Spermatogonial Divisions Research Articles

Tubulin TUBB4B Is Involved in Spermatogonia Proliferation and Cell Cycle Processes.

Tubb4b (tubulin β-4b chain) is essential for cell growth and development as a microtubule network protein. Previous studies have shown that TUBB4B affects mouse pronucleus migration, but the gene function has yet to be elucidated. To study TUBB4B-related functions in mouse reproductive development, we designed a single sgRNA in chromosome 2 and generated a knockout spermatogonia cell line of the β-tubulin isoform Tubb4b by the CRISPR/Cas9 system. Tubb4b-KO spermatogonia recognized abnormal lysosomal membranes and cell morphology defects. Compared to control mouse spermatogonia, the proliferation rate was significantly slower and cycling stagnated in the G1/0 population. Although spermatogonia lacking TUBB4B have abnormal divisions, they are not lethal. We detected the mRNA levels of the cell-regulating cyclins CyclinsD1, CyclinsE, Cdk2, Cdk4, P21, Skp2 and the cell growth factors C/EBP α, C/EBP β, and G-CSF in the spermatogonia of Tubb4b-KO and found that the expressions of CyclinsD1, Skp2 and cell growth factors were significantly reduced. Further analysis revealed that 675 genes were expressed differently after Tubb4b deletion and were enriched in negative regulation of cell population proliferation (GO:0008285), negative regulation of cell cycle G2/M phase transition (GO:1902750), and positive regulation of cell death (GO: 0010942). We also found that there is a common gene Cdkn1a (P21) in these three GO pathways related to cell proliferation and cell cycle, and both quantitative analysis and transcriptome sequencing results showed that the expression of this gene was up-regulated in Tubb4b knockout cells. This implies that Tubb4b may be involved in the division of spermatogonia with multiple cell cycle regulatory proteins. Overall, these data indicate that Tubb4b has a specific role in regulating spermatogonia proliferation and cell cycle.

A Potential Role for the Gsdf–eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective

AbstractGonadal soma–derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag “pull-down” assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor β–dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf–dynein and Gsdf–eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor β) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.

CG8005 Mediates Transit-Amplifying Spermatogonial Divisions via Oxidative Stress in Drosophila Testes.

The generation of reactive oxygen species (ROS) widely occurs in metabolic reactions and affects stem cell activity by participating in stem cell self-renewal. However, the mechanisms of transit-amplifying (TA) spermatogonial divisions mediated by oxidative stress are not fully understood. Through genetic manipulation of Drosophila testes, we demonstrated that CG8005 regulated TA spermatogonial divisions and redox homeostasis. Using in vitro approaches, we showed that the knockdown of CG8005 increased ROS levels in S2 cells; the induced ROS generation was inhibited by NAC and exacerbated by H2O2 pretreatments. Furthermore, the silencing of CG8005 increased the mRNA expression of oxidation-promoting factors Keap1, GstD1, and Mal-A6 and decreased the mRNA expression of antioxidant factors cnc, Gclm, maf-S, ND-42, and ND-75. We further investigated the functions of the antioxidant factor cnc, a key factor in the Keap1-cnc signaling pathway, and showed that cnc mimicked the phenotype of CG8005 in both Drosophila testes and S2 cells. Our results indicated that CG8005, together with cnc, controlled TA spermatogonial divisions by regulating oxidative stress in Drosophila.

Somatic ERK activation during transit amplification is essential for maintaining the synchrony of germline divisions in Drosophila testis

Transit amplification (TA) of progenitor cells maintains tissue homeostasis by balancing proliferation and differentiation. In Drosophila testis, the germline proliferation is tightly regulated by factors present in both the germline and the neighbouring somatic cyst cells (SCCs). Although the exact mechanism is unclear, the epidermal growth factor receptor (EGFR) activation in SCCs has been reported to control spermatogonial divisions within a cyst, through downstream activations of Rac1-dependent pathways. Here, we report that somatic activation of the mitogen-activated protein kinase (Rolled/ERK) downstream of EGFR is required to synchronize the mitotic divisions and regulate the transition to meiosis. The process operates independently of the Bag-of-marble activity in the germline. Also, the integrity of the somatic cyst enclosure is inessential for this purpose. Together, these results suggest that synchronization of germ-cell divisions through somatic activation of distinct ERK-downstream targets independently regulates TA and subsequent differentiation of neighbouring germline cells.

