C166 feeder layers. Cultures from 2 donors were passaged at 12 DIV and periodically as needed thereafter. Germ cell clumps consistently reestablished following each subculture and immunocytochemistry analysis confirmed maintenance of all 3 SSC markers. Cells were also positive for alkaline phosphatase activity. Cells that had been cryopreserved in culture medium with 5% (vol/vol) dimethyl sulphoxide after144 DIV (7 passages) were thawed and cultured for an additional 18 days. These cells continued to express SSC markers and form germ cell clusters. Taken together, these data demonstrate that C166 feeder cells can facilitate colony establishment and in vitro propagation of germ cell clumps in the domestic cat. This represents an important first step towards attainment and optimization of a long-term SSC culture system in the cat. This system would provide a mechanism to explore regulation of spermatogenesis, test species-specific drugs, and produce transgenic biomedical models.

VASA (DDX4) is a Putative Marker for Spermatogonia, Spermatocytes and Round Spermatids in Stallions.

Expression of the protein DDX4/MVH, or VASA, has been reported in germ cells of several species. The main objectives of this study were to (i) investigate VASA expression patterns in testicular cells of stallions at two different reproductive stages (pre-pubertal and post-pubertal) and (ii) evaluate the use of VASA antibody as a molecular marker for single germ cells from stallions. Testicular tissues were obtained from stallions and categorized as pre-pubertal and post-pubertal based on the formation of lumen and status of spermatogenesis on the cross section of seminiferous tubules. The results of Western blot showed a VASA protein band located at 76 kDa, indicating that the rabbit antibody has a cross-reactivity with horse testicular tissues. The result of immunolabelling showed that VASA was expressed in the cytoplasm of spermatogonia at both reproductive stages and in spermatocytes and round spermatids at the post-pubertal stage. GATA4-positive Sertoli cells and Leydig cells located in the interstitial space were not immunolabelled with VASA. These results suggest that VASA can be utilized as a molecular marker for germ cells of stallions at pre-pubertal and post-pubertal stages. Interestingly, immunolabelling intensity was significantly higher in pachytene spermatocytes compared to spermatogonia and round spermatid. VASA antibody was also effective for staining of single germ cell preparations. In conclusion, VASA protein expression can be used as a marker for identification of spermatogonia, spermatocytes and round spermatids in testicular tissues of stallions.

Genetics of the ovarian reserve.

Primordial follicles or non-growing follicles (NGFs) are the functional unit of reproduction, each comprising a single germ cell surrounded by supporting somatic cells. NGFs constitute the ovarian reserve (OR), prerequisite for germ cell ovulation and the continuation of the species. The dynamics of the reserve is determined by the number of NGFs formed and their complex subsequent fates. During the reproductive lifespan, the OR progressively diminishes due to follicle atresia as well as recruitment, maturation, and ovulation. The depletion of the OR is the major determining driver of menopause, which ensues when the number of primordial follicles falls below a threshold of ∼1,000. Therefore, genes and processes involved in follicle dynamics are particularly important to understand the process of menopause, both in the typical reproductive lifespan and in conditions like primary ovarian insufficiency, defined as menopause before age 40. Genes and their variants that affect the timing of menopause thereby provide candidates for diagnosis of and intervention in problems of reproductive lifespan. We review the current knowledge of processes and genes involved in the development of the OR and in the dynamics of ovarian follicles.

UTF1, a putative marker for spermatogonial stem cells in stallions.

Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of spermatogonia that express UTF1. Testicular samples were collected and categorized based on the age of the horses as follows: pre-pubertal (<1 yr), pubertal (1–1.5 yr), post-pubertal (2–3 yr), and adult (4–8 yr). Western blot analysis was utilized to determine the cross-activity of the UTF1 antibody to horse testes tissues. Immunohistochemistry was conducted to investigate the UTF1 expression pattern in germ cells at different reproductive stages. Whole mount staining was applied to determine the subpopulation of UTF1-positive spermatogonia. Immunohistological analysis showed that most germ cells in the pre-pubertal and pubertal stages were immunolabeled with UTF1, whereas only a few germ cells in the basal compartment of the seminiferous tubule cross-sections of post-pubertal and adult tissues were UTF1-positive. No staining was observed in the Sertoli or Leydig cells at any reproductive stages. Whole mount staining showed that As, Apr, and chains of 4, 8, 16 Aal spermatogonia were immunolabeled with UTF1 in the post-pubertal stallion tubule. Isolated single germ cells were also immunolabeled with UTF1. In conclusion, UTF1 is expressed in undifferentiated spermatogonia, and its antibody can be used as a putative marker for SSCs in stallions.

Primary rectal mature teratoma: a case report

This report describes a rare case of primary rectal mature teratoma in a 56-year-old woman. She was referred to the outpatient clinic with a large pedunculated rectal mass, which was found during a regular health check-up. Polypectomy was performed and microscopic findings showed various structures derived from all three germ cell layers. Epidermis, hair follicles, sebaceous glands, eccrine glands, and apocrine sweat glands, with some scattered melanophages and lymphocytes were present as ectodermal derivatives. Smooth muscle fibers, blood vessels, and fibrous and adipose tissues were found as mesodermal derivatives. In addition, thyroid follicles, mucinous glands, and bronchial respiratory epithelium with peribronchial glands were detected as endodermal derivatives. She is healthy and has shown no evidence of recurrence or distant metastasis for 25 months post-surgical resection. Primary rectal teratomas are generally benign and primarily affect women. Therefore, minimally invasive surgical procedures, such as endoscopic polypectomy for a pedunculated polyp and segmentectomy for a larger mass, are satisfactory in most cases. Induction of primary rectal teratomas has been suggested to occur mainly by errors in a single germ cell after the end of meiosis I; in addition, it has also been suggested that the difference in gender incidence may be associated with differences in sex chromosomes between males and females rather than with anatomical proximity between ovary and rectum.

Immunohistochemical Expression of Markers of Immaturity in Sertoli and Seminal Cells in Canine Testicular Atrophy

During maturation from fetal to adult testis, both Sertoli cells (SCs) and germ cells (GCs) switch from an immature to a mature immunophenotype. Immature canine SCs express cytokeratins (CKs), desmin (DES), vimentin (VIM), anti-Müllerian hormone (AMH) and inhibin (INH)-α, while mature SCs retain only expression of VIM. Immature GCs express placental alkaline phosphatase (PLAP), which is lost in spermatocytes. Re-expression of markers of immaturity has been observed in human atrophic testes and in human and canine testicular tumours. In human medicine, testicular atrophy is considered a risk factor for testicular cancer. In the present study 13 canine atrophic testes were examined immunohistochemically. VIM was expressed in the SCs of all cases, while CK, DES, INH-α and AMH were expressed in a variable percentage of SCs in two, five, five and eight cases, respectively. PLAP was expressed by single GCs in one case. Markers of immaturity are therefore expressed by SCs and GCs in canine atrophic testes. Similar results were reported previously in canine testicular neoplasia, suggesting that testicular atrophy may represent a risk factor for tumour development in the dog.

Comparative pathogenesis of type 1 (European genotype) and type 2 (North American genotype) porcine reproductive and respiratory syndrome virus in infected boar

