Pellegrino Rossi

Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation.

It is well established that the c-kit gene plays an essential role in the proliferation of differentiating spermatogonia in prepuberal mice. However, the mechanisms that regulate the onset of spermatogenesis, i.e. differentiation of spermatogonial stem cells and c-kit expression, are poorly understood. Here we identify a novel signal transduction system in mouse prepuberal testis regulating this developmental event, involving bone morphogenetic protein 4 (BMP4) and its transduction machinery. BMP4 is produced by Sertoli cells very early in the postnatal life and is successively down regulated in peri-puberal Sertoli cells. Its receptor Alk3 and the R-Smad Smad5 are specifically expressed both in proliferating primordial germ cells and in postnatal spermatogonia. BMP4 stimulation of cultured spermatogonia induces Smad4/5 nuclear translocation and the formation of a DNA-binding complex with the transcriptional coactivator p300/CBP. In vitro exposure of undifferentiated spermatogonia to BMP4 exerts both mitogenic and differentiative effects, inducing [3H]thymidine incorporation and Kit expression. As a result of the latter event, Kit-negative spermatogonia acquire sensitivity to Stem Cell Factor.

Repression of kit Expression by Plzf in Germ Cells

ABSTRACT Male mice lacking expression of Plzf, a DNA sequence-specific transcriptional repressor, show progressive germ cell depletion due to exhaustion of the spermatogonial stem cell population. This is likely due to the deregulated expression of genes controlling the switch between spermatogonial self-renewal and differentiation. Here we show that Plzf directly represses the transcription of kit, a hallmark of spermatogonial differentiation. Plzf represses both endogenous kit expression and expression of a reporter gene under the control of the kit promoter region. A discrete sequence of the kit promoter, required for Plzf-mediated kit transcriptional repression, is bound by Plzf both in vivo and in vitro. A 3-bp mutation in this Plzf binding site abolishes the responsiveness of the kit promoter to Plzf repression. A significant increase in kit expression is also found in the undifferentiated spermatogonia isolated from Plzf−/− mice. Thus, we suggest that one mechanism by which Plzf maintains the pool of spermatogonial stem cells is through a direct repression of kit expression.

Opposing effects of retinoic acid and FGF9 on Nanos2 expression and meiotic entry of mouse germ cells

In the mouse, three genes that are homologous to the Drosophila Nanos (Nos) gene have been identified. Deletion of one of these genes, Nanos2, results in male sterility, owing to loss of germ cells during fetal life. Before apoptosis, Nanos2-null gonocytes enter meiosis, suggesting that Nanos2 functions as a meiotic repressor. Here, we show that Nanos2 is continuously expressed in male germ cells from fetal gonocytes to postnatal spermatogonial stem cells. We observed that the promeiotic factor AtRA, an analog of retinoic acid (RA), downregulates NANOS2 levels, in both fetal and postnatal gonocytes, while promoting meiosis. Interestingly, FGF9, a growth factor crucial for sex differentiation and survival of fetal gonocytes, upregulates levels of NANOS2 in both male and female primordial germ cells (PGCs) and in premeiotic spermatogonia. This effect was paralleled by an impairment of meiotic entry, suggesting that FGF9 acts as an inhibitor of meiosis through the upregulation of Nanos2. We found that NANOS2 interacts with PUM2, and that these two proteins colocalize in the ribonucleoparticle and polysomal fractions on sucrose gradients, supporting the notion that they bind RNA. Finally, we found that recombinant NANOS2 binds to two spermatogonial mRNAs, Gata2 and Taf7l, which are involved in germ-cell differentiation.

Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase.

Microinjection in mouse eggs of tr‐kit, a truncated form of the c‐kit tyrosine kinase present in mouse spermatozoa, causes resumption of meiosis through activation of phospholipase Cγ1 (PLCγ1) and Ca2+ mobilization from intracellular stores. We show that the Src‐like kinase Fyn phosphorylates Tyr161 in tr‐kit and that this residue is essential for tr‐kit function. Fyn is localized in the cortex region underneath the plasma membrane in mouse oocytes. Using several approaches, we demonstrate that Fyn associates with tr‐kit and that the interaction requires Tyr161. The interaction between tr‐kit and Fyn triggers activation of the kinase as monitored by both autophosphorylation and phosphorylation of PLCγ1. Co‐injection of tr‐kit with the SH2 domain of Fyn, or pre‐treatment with a Fyn inhibitor, impairs oocyte activation, suggesting that activation of Fyn by tr‐kit also occurs in vivo. Finally, microinjection of constitutively active Fyn triggers oocyte activation downstream of tr‐kit but still requires PLC activity. We suggest that the mechanism by which tr‐kit triggers resumption of meiosis of mouse eggs requires a functional interaction with Fyn and phosphorylation of PLCγ1.