The Germline Linker Histone dBigH1 and the Translational Regulator Bam Form a Repressor Loop Essential for Male Germ Stem Cell Differentiation

Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. Here, we report the essential role of the germline linker histone dBigH1 during early spermatogenesis. Our results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression.

Macrophages Contribute to the Spermatogonial Niche in the Adult Testis.

The testis produces sperm throughout the male reproductive lifespan by balancing self-renewal and differentiation of spermatogonial stem cells (SSCs). Part of the SSC niche is thought to lie outside the seminiferous tubules of the testis; however, specific interstitial components of the niche that regulate spermatogonial divisions and differentiation remain undefined. We identified distinct populations of testicular macrophages, one of which lies on the surface of seminiferous tubules, in close apposition to areas of tubules enriched for undifferentiated spermatogonia. These macrophages express spermatogonial proliferation- and differentiation-inducing factors, such as colony-stimulating factor 1 (CSF1) and enzymes involved in retinoic acid (RA) biosynthesis. We show that transient depletion of macrophages leads to a disruption in spermatogonial differentiation. These findings reveal an unexpected role for macrophages in the spermatogonial niche in the testis and raise the possibility that macrophages play previously unappreciated roles in stem/progenitor cell regulation in other tissues.

Chk2 and P53 Regulate the Transmission of Healed Chromosomes in the Drosophila Male Germline

When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.

Cytological and karyological observations on two endemic giant pill-millipedesArthrosphaera(Pocock 1895) (Diplopoda: Sphaerotheriida) of the Western Ghats of India

Cytological features and karyomorphology of two endemic giant pill-millipedes (Arthrosphaera fumosa and A. magna) of the Western Ghats were studied based on the meiotic cell divisions using Giemsa staining technique. Different meiotic stages of spermatogonial cell divisions were compared and karyotypes were constructed from the metaphase stages. The diploid chromosome number, 2n = 30, in A. fumosa as well as A. magna with XY sex determination system was seen. Most of the meiotic chromosome behaviors were similar between the millipede species. All meiotic chromosomes in A. fumosa belonged to acrocentric type, while in A. magna 27 were acrocentrics, two (3rd and 10th) were sub-metacentrics and one (5th) was metacentric. The relative lengths of autosomes in A. fumosa and A. magna were 21.9–109.3 and 38.8–90.6, respectively. The X chromosome was longer than the Y chromosome in both millipedes. The relative lengths of the X chromosomes of A. fumosa and A. magna were 130.1 and 103.6, while for the Y chromosome ...

Germ Cell Specification Requires Zygotic Mechanisms Rather Than Germ Plasm in a Basally Branching Insect

Primordial germ cell (PGC) specification is a universal process across animals, but the molecular mechanisms specifying PGCs are remarkably diverse. In Drosophila, PGCs are specified by maternally provided, asymmetrically localized cytoplasmic factors (germ plasm). In contrast, historical literature on most other arthropods reports that PGCs arise from mesoderm during midembryogenesis, suggesting that an arthropod last common ancestor may have specified PGCs via zygotic mechanisms. However, there has been no direct experimental evidence to date for germ plasm-independent arthropod PGC specification. Here we show that in a basally branching insect, the cricket Gryllus bimaculatus, conserved germ plasm molecules are ubiquitously, rather than asymmetrically, localized during oogenesis and early embryogenesis. Molecular and cytological analyses suggest that Gryllus PGCs arise from abdominal mesoderm during segmentation, and twist RNAi embryos that lack mesoderm fail to form PGCs. Using RNA interference we show that vasa and piwi are not required maternally or zygotically for PGC formation but rather are required for primary spermatogonial divisions in adult males. These observations suggest that Gryllus lacks a maternally inherited germ plasm, in contrast with many holometabolous insects, including Drosophila. The mesodermal origin of Gryllus PGCs and absence of instructive roles for vasa and piwi in PGC formation are reminiscent of mouse PGC specification and suggest that zygotic cell signaling may direct PGC specification in Gryllus and other Hemimetabola.

