Die Frühentwicklung der Gonaden und die Ontogenese von Rete testis und Tubuli seminiferi recti beim Huhn (Gallus domesticus)1

The early development of the gonads, and the ontogenesis of the rete testis and tubuli seminiferi recti in the chicken (Gallus domesticus) The primordium of the gonads can be discerned in the 4-day chick embryo by the elevation of the celomic epithelium to form germinal epithelium, and by the arrival of the primordial germ cells. Already in the 4½ day chick embryo there appears on the left side of the body, as a result of the first proliferation of the germinal epithelium, a subepithelial mesenchymelike cell conglomeration, which has been erroneously labelled in the literature a “urogenital union”. In contrast to the opinion expressed in the literature such a union does not appear until the 10th day, when the mesenchymal cells change to forerunners of rete cells which after hatching differentiate into rete-epithelial cells. The rete testis consists of intra and extratesticular transverse cisterns and of an intercalated and partically subdivided longitudinal cistern. The tubuli seminiferi contori end on the intracapsular lingitudinal cisterns either directly, or indirectly by intercalated tubuli siminiferi recti or intratesticular transverse cisterns.     

Immunohistochemical Localization of Progesterone Receptor Isoforms in the Chick Pre-follicular Ovary

The distribution of progesterone receptor A and B isoforms in different cell types of the chick pre-follicular ovary was studied by immunohistochemistry. Newly hatched chicks were killed and the left ovary was removed, fixed and embedded in paraplast. Sections (5 microns thick) were made for the detection of progesterone receptor isoforms, using a technique of indirect immunoperoxidase. The results indicate that progesterone receptors were localized in the nuclei of germinal epithelium and germ cells of the ovarian cortex and in the interstitial and epithelial cells of the lacunar channels of the ovarian medulla. Undifferentiated cells did not present progesterone receptors. In all cell subpopulations progesterone receptor B was the predominantly expressed isoform. These data suggest that progesterone receptor isoforms are differentially expressed in the chick pre-follicular ovary and that progesterone effects in this tissue are mediated by the progesterone receptor B isoform.     

Attempt to produce nuclear transferred primordial germ cells using electrofusion in domestic chicken

As a step to develop a somatic nuclear transfer technique for avian species, an attempt to produce somatic nuclear transferred primordial germ cells (PGC) in the domestic chicken was carried out. Primordial germ cells and embryonic blood cells (EBC) were collected from 2‐day‐old embryos and the nuclei were transferred from EBC into PGC by electrofusion. The most efficient pearl chain was developed when a 350‐V/cm AC field was applied for 60 s. Cell fusion between PGC and EBC was most effective when 4‐kV/cm DC pulses, 60 µs pulse width, were applied three times to a cell suspension dispersed in 0.2 or 0.25 mol/L saccharose solution. The present results provide basic information for the production of somatic cell nuclear transferred chickens using PGC as the nuclear recipient.     

Scanning electron microscopy of circulating primordial germ cells in the chick embryo

A demonstration of circulating primordial germ cells in the stage 14 embryo was carried out. The definition of primordial cells was based upon specific criteria utilized by other workers and developed in this study. Evidence supports a passive transport of these cells to the germinal epithelium.     

Annulate Lamellae in the Germ Cells of Chick Embryo

The presence of annulate lamellae in the germ cells of the chick embryo, in stage 9 of H amburger /H amilton (1951), as well as in gonadal germ cells of male chick embryos which had been incubated for 8^1/2 days, both in normal and experimental conditions, suggests that these formations may be associated with some specific function in the development of this cell line.     

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre‐spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21–25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti‐human OCT‐4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT‐4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre‐spermatogonia would be observed at later stages of canine foetus development. We also showed that anti‐human OCT‐4 antibody can be useful to identify canine PGC in vivo.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Effect of follicle-stimulating hormone on different cell sub-populations in the ovary of newly hatched chicks treated during embryonic development

1. Cell sub-populations of the ovary of newly-hatched chicks were assessed following follicle stimulating hormone (FSH) treatment during embryonic development. Changes in cell number and the amount of oestradiol in serum were determined. 2. White Leghorn chick embryos received 1 mug FSH applied to the chorioallantoic membrane at 13, 15, and 17 d of incubation. Within 24 h after hatching, animals were killed and blood was collected. The left ovary was immediately removed then weighed and processed by an enzymatic-mechanical dissociation method for total cell count. An air-drying method was also used for meiotic preparations to study the germinal cells. 3. The pre-follicular ovary is able to respond to FSH by inducing an increase both in the serum oestradiol concentration and in the number of steroidogenic cells and of poorly differentiated cells of the ovarian medulla. 4. FSH increases the number of oogonia, which are responsible for a sharp increase in the total population of germ cells in the FSH-treated ovary. 5. It is possible that FSH acts to increase the proliferation of oogonia and a delay in the meiotic prophase through a change in the microenvironment rather than by a direct effect on germ cells.     

Regularities and Irregularities in the Structure of the Seminiferous Epithelium in the Domestic Fowl (Gallus domesticus)

A cellular association demarcated by two perpendiculars which were drawn between adjacent bundles of elongate spermatids from the tubular lumen to the basement membrane, was made the unit of histometrical observation in this study (provisionally called a “column”). Cell counting revealed that the average numbers per column of various types of germ cells do not show any significant differences among 5 fowls and between paired testes. The frequency of spermiogenic steps (numbered 1–8) was investigated in each column. A definite and common pattern was found in the frequency distribution in the 5 fowls observed. A relationship between spermiation and younger spermatid steps was also investigated in each column. The spermiation was found at different steps, but most frequently at step 2 (30.6 %). Based on these observations and referring to other author's information, an average time interval between two successive spermiations was calculated roughly at 3.3 ± 1.2 days. Theoretically, this value is equal to an average length of one epithelial cycle. Such a variable cycle may have caused irregular cellular associations in this species.     

Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.     

从原始生殖细胞分离和克隆兔的胚胎生殖细胞

研究采用 1 4～ 1 8d兔胎儿的生殖嵴及周围组织与其同源成纤维细胞共培养 ,低糖DMEM +1 0 %NBS +1 0 %FCS +1 0ng/mLLIF +1 0ng/mLSCF+0 1mol/Lβ 巯基乙醇 +1 0 0U/mL青霉素 +80U/mL链霉素作培养基 ,分离出兔原始生殖细胞 (PGC) ,克隆并多次传代。从原始生殖细胞 (PGC)中获得胚胎生殖细胞 (EG)细胞集落 ,1 4d胎儿原代观察到类EG细胞集落 ,传至 4代后丢失。 1 6d胎儿的类EG只传 2代 ,1 8d胎儿没有得到EG细胞集落。EG细胞具有干细胞的诸多特征 ,呈典型的团块状聚集生长 ,碱性磷酸酶 (AKP)染色呈阳性 ,在衰老饲养层的培养基中生长形成类胚体、上皮细胞、神经细胞和成纤维细胞等     

Technical note: viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos

The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring.     

水牛PGC和前精原细胞的体外培养

分别对取自50~95日龄水牛胎儿的原生殖细胞和前精原细胞进行体外培养,观察其生物学行为,并检测其碱性磷酸酶(AP)活性和Oct-4蛋白特性,探讨利用这些生殖细胞建立干细胞系的可行性和检测方法。结果表明水牛原生殖细胞及前精原细胞分别在体外培养时,均能形成细胞克隆;克隆与周围细胞分界明显,但克隆中细胞相互间界限不清;部分克隆有分隔现象,形如多个克隆共同组成一个大克隆;细胞克隆均至少能培养4代以上;原生殖细胞和前精原细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中部分克隆表现为AP假阳性。研究结果显示水牛原生殖细胞和前精原细胞均可用于建立干细胞系;体外培养时,AP活性和Oct-4蛋白不适宜用来检测这些细胞及其来源的细胞克隆。     

四氯联苯对鸡胚生殖新月原始生殖细胞分布的影响

为了探索四氯联苯(2,2′,5,5′)对鸡胚生殖新月原始生殖细胞(PGCs)分布的影响,本试验将四氯联苯注入种蛋的胚盘(第X期)处,在38℃,相对湿度60%,每2h45度转蛋的条件下孵化至原条期,通过不同方法检测原条期生殖新月明区、暗区及血液循环中的PGCs,探讨四氯联苯对胚盘生殖新月区PGCs分布的影响。结果表明,四氯联苯明显降低了原条期明区和血液中PGSs的数量(P<0.01),但对暗区PGCs的数量影响不明显。这说明四氯联苯对血液中PGCs的影响是由于降低了生殖新月明区PGCs的缘故,和暗区的PGCs无相关性。     

Biallele Expression of PEG10 Gene in Primordial Germ Cells Derived from Day 27 Porcine Fetuses

Primordial germ cells (PGCs) from day 27 porcine fetuses have often been isolated to establish pluripotent embryonic germ (EG) cell lines, but little is known regarding their imprinted gene status. In our study, we attempted to detect the imprinted gene expression of cloned embryos and EG cells derived from individual PGC of day 27 and day 35, using single nucleotide polymorphism (SNP) analysis of the paternally expression gene 10 (PEG10) as a sign of parental‐origin‐specific expression. The results showed biallelic gene expression of the SNP that occurred in EG cell colonies and almost all of the cloned blastocysts, demonstrating that aberrant imprinted gene expression of PEG10 occurs in the day 27 porcine PGCs, whereas monoallelic expression of the PEG10 gene occurs in all the PGC clones derived from day 35 PGCs. In addition, the same imprinted gene status was observed for blastocysts derived from both male and female PGCs, indicating that the parental genomic imprinting is erased in male and female germlines.     

