The sensitivity to X rays of mouse spermatogonia that are committed to differentiate and of differentiating spermatogonia.

In the CBA mouse the radiosensitivity of the undifferentiated spermatogonia that are committed to differentiate was determined by counting their more developed descendants 10 days after graded doses of X rays. Decreasing D0 values were found when these differentiating spermatogonia were derived from undifferentiated spermatogonia that were located in all likelihood in chains of increasing length. In stages IX and X of the epithelial cycle the radiosensitivity of these undifferentiated spermatogonia was characterized by a D0 of 2.2 Gy. This D0 value most likely belongs to the Asingle spermatogonia that form repopulating colonies which give rise to differentiating spermatogonia within the same epithelial cycle. In stages XII/I, where a D0 of 1.0 Gy was found, the dose-response curve is likely dominated by the Apaired spermatogonia present in these stages. In stages III to VII, the Aaligned spermatogonia transforming into A1 spermatogonia determine the radiosensitivity. During this period the D0 decreased from 0.7 to 0.4 Gy. Differentiating A1 to A3 and B spermatogonia had rather similar radiosensitivities of 0.4 to 0.5 Gy.     

The kinetics of the first wave of spermatogenesis in the newborn male mouse

A combination of autoradiography and air-dried techniques was used to calculate the duration of the major meiotic stages in the first wave of spermatogenesis in the newborn mouse. The data indicated that the entry into meiosis occurred asynchronously over 2 days, and the time required for each stage and the total cycle was constant. These time intervals were nearly identical with those estimated for adult animals in the present study and by other authors.     

Late onset of spermatogenesis and gain of fertility in POG-deficient mice indicate that POG is not necessary for the proliferation of spermatogonia

The germ cell-deficient (gcd) mouse mutation is a recessive, transgenic insertional mutation associated with the disruption of two Chr11 genes, Pog (proliferation of germ cells) and Vrk2 (vaccinia virus-related protein kinase 2). We have recently shown that like gcd/gcd mice, targeted Pog-/- males and females show virtually no spermatogenesis or oogenesis at 4-6 wk of age. Because Pog is deleted in gcd/gcd and Pog-/- mice, a comparison of the phenotypes of the two mouse models is appropriate. Here, we report that unlike in POG-deficient females, the germ cells in POG-deficient males eventually populate the seminiferous tubules at 9 wk, and fertility can be achieved by 12 wk. Homozygous gcd/gcd males did not show a similar degree of germ cell population, and most gcd/gcd males remained infertile at 16 and 22 wk of age. A comparison of the degree of germ cell deficiency at 13.5 days postcoitum and 1 day postpartum between Pog-/- and gcd/gcd males revealed that gcd/gcd males had far fewer germ cells than Pog-/- males at both time points. Our data suggest that Pog is essential for proper primordial germ cell proliferation in the embryonic stage but is not needed for spermatogonial proliferation after birth. Thus, the difference in the spermatogenetic potential in adult Pog-/- and gcd/gcd mice may result from the severity of germ cell deficiency rather than from the inability of gcd/gcd spermatogonia to proliferate efficiently. The greater deficiency of germ cells before the onset of spermatogenesis seen in gcd/gcd males compared to Pog-/- mice suggests either that the different background affects the outcome of Pog deletion or that Vrk2 has additional effects on germ cell development.     

Actin is present in a green alga that lacks cytoplasmic streaming

Summary Negative-staining of crude cytoplasmic extracts from cells of the green algaErnodesmis verticillata reveals the presence of numerous microfilaments. Rabbit skeletal muscle heavy-meromyosin binds to the microfilaments (in the absence of ATP) in typical arrowhead arrays. These results demonstrate that actin is present in this alga and it is suggested that actin may be involved in cytoplasmic contractions effecting wound healing, since cytoplasmic streaming does not occur in this organism.     