Upsetting the Dogma: Germline Selection in Human Males

Upsetting the Dogma: Germline Selection in Human Males

Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila

Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. Here, we show that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-βPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation.

Male gametogenesis without centrioles

The orientation of the mitotic spindle plays a central role in specifying stem cell-renewal by enabling interaction of the daughter cells with external cues: the daughter cell closest to the hub region is instructed to self-renew, whereas the distal one starts to differentiate. Here, we have analyzed male gametogenesis in DSas-4 Drosophila mutants and we have reported that spindle alignment and asymmetric divisions are properly executed in male germline stem cells that lack centrioles. Spermatogonial divisions also correctly proceed in the absence of centrioles, giving rise to cysts of 16 primary spermatocytes. By contrast, abnormal meiotic spindles assemble in primary spermatocytes. These results point to different requirements for centrioles during male gametogenesis of Drosophila. Spindle formation during germ cell mitosis may be successfully supported by an acentrosomal pathway that is inadequate to warrant the proper execution of meiosis.

HOW Is Required for Stem Cell Maintenance in the Drosophila Testis and for the Onset of Transit-Amplifying Divisions

The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric division to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic divisions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial divisions. HOW is normally expressed in a complementary pattern to Bam in the germline and bam mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Bam to the level required for differentiation, resulting in extra mitotic divisions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit-amplifying divisions.

Variation in the stock of testicular stem cells and in the yield of spermatogonial divisions in ram and bull testes.

Two types of stem spermatogonia- type A0 and A1 spermatogonia- have been observed in ram and bull testes. Type A0 spermatogonia are present in the impuberal testis. Their total number/testis does not vary greatly during life of bull. Type A0 spermatogonia give rise to type A1 spermatogonia which appear at the onset of spermatogenesis; their total number/testis increases during and after puberty. Total numbers of type A0 and A1 spermatogonia/testis are not correlated in the ram while total numbers of type A1 spermatogonia and Sertoli cells are correlated. Hemicastration performed in impuberal calves induces an increase in numbers of Sertoli cell/type A1 spermatogonia/testis. In adult ram, the total number of A0 and A1 spermatogonia decreases during the non sexual season and increase again in the sexual season. After hypophysectomy performed in adult rams, the number of A0 spermatogonia/testis is not modified while that of A1 spermatogonia decreases. In ram and bull, 6 spermatogonial generations were detected before the germ cells undergo meiosis. In the adult ram and bull, the type A1 spermatogonia originate mostly after divisions of A2 spermatogonia. Few of them arise from A0 spermatogonia. The yield of primary spermatocytes from spermatogonial divisions is very low, one third and one half of the expected number in the bull and ram respectively. In ram, thus yield positively correlated to LH plasma levels (0.5; P = 0.05). In hypophysectomized ram, this yield is nearly equal to zero.

Chromosomes and DNA bodies of Acheta desertus (Orthoptera). With parallels to Acheta domesticus.

The chromosome number of Acheta desertus (Orthoptera) was found to be 2n = 21 (male) and 2n = 22 (female) and the sex mechanism XO:XX. Acheta desertus has the same chromosome number as Acheta domesticus but different chromosome morphology. In both species a heteromorphic pair of autosomes appears in spermatogonial divisions. One of the autosomes of the pair shows negative heteropycnosis along its whole length while its partner resembles the other autosomes. This aberration is limited to the males and has been observed in all male crickets investigated but in a varying percentage of the total number of spermatogonial metaphases. One distinct DNA body as found in A. domesticus is also present in addition to the chromosomes in the oocytes of A. desertus. During early diplotene large masses of RNA-containing material accumulates at the periphery of the DNA body. As the oocytes proceed through diplotene this material reorganizes into small masses.

