Cell-cycle-dependent Colonization of Mouse Spermatogonial Stem Cells After Transplantation into Seminiferous Tubules

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5–10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.     

同源物种不同饲养层对鸡精原干细胞体外培养的影响

分别以鸡胚来源的不同细胞为饲养层培养精原干细胞,比较3种不同的饲养层对精原干细胞体外培养的影响。结果显示:精原干细胞在鸡胚成纤维细胞饲养层和鸡睾丸支持细胞饲养层上存活时间较长、生长增殖状况良好。在鸡胚成纤维饲养层上传至四代,每代的AKP阳性克隆率分别为45%、40%、36%和21%。在以睾丸支持细胞为饲养层进行培养时传至三代,每代的AKP阳性克隆率分别是40%、32%、和22%。而以鸡肝细胞作为饲养层,精原干细胞未见有克隆形成,不能进行传代培养,表明鸡胚肝细胞饲养层不适合培养精原干细胞。     

血清和饲养层对水牛精原干细胞体外培养的影响

本研究首先将水牛睾丸经二步酶消化法制成单细胞悬液,依次采用差速贴壁法和Percoll不连续密度梯度离心法分离和纯化精原细胞。在制备好的小鼠胎儿成纤维细胞饲养层上,采用含2．5％血清的培养液对精原细胞进行体外培养,观察血清和成纤维细胞对精原细胞生长的影响,并在体外成功培养了2周通过提取体外培养精原干细胞总FZNA,设计引物并对其进行基因鉴定和免疫细胞化学鉴定,证实体外培养所得的细胞仍保持有精原干细胞的特性,并且该克隆是处在未分化状态的精原干细胞形成的。上述研究可为体外建立水牛精原干细胞长期培养体系提供技术支撑。     

绵羊精原干细胞的分离纯化与鉴定

旨在探究一种简便高效的绵羊精原干细胞(SSCs)分离纯化方法,为后续研究SSCs奠定基础。本实验采用两步酶消化法对4月龄的绵羊睾丸进行消化处理,获得单细胞悬液。将睾丸细胞接种于0.2%的明胶预处理培养皿中,根据各细胞贴壁能力和速度的不同将SSCs纯化,并在纯化后用SSCs特异标记分子PLZF进行免疫荧光鉴定。结果表明,经免疫荧光染色得出存在PLZF阳性信号。这说明采用两步酶消化和差异贴壁法可以得到纯度较高的SSCs。     

Molecular and Cellular Characterization of Buffalo Bone Marrow‐Derived Mesenchymal Stem Cells

Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow‐derived MSCs (BM‐MSCs) at molecular and cellular level. Buffalo BM‐MSCs were isolated by Ficoll density gradient method and cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony‐forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM‐MSCs have the capacity to form plastic adherent clusters of fibroblast‐like cells and were successfully maintained up to 16^th passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT‐PCR analysis and protein localization study showed that buffalo BM‐MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM‐MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM‐MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy.     

Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.     

小鼠精原干细胞体外培养体系建立及功能鉴定

旨在通过探索ICR品系小鼠精原干细胞(spermatogonial stem cells,SSCs)的体外稳定培养,为别的品系小鼠和大动物的SSCs体外培养提供参考.本研究采集6?8日龄ICR乳鼠的睾丸,用两步酶消化法(胶原酶和胰蛋白酶)和差速贴壁法来纯化SSC细胞.用不同的饲养层(小鼠胎儿成纤维细胞(mouse em...     

Sperm‐TALP: An Alternative Extender for Retrieving and Diluting Epididymal Sperm in the Domestic Cat

Effects of sperm‐TALP (TALP) on the quality of fresh‐extended and frozen‐thawed epididymal cat sperm were evaluated. The epididymides suspended in Tris–glucose–citrate solution (Tris), a conventional medium, and TALP were cut into small pieces to recover epididymal sperm. In experiment 1, the sperm pellets remained after centrifugation were re‐suspended (1 : 2, v/v) in Tris and TALP. The sperm quality in all four groups, that is, sperm retrieved with Tris (I and II) or TALP (III and IV) and diluted with Tris (I and III) or TALP (II and IV) was assessed. The sperm motility at the 0‐h incubation in TALP–TALP was superior to that of the rest (p < 0.001 to p = 0.04). At the 2‐h incubation, the motility in Tris/TALP–TALP was greater than that in Tris/TALP–Tris (p ≤ 0.001). In experiment 2, after centrifugation, the sperm pellets were added with freezing extenders and frozen. The thawed sperm previously retrieved from the epididymides with Tris and TALP were allotted so as not to further diluted (Tris/TALP–O) and to further diluted (1 : 1, v/v) with Tris (Tris/TALP–Tris) and TALP (Tris/TALP–TALP) and were evaluated the quality. At both incubation times, the motility of frozen‐thawed sperm recovered with TALP (TALP–O/Tris/TALP) was comparable with or significantly higher than that in the Tris groups (Tris–O/Tris/TALP; p = 0.003 to p > 0.05). The motility and viability of thawed sperm in Tris–Tris were significantly decreased during the 2‐h incubation (p = 0.007 for the motility and p = 0.01 for the viability). In both experiments, neither type of diluent (Tris vs TALP) nor incubation time (0 vs 2 h) significantly affected the sperm membrane integrity under hypo‐osmotic condition (p > 0.05). According to beneficial effects on the quality of fresh‐extended and frozen‐thawed sperm demonstrated, sperm‐TALP could be used as an alternative medium for recovering sperm from the epididymides and for diluting epididymal sperm in the domestic cat.     

