Christopher J. Payne

The Role of Maternal VegT in Establishing the Primary Germ Layers in Xenopus Embryos

VegT is a T-box transcription factor whose mRNA is synthesized during oogenesis and localized in the vegetal hemisphere of the egg and early embryo. We show that maternally expressed VegT controls the pattern of primary germ layer specification in Xenopus embryos. Reduction of the maternal store completely alters the fates of different regions of the blastula so that animal cell fate is changed from epidermis and nervous system to epidermis only, equatorial cell fate is changed from mesoderm to ectoderm, and vegetal cell fate is changed from endoderm to mesoderm and ectoderm. Vegetal cells lose their capacity both to form endoderm and to release mesoderm-inducing signals. These results show that a single maternally expressed gene controls the patterning of the Xenopus blastula.

Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice.

We have generated a transgenic mouse line,Tg(Stra8‐cre)1Reb (Stra8‐cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell‐specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early‐stage spermatogonia and is detected through preleptotene‐stage spermatocytes. To further define when cre becomes active, we crossed Stra8‐cre males with Tg(ACTB‐Bgeo/GFP)21Lbe (Z/EG) reporter females and compared the expression of enhanced green fluorescent protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF—a marker for undifferentiated spermatogonia. Co‐expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8‐cre;Z/EG males and females with wild‐type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8‐cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell‐specific activity of Stra8‐cre makes this mouse line a unique resource to study testicular germ cell development. genesis 46:738–742, 2008.

Insulin reduces norepinephrine transporter mRNA in vivo in rat locus coeruleus.

Acute and chronic in vitro insulin treatment can inhibit the uptake of norepinephrine (NE) by adult rat brain synaptosomes and slices, fetal neuronal cultures, and PC12 cells. In the present study we tested whether chronic in vivo insulin treatment could alter the biosynthetic capacity of rat locus coeruleus neurons for the NE transporter protein (NET). Chronic third ventricular insulin treatment resulted in a suppression of NET mRNA to about one third of the level of vehicle-treated controls. Our finding suggests that insulin may play a regulatory role in the synthesis of this transporter, thereby modulating activity in CNS noradrenergic pathways.

Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization

Fertilization is complete once the parental genomes unite, and requires the migration of the egg nucleus to the sperm nucleus (female and male pronuclei, respectively) on microtubules within the inseminated egg. Neither the molecular mechanism of pronucleus binding to microtubules nor the role of motor proteins in regulating pronuclear motility has been fully characterized, and the failure of zygotic development in some patients suggests that they contribute to human infertility. Based on the minus-end direction of female pronuclear migration, we propose a role for cytoplasmic dynein and dynactin in associating with the pronuclear envelope and mediating genomic union. Our results show that dynein intermediate and heavy chains preferentially concentrate around the female pronucleus, whereas dynactin subunits p150Glued, p50 and p62 localize to the surfaces of both pronuclei. Transfection of antibodies against dynein and dynactin block female pronuclear migration in zygotes. Both parthenogenetic activation in oocytes and microtubule depolymerization in zygotes significantly reduce the localization of dynein to the female pronucleus but do not inhibit the pronuclear association of dynactin. When immunoprecipitated from zygotes, p150Glued associates with nuclear pore complex proteins, as well as the intermediate filament vimentin and dynein. Antibodies against nucleoporins and vimentin inhibit pronuclear apposition when transfected into zygotes. We conclude that preferentially localized dynein and perinuclear dynactin associate with the nuclear pore complex and vimentin and are required to mediate genomic union. These data suggest a model in which dynein accumulates and binds to the female pronucleus on sperm aster microtubules, where it interacts with dynactin, nucleoporins and vimentin.

Non-random chromosome positioning in human sperm and sex chromosome anomalies following intracytoplasmic sperm injection.

Chromosome paint for chromosome 18 hy b ridised in more than 80% (190/234) of sperm examined, and chromosome paint for X chromosome hy b ridised in just over 40% ( 2 3 0 / 5 5 3 ) . The sperm tail was used as a reference to identify apical or basal position. Chromosome 18 was found near the s p e rm tail in almost 60% of the sperm (113/190), while the X chromosome was found near the sperm tail in less than 40% ( 8 8 / 2 3 0 ) . This shows that chromosome 18 is preferentially l o c ated in the basal region of the sperm nucleus, while the X chromosome is preferentially located in the apical regi o n . The placement of chromosomes within the sperm nucleus m ay have implications for assisted reproductive technologi e s ( A RT ) , and may help explain the apparent increase in sexchromosome aberr ations in ICSI offspri n g . This increase could be due to the fact that the X chromosome in sperm nuclei preferentially localises in the apical regi o n .I n such n u c l e i , the S-phase of the cell cycle may be delayed due to the decreased rate of nuclear decondensation in the apical regi o n of the sperm head, h i n d e ring progression to the onset of firs t mitosis in the zygote. This may lead to mitotic err o rs in the d i s t ri bution of the X chromosome during first cleava g e , implying that such positioning of the sex chromosome could lead to the increase in sex-chromosome anomalies follow i n g I C S I . All animal procedures were approved by the ORPRC Institutional A n i m a l Care and Use Committee. Human sperm were obtained from the Fo l l a s L a b o r at o ries after Institutional Review Board approva l .This research wa s sponsored by grants to GS by the NIH.

MicroRNA 146 (Mir146) Modulates Spermatogonial Differentiation by Retinoic Acid in Mice

ABSTRACT Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3′ untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation.

Sin3a is required by sertoli cells to establish a niche for undifferentiated spermatogonia, germ cell tumors, and spermatid elongation.

Microenvironments support the maintenance of stem cells and the growth of tumors through largely unknown mechanisms. While cell‐autonomous chromatin modifications have emerged as important determinants for self‐renewal and differentiation of stem cells, a role for non‐cell autonomous epigenetic contributions is not well established. Here, we genetically ablated the chromatin modifier Swi‐independent 3a (Sin3a) in fetal Sertoli cells, which partly comprise the niche for male germline stem cells, and investigated its impact on spermatogenic cell fate and teratoma formation in vivo. Sertoli cell‐specific Sin3a deletion resulted in the formation of few undifferentiated spermatogonia after birth while initially maintaining spermatogenic differentiation. Stem cell‐associated markers Plzf, Gfra1, and Oct4 were downregulated in the mutant fetal gonad, while Sertoli cell markers Steel and Gdnf, which support germ cells, were not diminished. Following birth, markers of differentiating spermatogonia, Kit and Sohlh2, exhibited normal levels, but chemokine‐signaling molecules chemokine (C‐X‐C motif) ligand 12 (CXCL12)/stromal cell‐derived factor 1 (SDF1) and chemokine (C‐X‐C motif) receptor 4 (CXCR4), expressed in Sertoli cells and germ cells, respectively, were not detected. In the juvenile, mutant testes exhibited a progressive loss of differentiating spermatogonia and a block in spermatid elongation, followed by extensive germ cell degeneration. Sertoli cell‐specific Sin3a deletion also suppressed teratoma formation by fetal germ cells in an in vivo transplantation assay. We conclude that the epigenome of Sertoli cells influences the establishment of a niche for germline stem cells as well as for tumor initiating cells. STEM CELLS 2010;28:1424–1434

MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells

Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3.

Transcription and imprinting dynamics in developing postnatal male germline stem cells

Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1(+); high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1(+); moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT(+)). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor-ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles.

Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia

Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.