Distribution of type A spermatogonia in the mouse is not random

The distribution of type A spermatogonia was studied using drawings of cross-sectioned tubules at various stages of the spermatogenic cycle of perfusion-fixed, epoxy-embedded mouse testis. Spermatogonia were classified as either positioned opposite the interstitium or opposite the region where two tubules make contact or in a defined, intermediate region at which the two tubules diverged. At stage V, the population of type A spermatogonia, comprised of A(s) through A(al) cells, is randomly positioned around the periphery of the seminiferous tubule. The A(s) through A(al) population becomes nonrandomly distributed beginning at stage VI, being located primarily in regions where the tubule opposes the interstitium, and remains nonrandom through stage III of the next cycle. The A(1) spermatogonia of stage VII, derived from most A(pr) and A(al) spermatogonia, and the A(2) spermatogonia of stage IX, derived from the A(1) spermatogonia, are also nonrandomly positioned opposing the interstitium. However, the A(3) population of stage XI becomes randomly distributed around the tubule. To our knowledge, these are the first data to show that the more primitive spermatogonial types (A(s) to A(al)) move to specific sites within the seminiferous tubule. Division of the regularly spaced, more primitive spermatogonia (A(s) to A(al)) leads to the spread of their progeny (A(1) to A(4)) laterally along the base of the seminiferous tubule. The lateral spread from more or less evenly spaced foci ensures that spermatogenesis is conducted uniformly around the entire tubule. The data also suggest that the position of a seminiferous tubule in the mouse is stabilized in relationship to other seminiferous tubules.     

CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes

In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.     

Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function.

Recent studies have shown that the dominant white spotting (W) locus encodes the proto-oncogene c-kit, a member of the tyrosine kinase receptor family. One symptom of mice bearing mutation within this gene is sterility due to developmental failure of the primordial germ cells during early embryogenesis. To elucidate the role of the c-kit in gametogenesis, we used an anti-c-kit monoclonal antibody, ACK2, as an antagonistic blocker for c-kit function to interfere with the development of male and female germ cells during postnatal life. ACK2 enabled us to detect the expression of c-kit in the gonadal tissue and also to determine the functional status of c-kit, which is expressed on the surface of a particular cell lineage. Consistent with our immunohistochemical findings, the intravenous injection of ACK2 into adult mice caused a depletion in the differentiating type A spermatogonia from the testis during 24-36 h, while the undifferentiated type A spermatogonia were basically unaffected. Intraperitoneal injections of ACK2 into prepuberal mice could completely block the mitosis of mature (differentiating) type A spermatogonia, but not the mitosis of the gonocytes and primitive type A spermatogonia, or the meiosis of spermatocytes. Our results indicate that the survival and/or proliferation of the differentiating type A spermatogonia requires c-kit, but the primitive (undifferentiated) type A spermatogonia or spermatogenic stem cells are independent from c-kit. Moreover, the antibody administration had no significant effect on oocyte maturation despite its intense expression of c-kit.     

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis

Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.     

Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis

Summary The structure of guppy (Poecilia reticulata) spermatogonia and spermatocytes has been studied using electron microscopy. The spermatogonia, situated at the apex of the seminiferous tubule, are almost all surrounded by a network of Sertoli cells; they have very diffuse chromatin and one or two large nucleoli. The cytoplasm contains relatively few organelles, although annulate lamellae are found. The mitochondria have few cristae and are concentrated at one pole of the cell; they are sometimes found with intermitochondrial cement. These spermatogonia are separated from each other, having no intercellular bridges or inclusion in Sertoli cells, and are relatively undifferentiated; they correspond to stem cells. The spermatogonia beneath the apex are organized into cysts. First-generation spermatogonia are more dense and heterogeneous, their nuclei becoming smaller and their chromatin becoming denser during successive generations. In spermatocytes, the synaptinemal complex exists as a modified form until metaphase. The concentration of organelles in the cytoplasm increases and the organelles become more diversified as spermatogenesis progresses. Many cytoplasmic bridges are observed (several per cell), indicating that the cells remain in contact after several divisions. These changes in germ cell structure have been related to some of the characteristic features of spermatogenesis in guppy, e.g. the large number of spermatogonial generations and the complexity of spermiogenesis.     

Yeast clathrin has a distinctive light chain that is important for cell growth

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.     

Strain differences in the radiosensitivity of mouse spermatogonia

The radiosensitivity of spermatogonia was found to be greater by up to a factor of 2 in C3H mice than in B6D2F1 mice, whether assessed for the highly sensitive spermatogonia (types A2 to In) or the much more resistant clonogenic spermatogonia which repopulate tubules. The latter were similarly resistant in the B6D2F1 hybrid and in the DBA2 parent, but were much more sensitive in the C57BL parent strain. A difference in sensitivity by up to a factor of 2 results in a variation by a factor of 10 or more in the level of survival of clonogenic cells after high doses. This variation is also observed when comparing data in the literature from different authors using various strains of mice. Using the radiosensitizer misonidazole, it was shown that hypoxia did not play a major role in the lesser sensitivity demonstrated in B6D2F1 mice. The variation in sensitivity is similar to the range reported in the literature for reciprocal translocations.