Mycobacterioses in the Domestic Carnivora—Present-day Epidemiology of Tuberculosis in the Cat and Dog

Abstract— The present situation with mycobacterioses in domestic carnivores (dogs and cats) is reviewed with particular reference the current epidemiology of tuberculosis in small animals in France. Animal infections with atypical mycobacteria are dealt with as well as particular emphasis being placed on the creation of preventive health programmes and the compulsory notification of cases.
Résumé— L'étude de la présence des mycobactérioses chez les carnivores domestiques (chiens et chats) est faite, en attachant une importance toute particulière à I'épidémiologie actuelle de la tuberculose chez les petits animaux en France.
Les infections animales comportant des mycobactéries typiques y sont traitées, et la création de programmes sanitaires préventifs ainsi que I'établissement d'une loi obligeant ä mentionner des cas similaires y sont tout particuliërement soulignés.
Zusammenfassung— Die gegenwärtige Sachlage mit Mykobakteriosen in fleischfressenden Haustieren (Hunde und Katzen) wird unter besonderer Berücksichtigung der jetzigen Epidemiologic von Tuberkulose kleiner Tiere in Frankreich untersucht. Tierinfektionen durch typische Mykobakterien werden beschrieben, ebenso wie besonderer Nachdruck auf die Schaffung vorbeugender Gesundheitsprogramme und zwangsläufiger Mitteilungsvorschriften von Krankheitsfällen gelegt.     

Expression of Survivin,Bcl‐2 and Bax Proteins in the Domestic Cat (Felis catus) Endometrium During the Oestrus Cycle

Apoptosis has been shown to be an important regulator of endometrium function. To clarify the regulation of apoptosis in the cat endometrium during the normal oestrus cycle, the expressions of the apoptosis‐related proteins (Bcl‐2 and Bax) and their correlation to the inhibitor of apoptosis protein Survivin were analysed using immunohistochemistry. The TUNEL technique (TdT‐mediated dUTP nick end labelling) was also used to detect DNA fragmentation characteristic of apoptotic cells. The results demonstrated that TUNEL labelling is not effective for the detection of apoptosis in cat endometrium. Survivin was expressed in the luminal and glandular epithelial cells of cat endometrium during all phases of the oestrus cycle. Survivin was localized in both the cytoplasm and nuclei of superficial and deep uterine gland cells during the luteal phase, while only cytoplasmic staining was observed during the follicular and anoestrus phases. Bax immunoreactivity in the cytoplasm of luminal and glandular epithelial cells as well as the smooth muscle cells of blood vessels was weak in the anoestrus phase. Compared with anoestrus, the intensity of Bax immunostaining was moderate in the follicular phase and increased dramatically in the luteal phase. Bcl‐2 immunostaining in the cytoplasm of luminal and glandular epithelial cells was moderate in the anoestrus phase. During the early follicular phase, cytoplasmic Bcl‐2 immunostaining was detected mostly in glandular epithelial cells. In the mid‐follicular phase, in glands, the amount of Bcl‐2 protein increased progressively from the superficial to the deep layer. In contrast, the expression of Bcl‐2 decreased in the secretory phase, being very low or absent in the mid‐ and late luteal phases. The overall results suggest that Survivin, Bax and Bcl‐2 proteins may cooperatively contribute to cell apoptosis and cell proliferation in the cat uterus during the oestrus cycle.     

Neurogenic Differentiation of EGFP Gene Transfected Amniotic Fluid‐Derived Stem Cells from Pigs at Intermediate and Late Gestational Ages

The aims of this study were (i) to determine whether amniotic fluid‐derived stem cells (amniotic fluid‐derived stem; AFS cells) could be isolated from pigs at intermediate and late gestational ages, and (ii) to determine if these AFS cells could be differentiated in vitro into neural lineages following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Amniotic fluid‐derived stem cells were isolated from embryonic day 60 and day 110 porcine amniotic fluid respectively, and transfected with EGFP gene using lipofection. The transfected AFS cells were induced to differentiate into cells of neuronal lineages. Markers associated with undifferentiated AFS cells and their neural derivatives were tested by polymerase chain reaction. The results demonstrated that porcine AFS cells could be isolated at intermediate and late gestational ages and that transfected AFS expressed EGFP and could be induced to differentiate in vitro. Undifferentiated AFS cells expressed POU5F1, THY1 and SOX2, while following differentiation cells expressed markers for astrocytes (GFAP), oligodendrocytes (GALC) and neurons (NF, ENOS and MAP2).     

