Further evidence for the proposed way of spermatogonial stem cell renewal in the rat and the mouse

Summary By means of ³H-thymidine and autoradiography it could be established that in rats and mice the A₁, A₂ and A₃ spermatogonia do not give rise to a significant number of stem cells.The number of A₀ spermatogonia was found to be circa 20% of the number of A₀+A₁ spermatogonia in the rat and circa 10% in the mouse.The author wishes to thank Prof. Dr. M. T. Jansen and Dr. M. F. Kramer for helpful discussions and Mr. J. G. van Essen for technical assistance.     

Renewal and proliferation of spermatogonia during spermatogenesis in the Japanese quail,Coturnix coturnix japonica

Summary Four different types of spermatogonia were identified in the seminiferous tubules of the Japanese quail: a dark type A (A_d), 2 pale A type (A_p1 and A_p2), and a type B. A model is proposed describing the process of spermatogonial development in the quail. The A_d spermatogonia are considered to be the stem cells. Each divides to produce a new A_d spermatogonium and a A_p1 spermatogonium during Stage IX of the cycle of the seminiferous epithelium. An A_p1 spermatogonium produces two A_p2 spermatogonia during Stage II of the cycle, A_p2 spermatogonia produce four type B spermatogonia during Stage VI of the cycle, and type B spermatogonia produce eight primary spermatocytes during Stage III of the cycle. Consequently, 32 spermatids can result from each division of an A_d spermatogonium. Spermatogonial development in the quail differs from the process described in mammals in that there are fewer mitotic divisions and they are all synchronized with the cycle of the seminiferous epithelium. It is suggested that the fewer mitotic divisions explain why a smaller area of the seminiferous tubule is occupied by a cellular association in the quail than in mammals like the rat, ram and bull. The duration of spermatogenesis from the division of the A_d spermatogonia to sperm release from the seminiferous epithelium was estimated to be 12.77 days.     

Spermatogonial Multiplication In the Chinese Hamster. Iv. Search For Long Cycling Stem Cells

ABSTRACT In the Chinese hamster, 17 days, i. e. one cycle of the seminiferous epithelium, after two injections of [³H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (A_s). These labelled A_s spermato-gonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [³H]TdR. In the steady state, half of the divisions of the A_s will be self-renewing and the other half will give rise to A_pr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. the following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i. e. newborn stem cells that still have to migrate away. mostly during G₁, from their sister cells and are scored as A_pr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the A_s, A_pr and A_al spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of A_s that give rise to new A_s for the next epithelial cycle are similar to those of the A_s that will divide into A_pr spermatogonia during the same epithelial cycle. Grain counts revealed that more [³H]TdR is incorporated into A_s, A_pr and A_al spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Puma and Trail/Dr5 Pathways Control Radiation-Induced Apoptosis in Distinct Populations of Testicular Progenitors

Spermatogonia- stem cells and progenitors of adult spermatogenesis- are killed through a p53-regulated apoptotic process after γ-irradiation but the death effectors are still poorly characterized. Our data demonstrate that both intrinsic and extrinsic apoptotic pathways are involved, and especially that spermatogonia can be split into two main populations, according to apoptotic effectors. Following irradiation both Dr5 and Puma genes are upregulated in the α₆-integrin-positive Side Population (SP) fraction, which is highly enriched in spermatogonia. Flow cytometric analysis confirms an increased number of Dr5-expressing SP cells, and Puma-β isoform accumulates in α₆-integrin positive cellular extracts, enriched in spermatogonia. Trail−/− or Puma−/− spermatogonia display a reduced sensitivity to radiation-induced apoptosis. The TUNEL kinetics strongly suggest that the extrinsic and intrinsic pathways, via Trail/Dr5 and Puma respectively, could be engaged in distinct subpopulations of spermatogonia. Indeed flow cytometric studies show that Dr5 receptor is constitutively present on more than half of the undifferentiated progenitors (Kit⁻ α₆ ⁺ SP) and half of the differentiated ones (Kit⁺ α₆ ⁺ SP). In addition after irradiation, Puma is not detected in the Dr5-positive cellular fraction isolated by immunomagnetic purification, while Puma is present in the Dr5-negative cell extracts. In conclusion, adult testicular progenitors are divided into distinct sub-populations by apoptotic effectors, independently of progenitor types (immature Kit-negative versus mature Kit-positive), underscoring differential radiosensitivities characterizing the stem cell/progenitors compartment.     