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) now has two main genotypes, genotype 1 (European) and genotype 2 (North American). There is a lack of data on the comparison of pathogenicity of the two genotypes in boars. The objectives of the present study were to evaluate the amount of PRRSV present in semen over time and compare the viral distribution and microscopic lesions of type 1 and type 2 PRRSV-infected boars.MethodsTwenty-four 8-month-old PRRSV-naïve Duroc boars were randomly allocated to 3 treatment groups. The boars in groups 1 (n = 9) and 2 (n = 9) were intranasally inoculated with type 1 or type 2 PRRSV, respectively. The boars in groups 1 (n = 6) served as negative controls. Semen and blood samples were collected up to 35 days post-inoculation (dpi), and necropsies were performed on 14, 21, and 35 dpi.ResultsThere were no significant differences in the genomic copy number of PRRSV, microscopic testicular lesion score, number of PRRSV-positive germ cells, or number of apoptotic cells between the type 1 and type 2 PRRSV-infected boars throughout the experiment. Histopathological changes were manifested by the desquamation of spermatocytes and the presence of multinucleated giant cells in seminiferous tubules of both type 1 and type 2 PRRSV-infected boars. The distribution of PRRSV-positive cells was focal; the virus was found in single germ cells or small clusters of germ cells, localized to the spermatogonia, spermatocytes, spermatids, and non-sperm cells in type 1 and type 2 PRRSV-infected boars.ConclusionsThe results of this study demonstrated that two genotypes of PRRSV do not have significantly different virulence toward the male reproductive system of pigs.

DBre1/dSet1-dependent pathway for histone H3K4 trimethylation has essential roles in controlling germline stem cell maintenance and germ cell differentiation in the Drosophila ovary

The Drosophila ovarian germline stem cells (GSCs) constantly experience self-renewal and differentiation, ensuring the female fertility throughout life. The balance between GSC self-renewal and differentiation is exquisitely regulated by the stem cell niche, the stem cells themselves and systemic factors. Increasing evidence has shown that the GSC regulation also involves epigenetic mechanisms including chromatin remodeling and histone modification. Here, we find that dBre1, an E3 ubiquitin ligase, functions in controlling GSC self-renewal and germ cell differentiation via distinct mechanisms. Removal or knock down of dBre1 function in the germline or somatic niche cell lineage leads to a gradual GSC loss and disruption of H3K4 trimethylation in the Drosophila ovary. Further studies suggest that the defective GSC maintenance is attributable to compromised BMP signaling emitted from the stem cell niche and impaired adhesion of GSCs to their niche. On the other hand, dBre1-RNAi expression in escort cells causes a loss of H3K4 trimethylation and accumulation of spectrosome-containing single germ cells in the germarium. Reducing dpp or dally levels suppresses the germ cell differentiation defects, indicating that dBre1 limits BMP signaling activities for the differentiation control. Strikingly, all phenotypes observed in dBre1 mutant ovaries can be mimicked by RNAi-based reduced expression of dSet1, a Drosophila H3K4 trimethylase. Moreover, genetic studies favor that dBre1 interacts with dSet1 in controlling GSC maintenance and germ cell differentiation. Taken together, we identify a dBre1/dSet1-dependent pathway for the H3K4 methylation involved in the cell fate regulation in the Drosophila ovary.

Cellular Origin of Testis-Derived Pluripotent Stem Cells: A Case for Very Small Embryonic-Like Stem Cells

It has been suggested that testicular germ stem cells represent the only adult body stem cells that dedifferentiate and reprogram into a pluripotent state without any genetic modification. Emerging debate about the authenticity of embryonic stem cell (ES)-like cells derived from adult testicular tissue has prompted us to put forth this letter. We wish to reinforce our findings that pluripotent very small embryonic-like stem cells (VSELs) exist as a small population in adult mammalian testis and may result in ES-like colonies. Because of their small size, it is felt that VSELs could be contaminating the initial cells used for seeding, although efforts were made to place a single germ cell per well in a 96-well plate for clonal expansion, or magnetic activated cell sorting (MACS)-sorted α6 integrin positive cells were used. On a similar note, it is felt that the presence of VSELs in various tissues along with mesenchymal stem cells (MSCs) may provide an alternative explanation to the transdifferentiation potential of MSCs. We conclude that like Oct-4 biology, presence of VSELs in adult body tissues has somewhat surprised stem cell biologists.