New Insights into the Neural Differentiation Potential of Canine Adipose Tissue‐Derived Mesenchymal Stem Cells

Adipose tissue‐derived stem cells (ASCs) can be obtained from different adipose tissue sources within the body. It is an abundant cell pool, easily accessible, suitable for cultivation and expansion in vitro and preparation for therapeutic approaches. Amongst these therapeutic approaches are tissue engineering and nervous system disorders such as spinal cord injuries. For such treatment, ASCs have to be reliably differentiated in to the neuronal direction. Therefore, we investigated the neural differentiation potential of ASCs using protocols with neurogenic inductors such as valproic acid and forskolin, while dog brain tissue served as control. Morphological changes could already be noticed 1 h after neuronal induction. Gene expression analysis revealed that the neuronal markers nestin and βIII‐tubulin as well as MAP2 were expressed after induction of neuronal differentiation. Additionally, the expression of the neurotrophic factors NGF, BDNF and GDNF was determined. Some of the neuronal markers and neurotrophic factors were already expressed in undifferentiated cells. Our findings point out that ASCs can reliably be differentiated into the neuronal lineage; therefore, these cells are a suitable cell source for cell transplantation in disorders of the central nervous system. Follow‐up studies would show the clinical benefit of these cells after transplantation.     

Confocal Imaging of Trans‐epithelial Trafficking by Immune and Umbilical Cord Stem Cells in the Neonatal Porcine Intestine

Maternal colostral leucocytes (CL) and peripheral blood mononuclear cells (PBMC) enter the neonatal circulation after ingestion in pigs and cattle. Porcine umbilical cord matrix stem cells (PUCs) are relatively non‐immunogenic after initial allogeneic transplantation. Using intestinal explant cultures incubated with labelled cells and confocal microscopy, we demonstrated trans‐epithelial trafficking of exogenous CL, PBMC and PUCs below the luminal surface after 72 h of culture. We orally administered PBMC and PUCs to pre‐colostral neonatal pigs and tracked their location 8 or 24 h later. Both PBMC and PUCs were found in the intestinal wall of all samples. Exposure to 25% of acellular colostrum had no detected effect on trafficking. Labelled PUCs and PBMC were detected on the surface of the epithelium and in the lamina propria 8 h post‐treatment and PBMC were also in the superficial submucosa. At 24 h, PUCs and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa and deep submucosa. Our findings show the potential of PUCs for allogeneic engraftment in the neonatal intestine and may lead to cell‐based delivery of therapeutics.     

Impact of Ultra‐Rapid Freezing on the Motility,Morphology, Viability and Acrosome Integrity of Epididymal Cat Sperm Diluted in Sucrose‐Based Extenders

The objective of the present study was to investigate the influence of different sucrose‐based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra‐rapid freezing method. Nine cats were castrated, and collected semen was diluted 1 : 1 with Dulbecco`s phosphate‐buffered saline‐BSA1%‐based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6 m ). After ultra‐rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p < 0.01) and morphologically normal (p < 0.01) sperm was higher in the sucrose‐supplemented groups than in the sucrose‐free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4 m sucrose, spermatozoa viability showed higher values (57.0 ± 4.7; p < 0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra‐rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4 m of sucrose was used. This is the first report on sperm ultra‐rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.     

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

Ultrasonographic Percutaneous Anatomy of the Atlanto‐Occipital Region and Indirect Ultrasound‐Guided Cisternal Puncture in the Dog and the Cat

Cisternal puncture in dogs and cats is commonly carried out. This article describes the percutaneous ultrasound anatomy of the cisternal region in the dog and the cat and an indirect technique for ultrasound‐guided cisternal puncture. Ultrasound images obtained ex vivo and in vivo were compared with anatomic sections and used to identify the landmarks for ultrasound‐guided cisternal puncture. The ultrasound‐guided procedure was established in cadavers and then applied in vivo in seven dogs and two cats. The anatomic landmarks for the ultrasound‐guided puncture are the cisterna magna, the spinal cord, the two occipital condyles on transverse images, the external occipital crest and the dorsal arch of the first cervical vertebra on longitudinal images. Using these ultrasound anatomic landmarks, an indirect ultrasound‐guided technique for cisternal puncture is applicable in the dog and the cat.     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.