The ultrastructure of the nuclei and the behaviour of the sex chromosomes of human spermatogonia

Summary The nuclear structure of human spermatogonia has been studied with electron microscopical and histochemical methods. Type B spermatogonia have chromatin clumps without any special ultrastructure and several nucleoli. Five different types of nuclear bodies, and besides, a nuclear vacuole, have been observed in type A spermatogonia. Type I bodies are typical nucleoli consisting of three regions: amorphous, fibrillar and granular. Type II, III and V are considered to be atypical nucleoli. Type IV bodies are small chromatin condensations. Type I bodies are the only ones in which RNA was demonstrated by light histochemical techniques and no PAS positive material was found inside the nuclei. The absence of any special ultrastructure in the chromatin from spermatogonia, and the small mass of the chromatin condensations, show that the human X chromosome and perhaps the Y chromosome are not heteropycnotic in the interphasic nuclei of human spermatogonia.Abbreviations Used RNA ribonucleic acid - gonia spermatogonia This work has been supported by a grant (No. 2623) of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, and partially by a grant (C.M. 6522) from the Population Council.We wish to thank Professor R. E. Mancini for his suggestions during this investigation and his support for its achievement, and to Dr. J. C. Lavieri for providing the biopsies.     

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

Background  

Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into A_s (single), A_pr (paired), and A_al (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

Essential role of mouse Dead end1 in the maintenance of spermatogonia

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of A_single and A_paired spermatogonia were preferentially decreased as compared with those of A_aligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.     

The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3

Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markers of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A_s to A_pr cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A_s to A_al) and differentiating A₁ spermatogonia. In our present study, we find that A_s and A_pr can be basically classified into three categories: (1) GFRα1⁺Nanos2⁺Nanos3⁻Ngn3⁻, (2) GFRα1⁺Nanos2⁺Nanos3⁺Ngn3⁻, and (3) GFRα1⁻Nanos2 ^± Nanos3⁺Ngn3⁺. We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells.     

In vitro effects of ecdysterone on the spermatogonial cell cycle in Locusta

The effect of ecdysterone on specific phases of the cell cycle of Locusta migratoria migratorioides spermatogonia was assayed in vitro. An increase in labeling index was noted, indicating a decrease in duration of the G₁ phase. On addition of the hormone to adapted organs in vitro, spermatogonial mitotic index increases rapidly, then declines to a basal level within a time period which approximates that of the G₂ phase. Such a response indicates a removal of a G₂/M inhibition by the hormone.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Considerations on the neoblasts of planarians on the basis of certain karyological evidence

The problem of the sexual differentiation of the neoblasts of planarians is approached on the basis of karyological evidence assembled by the author and by his collaborators. In the polyploid and pseudogamic biotypes of Dugesia benazzii, D. lugubris and Polycelis nigra, the transformation of the neoblasts into oogonia or spermatogonia is accompanied by variations in the chromosome set. These variations may be considered to be dependent upon the gradient of sexuality — that is, of the territorial influences that determine the evolution in a female or a male direction. Significant evidence is also inferred from the chromosome cycle of hybrids between amphigonic diploid biotypes and polyploid biotypes. A new direction of research, based on the comparison between anterior and posterior regenerated segments of experimental polyploids with variable chromosome sets aims at establishing possible regional differences in the set of the neoblasts.Dedicated to Prof. Dr. J. Seiler on the occasion of his eightieth birthday.     

Nuclear membranous whorls

Summary Membranous whorls have been seen in the nuclei of peritoneal and testicular cells which had been subjected to various experimental manoeuvres. It seems likely that this is an early manifestation of cell degeneration which is demonstrated readily only by glutaraldehyde fixation, and to that extent can be regarded as a glutaraldehyde artifact. Acknowledgements. This work was supported by grants from the Medical Research Council, and the University of Sheffield Tuberculosis Research Fund, and by a grant to the Department from Unilever Ltd.I am grateful to Professor R. Barer for his advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, to Dr. E. J. Clegg for permission to use material from joint experiments. Technical and photographic assistance was provided by Messrs. P. GarLick and L. Murgatroyd and by Miss M. Tune.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

Comparative immunochemical studies with antisera to sheep prolactin and bovine growth hormone on anterior pituitaries of ox,sheep, rats and mice

