Characterization of a novel spermatogenic cell antigen specific for early stages of germ cells in mouse testis

To study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N-glycanase, suggesting the presence of asparagine-linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present. © 1995 Wiley-Liss, Inc.     

In-vitro proliferation of germ cells and supporting cells in the neonatal mouse testis

Summary Testicular cells were prepared from neonatal (48 h after birth) mice by enzymatic dissociation and were cultured in serum-supplemented medium to investigate cell proliferation in vitro. The cultured cells were composed mostly of germ cells, identified by immunocytochemistry using a germ cell-specific antiserum, and supporting (immature Sertoli) cells. After 36 h in culture, the cells were pulse-labeled with ³H-thymidine and fixed at 2-h intervals for 36 h after labeling. Numbers of labeled and unlabeled metaphases of germ cells and supporting cells were counted, and percent labeled metaphases for both cell types were determined for cell-cycle analysis. The results indicate that germ cells, as well as supporting cells, incorporate ³H-thymidine and progress through the cell cycle in vitro. From the curve of the percent labeled metaphases for the supporting cells, the total cell cycle and intervals of DNA synthesis were estimated to be 27.2 h and 13.2 h, respectively.     

Ultrastructural and immunohistochemical studies on steroid-secreting cells of the testis and ovary of normal and 3-methylcholanthrene-treated mice

Summary The testis and ovary of normal and 3-methylcholanthrene-treated mice were studied ultrastructurally and immunohistochemically in order to learn whether steroid-secreting cells of the gonads are involved in drug metabolism. The steroid-secreting cells, i.e., Leydig cells of the testis, and theca interna cells, interstitial gland cells, and corpus luteum cells of the ovary of 3-methylcholanthrene-treated mice, show a strong positive reaction to the antiserum against, hepatic microsomal cytochrome P-450, of liver which is the terminal oxidase of the drug-metabolizing enzyme complex. In addition, it was found that elements of smooth endoplasmic reticulum (SER) in drug-treated mice become well developed as compared with those in control animals. These findings indicate that the steroid secreting cells in testis as well as ovary are involved in the metabolism of both endogenous and exogenous chemical compounds.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

p38 MAPK在小鼠睾丸不同发育阶段的表达和定位

为探讨丝裂原活化蛋白激酶p38 MAPK在小鼠睾丸不同发育阶段的表达,应用蛋白质免疫印迹杂交技术和免疫组织化学SABC法检测1至7周龄小鼠睾丸p38 MAPK的表达、定位及发育变化,并通过图像分析技术对免疫组织化学结果进行统计学分析。免疫印迹杂交发现,p38 MAPK在2～7周龄小鼠睾丸中均有表达。免疫组织化学结果显示,在2周龄小鼠睾丸曲细精管上皮中即可观察到p38 MAPK免疫阳性反应,免疫反应阳性细胞为精原细胞;3、4、5周龄小鼠睾丸仅有个别曲细精管上皮可见p38 MAPK免疫阳性反应;6、7周龄小鼠睾丸中p38 MAPK表达较丰富,免疫反应阳性细胞为精原细胞和初级精母细胞,免疫阳性反应物均主要位于细胞核内。在7周龄小鼠睾丸中还可见到部分间质细胞的细胞质亦呈p38 MAPK阳性。这些结果提示,p38 MAPK可能对生精细胞的增殖分化具有调控作用。     

Differential expression of ubiquitin-conjugating enzyme E2r in the developing ovary and testis of penaeid shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

In order to identify genes involved in oogenesis and spermatogenesis in penaeid shrimp Marsupenaeus japonicus, a modified annealing control primer (ACP) system was adapted to identify genes differentially expressed in ovary and testis at different developmental stages. By using 20 pairs of ACP primers, 8 differentially expressed genes were obtained. One of these genes is ubiquitin-conjugating enzyme E2r (UBE2r). Bioinformatics analyses show that this gene encodes a protein of 241 amino acids with a predicted molecular mass of 27.4 kDa. Real time PCR analyses demonstrated that the expression level changed significantly in the developing testis and ovary. In the stage 2 of testis, it reached its highest expression level, the lowest expression level present in the stage 1 of ovary. The significantly different expression levels in developing testis and ovary suggest that UBE2r has an important role in oogenesis and spermatogenesis. This article is the first report of UBE2r in crustaceans and also is the first report showing that UBE2r is differentially expressed at different stages of the developing ovary and testis in an animal.     

Germ-cell death during prespermatogenesis in the testis of the golden hamster

Summary Degenerating prespermatogonial germ cells in the testis of the immature golden hamster [aged 14 days post conceptionem (dpc) to 13 days post partum [dpp)] were studied with regard to their morphology and temporal incidence. Judged by their ultrastructural features, these cells clearly take the form of apoptosis and finally are subjected to phagocytosis by neighboring Sertoli cells; only a few germ cells of a presumably incipient, partly variant degenerative morphology cannot, at present, be assigned to the apoptotic mode of cellular death. Degenerating prespermatogonia occur between the 14th dpc and 3rd dpp and again, after an interval in which no such cells are found, from the 9th dpp onwards. This pattern reveals a striking parallelism to the phases of proliferation of these cells, viz., the appearance of M- and T₂-prespermatogonia. Both this obvious temporal association of proliferation and degeneration and the classification of prespermatogonial death as apoptosis suggest some developmental significance of the degenerative phenomena investigated.     