Expression of p63 in the mouse primordial germ cells

Proteins encoded by p63 gene a have structural similarity with tumor suppressor p53, and were thought to induce cell cycle arrest and apoptosis during development. The p63 proteins are also expressed in the basal cells of many epithelial tissues in the adult, and supposed to play important roles in maintaining the epidermal stem cells. Previously, we reported the p63 expression in the testis of mouse embryos, suggesting their involvement in the growth arrest and apoptosis of testicular germ cells (Nakamuta and Kobayashi, J. Vet. Med. Sci. 65:853-856). In this study, we investigated the timing of this p63 expression in the germ cells during migration and colonization to the gonads. Immunohistochemical analysis of mice from embryonic day (E) 7.5 to E12.5 demonstrated that p63 positive reactivity was seen as early as E8.5 when the founder cells of germ cells, primordial germ cells (PGCs), were located in the hind gut epithelium, but PGCs were negative for p63 at E7.5 when they first appeared. p63 is expressed as six isoforms, resulting from alternative splicing at C-terminus and by the use of two promoters that generate variations at N-terminal end. RT-PCR analyses suggested that different types of p63 mRNAs were likely to be expressed in PGCs during development. These results imply that p63 may be involved in the regulation of PGC development by controlling the gene expression required for their migration and colonization to the gonads.     

Autoradiographische Untersuchungen zur Kinetik der Präspermatogenese und Spermatogenese der Wistarratte

Inhalt Mit der “Methode der markierten Mitosen” (Quastler und Shermann , 1959) sowie der “Methode der Doppelmarkierung” (Hilscher und Maurer , 1962) lassen sich die Generationszeiten und Teilphasen der männlichen Keimzellen bestimmen. Dadurch wird es möglich, die einzelnen Etappen der “Präspermatogenese” und “Spermatogenese” exakt zeitlich zu erfassen, wie an einigen Beispielen gezeigt wird. Als Versuchstiere dienten Wistarratten einer Zucht (Möller, Haan). Contents Using the method of labeled mitoses (Quastler & Sherman, 1959) and that of double labeling (Hilscher & Maurer, 1962) the generation times and sub phases of the male germ cells can be determined. By these means it is possible to measure the exact time required for the different stages of prespermatogenesis and spermatogenesis. A number of examples are given. Wistar rats of the same stock were used as experimental animals (Müller, Haan).     

X-Chromosome monosomy (37,XO) in a Burmese cat with gonadal dysgenesis

A 37,XO-chromosome complement was detected in a 21/2-year-old sable Burmese cat examined because of primary anestrus. The cat was smaller than its littermates; other somatic abnormalities associated with the XO karyotype in other species were not present. The ovaries, which did not respond to gonadotropin stimulation, contained inactive germinal epithelium (lacking follicles and primordial germ cells), which was similar to that of adult human patients with XO-gonadal dysgenesis.     

Lectin Histochemistry as a Tool to Identify Apoptotic Cells in the Seminiferous Epithelium of Syrian Hamster (Mesocricetus auratus) Subjected to Short Photoperiod

Lectins have been widely used to study the pattern of cellular glycoconjugates in numerous species. In the process of cellular apoptosis, it has been observed that changes occur in the membrane sugar sequences of these apoptotic cells. The aim of our work was to identify which lectins, out of an extensive battery of the same (PNA, SBA, HPA, LTA, Con‐A, UEA‐I, WGA, DBA, MAA, GNA, AAA, SNA), show affinity for germinal cells in apoptosis, at what stage of cell death they do so and in which germinal cell types they can be detected. For this, we studied testis sections during testicular regression in Syrian hamster (Mesocricetus auratus) subjected to short photoperiod. Several lectins showed an affinity for the glycoconjugate residues of germ cells in apoptosis: Gal β1,3‐GalNAcα1, α‐d ‐mannose, N‐acetylgalactosamine and l ‐fucose. Furthermore, lectin specificity was observed for some specific germinal cells and in certain stages of apoptosis. It was also observed that one of these lectins (PNA) showed affinity for Sertoli cells undergoing apoptosis. Therefore, we conclude that the use of lectin histochemistry could be a very useful tool for studying apoptosis in the seminiferous epithelium because of the specificity shown towards germinal cells in pathological or experimentally induced epithelial depletion models.