Summary Rabbit antisera to sheep prolactin and bovine growth hormone were used in the indirect fluorescent antibody technique on cryostat sections of anterior pituitaries of sheep, ox, rats and mice. It is demonstrated that in sheep and ox prolactin and growth hormone are manufactured by different acidophilic pituitary cells. Though the antisera do not precipitate the analogous hormones of rats and mice in gel diffusion tests, evidence is given for the specificity of the cross-reaction of the antisera with the analogous murine hormones in situ as found with the fluorescent antibody technique.We are grateful to Dr. J. D. H. Homan of Organon, Oss (The Netherlands) for the supply of bovine growth hormone and for kindly giving us the information concerning this preparation.We should like to thank Dr. F. J. A. Prop for the prolactin assays, Dr. H. G. Kwa for providing the mouse pituitary tumours and Dr. L. M. Boot for the mouse pituitary isograft in the kidney, Mrs. J. J. Geiger-Koedijk for the conventional staining of pituitary sections, and Mr. J. van der Kamp for the photography.     

Histochemical localization of adenosinetriphosphatase in the testicular and pre-testicular nephridia of the Indian leech Hirudinaria granulosa (Savigny)

Summary Adenosinetriphosphatase has been histochemically demonstrated in the initiallobe cells and the central canal in its first round in the pre-testicular and testicular nephridia of the Indian leech Hirudinaria granulosa. The apical-lobe cells are positive in the pre-testicular and negative in the testicular nephridia. The functional significance of the enzyme at these sites has been discussed.I wish to express my gratitude to Dr. H. B. Tewari, under whose guidance the present work has been carried out. I am also indebted to Dr. M. L. Dhar, Director, Central Drug Research Institute, Lucknow; Dr. P. S. Krishnan, Professor of Biochemistry, University of Lucknow; and Dr. (Miss) Usha Gupta, Department of Pathology, University of Lucknow, for the hospitality which they have accorded me in their laboratories.     

Spermatogonial multiplication in the Chinese hamster. IV. Search for long cycling stem cells

In the Chinese hamster, 17 days, i.e. one cycle of the seminiferous epithelium, after two injections of [3H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (As). These labelled As spermatogonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [3H]TdR. In the steady state, half of the divisions of the As will be self-renewing and the other half will give rise to Apr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. The following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i.e. newborn stem cells that still have to migrate away, mostly during G1, from their sister cells and are scored as Apr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the As, Apr and Aal spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of As that give rise to new As for the next epithelial cycle are similar to those of the As that will divide into Apr spermatogonia during the same epithelial cycle. Grain counts revealed that more [3H]TdR is incorporated into As, Apr and Aal spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

On the glycocalyx,the external coat of the plasma membrane,of some secretory cells

Summary The free surface of epithelial cells of secretory organs (human placenta, lactating mammary gland of the rat, choroid plexus of man and rat) and of the accessory organs of the genital tract of the male rat is characterized by a plasmalemmal differentiation named glycocalyx or surface mucous coat. This structure is built up by filamentous or globular substructures.Two main ultrastructural types of the glyeocalyx were observed: 1) The filamentous type such as in the rat epididymis, which resembles the cat intestinal glyeocalyx (Ito, 1965) and that one of human transitional epithelium (Monis and Zambrano, 1968), and 2) The globular type, as observed in the lumen of the lactating mammary gland of the rat.Sialic acid was demonstrated histochemically in the luminal glyeocalyx of all organs studied. In addition, the glyeocalyx of acinar cells of the lactating mammary gland contains sulfate and phosphate groups which were identified by histochemical technics, using enzymatic digestion procedures, suggesting the chemical heterogeneity of this glyeocalyx.Present investigations follow the working hypothesis that the complex carbohydrates of glycocalyces become part of the product of activity of secreting cells.We thank Mr. Luis Iwakawa, Miss Silvia Falcón, Miss Elsa M. Orgnero for technical help, Miss Graciela Aliaga for secretarial assistance. Photography by Mr. H. Magnani. Dr. Hugo F. Carrer cooperated in the initial stages of this investigation.The authors acknowledge the use of the electron microscope of the Department of Pathology, Córdoba University Medical School, for which they thank Prof. E. Mosquera and Dr. E. Hliba. Dr. Hliba photographed picture number 4.     

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics Chromium^TM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (A_pale and A_dark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, A_pale and A_dark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.