Estrogens in testicular physiology: GPR30 – a novel g-protein estradiol receptor conveys estradiol action on early spermatogonial divisions and spermiogenesis in rodents and humans

Estrogens in testicular physiology: GPR30 – a novel g-protein estradiol receptor conveys estradiol action on early spermatogonial divisions and spermiogenesis in rodents and humans

Les modalités du cycle sexuel du poulpe Octopus vulgaris du Sud marocain (Tantan, Boujdour)

The reproduction of Octopus vulgaris between Tan Tan and Boujdour is studied. The samples used are sorted monthly from commercial catches of coastal trawlers operating in this area from December 2001 to May 2003. The study shows that sexual maturity is in advance for males than for females. Two spawning periods are made out by the follow-up of the RGS: a spring period from March to July, and an autumnal period, which is less intense, between September and October. The division of spermatogonia and spermatocytes reaches its maximal intensity in July and December, whereas the spermiogenesis is active until the time of mating. The vitellogenesis starts in mid-December and extends until the following spawning period. This reveals a later gonadic development for females than for males; approximately seven months for females and three months for males. To cite this article: F.H. Idrissi et al., C. R. Biologies 329 (2006).

Novel Class of Glycosphingolipids Involved in Male Fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.

Aurora B expression in normal testis and seminomas.

Aurora/Ipl1-related kinases are a conserved family of proteins that have multiple functions during mitotic progression. High levels of Aurora kinases are characteristic of rapidly dividing cells and tumours. Aurora B encodes a protein that associates with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. In this study the expression and the localisation of Aurora B throughout germinal epithelial progression in normal testis and its neoplastic counterpart were analysed. Immunocytochemistry and RT-PCR analysis of mouse germinal epithelium cells showed the presence of Aurora B in spermatogonia and occasionally in spermatocytes. Western blot analysis revealed the typical Aurora B isoform ( approximately 41 kDa) in the same cellular types. A similar distribution was observed in human testis by immunohistochemistry. Moreover, the distribution and the expression of Aurora B were investigated in neoplasms derived from germ cells. Surgical samples of seminomas were analysed, and a high percentage of Aurora B positive cells (51%) was detected; the expression of Aurora B was significantly related to the MIB-1 proliferation marker (R=0.816). The data presented here demonstrate that Aurora B expression occurs in spermatogonial division. Furthermore, our results indicate that the expression of Aurora B is a consistent feature of human seminomas.

B2 bradykinin receptor mediates the stimulatory effect of bradykinin on rat germ cell proliferation in vitro

The contribution of B1 and B2 bradykinin receptors to germ cell proliferation was studied by using in vitro organ cultures of testicular fragments of 3.5-day-old rats in the presence of 3H thymidine. Different combinations of agonists and antagonists of B1 and B2 receptors exerted differential mitogenic effect on pro-spermatogonial cells. Application of bradykinin (B2 receptor agonist) alone induced a threefold increase in germ cell labelling index compared with the control, whereas des-Arg9-bradykinin (B1 receptor agonist) caused weak stimulation (24%) on spermatogonial mitotic activity. The bradykinin-induced germ cell proliferation was significantly affected by B2 receptor antagonist (HOE-140) but not by B1 receptor antagonist. When B1 or B2 receptor antagonists were applied with des-Arg9-bradykinin, the germ cell labelling indices were nonsignificantly different compared with those of B1 receptor agonist only. The present findings suggest that B2 receptor is involved in mediating the stimulatory effect of bradykinin on germ cell proliferation and therefore bradykinin might be an important local testicular factor in the regulation of spermatogonial division and germ cell number.