Follicle cells and germ line cells both affect polarity in dicephalic chimeric follicles of Drosophila

Summary In aberrant egg follicles of the pattern mutant dicephalic (dic) the oocyte is wedged in between two groups of nurse cells, and this condition may give rise to embryos which express anterior traits at both ends. We have analysed the role of the dic genotype of the germ line cells and the surrounding somatic follicle cells in the formation of the dic follicular phenotype. By means of pole cell transplantations into Fs (1) K 1237 hosts (this cell-autonomous mutation causes degeneration of the host's germ line cells early in oogenesis), we constructed chimeras in which either the follicle cells, the germ line cells, or both were homozygous for the dic mutation. In all three combinations the dic phenotype was expressed but not in controls with dic ⁺ in both germ line cells and follicular epithelium. Since follicles with the dic phenotype may be produced if either the germ line cells or the follicle cells lack dic ⁺ gene activity we suggest that cellular interactions between both cell types are required for the correct positioning of the oocyte at the follicle's posterior pole.     

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

Evaluation of immunohistochemical markers of germ cells' proliferation in the developing rat testis: a comparative study

Germ cells' proliferation during testicular organogenesis in Wistar rat embryos and neonates [14.5, 18.5, 20.5 days post conception (dpc), birth (day 0), 1, 3, 5, 7 days post partum (dpp)] was evaluated via immunohistochemistry, using the PCNA and Ki-67 nuclear antibodies. Estimation of the reactive/total cell ratio, per visual field [labeIing index (LI)] was achieved using the Image Pro Plus Software. Immunostaining of the fetal testis, with both antibodies, revealed increasing germ cells' numbers between 14.5 dpc and birth. From birth onwards, a sharp decline of germ cells' population was observed in the first 3 days of postnatal life. Then, a transient increase of the LI, between 3 and 5 dpp, was noted. Afterwards, proliferation of germ cells ceased. These results indicate that, during fetal and neonatal life, two peaks of proliferative activity of germ cells are noticed. Following estimation of the LI for both PCNA and Ki-67, a prominent labeling for the first antibody was observed throughout the examined period. Ki-67 staining follows a similar pattern, showing, however, significant fluctuation in the obtained values, in comparison to PCNA. The significant differences observed don't seem to be simply a result of the different half lives of the two markers, but rather a consequence of additional underlying cellular activity associated with PCNA, such as DNA repair.     

Intercellular bridges between germ cells in the immature golden hamster testis: evidence for clonal and non-clonal mode of proliferation

Summary Intercellular bridges of prespermatogonia and of the first A-spermatogonia in the maturing testes of newborn to 17-day-old golden hamsters have been studied by electron microscopy. Incomplete cytokinesis of dividing M- and T₂-prespermatogonia and A-spermatogonia produces these bridges, which undergo different developmental fates. Bridges of the first A-spermatogonia are stable beyond subsequent mitoses of these cells; this gradually leads to the formation of bridge-connected groups of synchronously developing germ cells. Thus, the clonal mode of male germ cell proliferation is already established in this period of testis maturation. During mitoses, pre-existing bridges reversibly develop structural modifications, i.e. considerable elongation and formation of a bridge-partitioning complex. In contrast, intercellular bridges of prespermatogonia are mostly severed and become lost during subsequent mitoses of the cells involved; this results in separation of the germ cells and represents a mainly non-clonal mode of M- and T₂-prespermatogonial proliferation. Here, too, pre-existing bridges elongate and develop the bridge-partitioning complex during subsequent mitoses of the joined cells, but this is superposed and interrupted by the simultaneous process of disconnection of the bridges.Parts of this study were presented at the 85th meeting of the Anatomische Gesellschaft in Munich, April 1990     

Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela,Amphibia)

Summary In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.     

DFL, a FLORICAULA/LEAFY homologue gene from Dendranthema lavandulifolium is expressed both in the vegetative and reproductive tissues

FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species. The inflorescence organization of chrysanthemum differs from typical dicotyledons such as Arabidopsis and Antirrhinum as clear sepals are absent, and instead, a pappus, a rudimentary sepal, is formed. To understand the mechanism of reproduction of chrysanthemum at the molecular level, DFL, a FLORICAULA/LEAFY homologous gene, was cloned from Dendranthema lavandulifolium, which is one of the original species of chrysanthemum. The DFL gene consists of a 1,236-bp open reading frame and encodes a putative protein of 412 amino acids, which is 63% identical to LFY and 70% to FLO. The expression patterns of DFL during the flower development were analyzed, and RT-PCR results showed that DFL was strongly expressed in the flower bud. In situ hybridization experiments showed that it is strongly expressed in the inflorescence bract, petal and stamen primordial tissues throughout the inflorescence development. Its expression signals were also detected in stems, leaf primordial tissues and developing inflorescence bracts.     

Timing of establishment of paternal methylation imprints in the mouse

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.