Role of c-kit in mammalian spermatogenesis

The tyrosine-kinase receptor c-kit and its ligand, stem cell factor (SCF), are essential for the maintenance of primordial germ cells (PGCs) in both sexes. However, c-kit and a postmeiotic- specific alternative c-kit gene product play important roles also during post-natal stages of spermatogenesis. In the adult testis, the c-kit receptor is re-expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas SCF is expressed by Sertoli cells under FSH stimulation. SCF stimulates DNA synthesis in type A spermatogonia cultured in vitro, and injection of anti-c-kit antibodies blocks their proliferation in vivo. A point mutation in the c-kit gene, which impairs SCF-mediated activation of phosphatydilinositol 3-kinase, does not cause any significant reduction in PGCs number during embryonic development, nor in spermatogonial stem cell populations. However males are completely sterile due to a block in the initial stages of spermatogenesis, associated to abolishment of DNA-synthesis in differentiating A1-A4 spermatogonia. With the onset of meiosis c-kit expression ceases, but a truncated c-kit product, tr-kit, is specifically expressed in post-meiotic stages of spermatogenesis, and is accumulated in mature spermatozoa. Microinjection of tr-kit into mouse eggs causes their parthenogenetic activation, suggesting that it might play a role in the final function of the gametes, fertilization.

A novel c-kit transcript, potentially encoding a truncated receptor, originates within a kit gene intron in mouse spermatids.

We have cloned a novel c-kit mRNA of 3.2 kb expressed in postmeiotic male germ cells. This transcript initiates in the genomic region immediately upstream of the exon coding for the second box of the split c-kit tyrosine kinase domain. The open reading frame (ORF) contains 12 novel amino acids in frame with the C-terminal 190 amino acids of the c-kit protein. It lacks therefore the upstream region in the 5.5-kb c-kit mRNA encoding the extracellular and transmembrane domain, the ATP-binding site and the kinase insert domain present in the c-kit protein.

Expression of a Truncated Form of the c-Kit Tyrosine Kinase Receptor and Activation of Src Kinase in Human Prostatic Cancer

A truncated form of the c-Kit tyrosine kinase receptor, originally identified in mouse haploid germ cells, is aberrantly expressed in human cancer cell lines of various origin. This alternative transcript originates in the 15th intron of the human c-kit gene. We have previously demonstrated that sperm-carried mouse truncated c-Kit (tr-Kit) is a strong activator of the Src-family tyrosine kinases both in transfected cells and in mouse oocytes. In the present work, we report that human tr-Kit mRNA and protein are expressed in LNCaP prostatic cancer cells. We have identified two regions in the 15th and 16th introns of the human c-kit gene that show homology with sequences in the spermatid-specific tr-Kit promoter within the 16th intron of mouse c-kit. We also show that nuclear factors present in LNCaP cells bind to discrete sequences of the mouse tr-Kit promoter. Moreover, Western blot analysis of 23 primary prostate cancers indicated that tr-Kit was expressed in approximately 28% of the tumors at less advanced stages (Gleason grade 4 to 6) and in 66% of those at more advanced stages (Gleason grade 7 to 9), whereas it was not expressed in benign prostatic hypertrophies. Sequencing of the cDNA for the truncated c-Kit, amplified from both LNCaP cells and neoplastic tissues, confirmed the existence in prostate cancer cells of a transcript arising from the 15th intron of human c-kit. We also show that tr-Kit-expressing LNCaP cells and prostatic tumors have higher levels of phosphorylated/activated Src than tr-Kit-negative PC3 cells or prostatic tumors, and that transfection of tr-Kit in PC3 cells caused a dramatic increase in Src activity. Interestingly, we found that Sam68, a RNA-binding protein phosphorylated by Src in mitosis, is phosphorylated only in prostate tumors expressing tr-Kit. Indeed, both activation of Src and phosphorylation of Sam68 were observed in all of the three grade 7 to 9 tumors analyzed that expressed tr-Kit. Our data describe for the first time the existence of a truncated c-Kit protein in primary tumors and show a correlation between tr-Kit expression and activation of the Src pathway in the advanced stages of the disease. Thus, these results might pave the way for the elucidation of a novel pathway in neoplastic transformation of prostate cells.

Gynaecomastia in men with chronic myeloid leukaemia after imatinib

cKit and platelet-derived growth-factor receptor (PDGFR) are receptor tyrosine kinases expressed in the testis, are involved in testosterone production, and are inhibited by imatinib. We measured hormone concentrations in 38 men receiving imatinib for chronic myeloid leukaemia at baseline and during treatment. Mean follow-up was 23.6 months (SD 7.5). We noted seven cases of gynaecomastia (18%, 95% CI 6-30%). A comparison of hormone concentrations in 21 patients before and during treatment showed that patients who developed gynaecomastia had a reduction in free testosterone concentrations of 29.53 pmol/L (95% CI 11.63-47.43), while patients who did not had a decrease of 6.36 pmol/L (-1.02 to 13.74). In most men with chronic myeloid leukaemia studied here, imatinib was associated with a reduction in the production of testicular hormones and in some, with the development of gynaecomastia.

Activation of the Mitogen-activated Protein Kinase ERK1 during Meiotic Progression of Mouse Pachytene Spermatocytes

Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OA-stimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G2/M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G2/M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.

SOHLH1 and SOHLH2 control Kit expression during postnatal male germ cell development

How Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kit-expressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo co-immunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression.