Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.     

The Caenorhabditis elegans Ste20 kinase, GCK-3, is essential for postembryonic developmental timing and regulates meiotic chromosome segregation

Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.     

Expression of TTK, a novel human protein kinase, is associated with cell proliferation.

We have isolated the full-length sequence for a unique human kinase, designated TTK. TTK was initially identified by screening of a T cell expression library with anti-phosphotyrosine antibodies. The kinases most closely related to TTK are the SPK1 serine, threonine and tyrosine kinase, the Pim1, PBS2, and CDC2 serine/threonine kinases, and the TIK kinase which was also identified through screening of an expression library with anti-phosphotyrosine antibodies. However, the relationships are distant with less than 25% identity. Nevertheless, TTK is highly conserved throughout phylogeny with hybridizing sequences being detected in mammals, fish, and yeast. TTK mRNA is present at relatively high levels in testis and thymus, tissues which contain a large number of proliferating cells, but is not detected in most other benign tissues. Freshly isolated cells from most malignant tumors assessed expressed TTK mRNA. As well, all rapidly proliferating cell lines tested expressed TTK mRNA. Escherichia coli expressing the complete kinase domain of TTK contain markedly elevated levels of phosphoserine and phosphothreonine as well as slightly increased levels of phosphotyrosine. Taken together, these findings suggest that expression of TTK, a previously unidentified member of the family of kinases which can phosphorylate serine, threonine, and tyrosine hydroxyamino acids, is associated with cell proliferation.     

Aurora kinase B modulates chromosome alignment in mouse oocytes

The elevated incidence of aneuploidy in human oocytes warrants study of the molecular mechanisms regulating proper chromosome segregation. The Aurora kinases are a well‐conserved family of serine/threonine kinases that are involved in proper chromosome segregation during mitosis and meiosis. Here we report the expression and localization of all three Aurora kinase homologs, AURKA, AURKB, and AURKC, during meiotic maturation of mouse oocytes. AURKA, the most abundantly expressed homolog, localizes to the spindle poles during meiosis I (MI) and meiosis II (MII), whereas AURKB is concentrated at kinetochores, specifically at metaphase of MI (Met I). The germ cell‐specific homolog, AURKC, is found along the entire length of chromosomes during both meiotic divisions. Maturing oocytes in the presence of the small molecule pan‐Aurora kinase inhibitor, ZM447439 results in defects in meiotic progression and chromosome alignment at both Met I and Met II. Over‐expression of AURKB, but not AURKA or AURKC, rescues the chromosome alignment defect suggesting that AURKB is the primary Aurora kinase responsible for regulating chromosome dynamics during meiosis in mouse oocytes. Mol. Reprod. Dev. 76: 1094–1105, 2009. © 2009 Wiley‐Liss, Inc.     

A novel serine/threonine kinase gene,Gek1, is expressed in meiotic testicular germ cells and primordial germ cells

We have isolated a novel serine/threonine kinase gene designated Gek1 from mouse primordial germ cell-derived embryonic germ cell. Gek1 is preferentially expressed in meiotic testicular germ cells and primordial germ cells. Gek1 mRNA is also detected in several other tissues, including hematopoietic organs in adult mice and central nervous system in embryos. The Gek1 cDNA encodes a protein with the consensus sequence of the catalytic domain of protein kinases in its N-terminal region. The deduced amino acid sequence of Gek1 in the kinase domain is related to those encoded by the Saccharomyces cerevisiae STE20, CDC15, and Drosophila melanogaster ninaC. The patterns of expression and the structural features of Gek1 suggest that the gene product is involved in signal transduction or nuclear division of germ cells and other proliferating cells. We also show that Gek1 locates on chromosome 11, near the wr locus, showing neuronal and reproductive defects. © 1996 Wiley-Liss, Inc.     

Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.     

Retinoic Acid signalling and the control of meiotic entry in the human fetal gonad

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8-9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis.     

ATRA and KL promote differentiation toward the meiotic program of male germ cells.

While it is known that Retinoic Acid (RA) induces meiosis in mouse female fetal gonads, the mechanisms which regulate this process during spermatogenesis are poorly understood. We show that the All trans RA derivative (ATRA) and Kit Ligand (KL) increase meiotic entry of postnatal mouse spermatogonia in vitro without synergism. Competence to enter meiosis is reached by spermatogonia only at the stage in which they undergo Kit-dependent divisions. Besides increasing Kit expression in spermatogonia, ATRA also upregulates KL expression in Sertoli cells. Both ATRA and KL increase the expression of Stimulated by Retinoic Acid Gene 8 and Dmc1, an early meiotic marker. A specific Kit tyrosine kinase inhibitor prevents the increase in the number of meiotic cells induced by both the two factors, suggesting that they converge on common Kit-dependent signalling pathways. Meiotic entry induced by ATRA and KL is independent from their ability to affect germ cell viability, and is mediated by the activation of PI3K and MAPK pathways through Kit autophosphorylation. ATRA-induced phosphorylation of the two downstream kinases is mediated by a non-genomic mechanism.

These data suggest that RA may control the timing of meiosis by influencing both the somatic and the germ cell compartment of the postnatal testis through the activation of the KL/Kit system.     

Estrogen-induced gonadal sex reversal in the tammar wallaby

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.