Meiotic wave adds extra asymmetry to the development of female chicken gonads

Development of female gonads in the chicken is asymmetric. This asymmetry affects gene expression, morphology, and germ cell development; consequently only the left ovary develops into a functional organ, whereas the right ovary remains vestigial. In males, on the other hand, both gonads develop into functional testes. Here, we revisited the development of asymmetric traits in female (and male) chicken gonads between Hamburger Hamilton stage 16 (HH16) and hatching. At HH16, primordial germ cells migrated preferentially to the left gonad, accumulating in the left coelomic hinge between the gut mesentery and developing gonad in both males and females. Using the meiotic markers SYCP3 and phosphorylated H2AFX, we identified a previously undescribed, pronounced asymmetryc meiotic progression in the germ cells located in the central, lateral, and extreme cortical regions of the left female gonad from HH38 until hatching. Moreover, we observed that-in contrast to the current view-medullary germ cells are not apoptotic, but remain arrested in pre-leptotene until hatching. In addition to the systematic analysis of the asymmetric distribution of germ cells in female chicken gonads, we propose an updated model suggesting that the localization of germ cells-in the left or right gonad; in the cortex or medulla of the left gonad; and in the central part or the extremities of the left cortex-has direct consequences for their development and participation in adult reproduction

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

BACKGROUND: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives

JMJD1C Exhibits Multiple Functions in Epigenetic Regulation during Spermatogenesis.

Jmjd1C is one of the Jmjd1 family genes that encode putative demethylases against histone H3K9 and non-histone proteins and has been proven to play an indispensable role in mouse spermatogenesis. Here, we analyzed a newly-bred transgenic mouse strain carrying a Jmjd1C loss-of-function allele in which a β-geo cassette was integrated into the intron of the Jmjd1C locus. Jmjd1C gene-trap homozygous testes exhibited malformations in postmeiotic processes and a deficiency in the long-term maintenance of undifferentiated spermatogonia. Some groups of spermatids in the homozygous testis showed abnormal organization and incomplete elongation from the first wave of spermatogenesis onwards. Moreover, histone H4K16 acetylation, which is required for the onset of chromatin remodeling, appeared to be remarkably decreased. These effects may not have been a result of the drastic decrease in gene expression related to the events but instead may have been due to the lack of interaction between JMJD1C and its partner proteins, such as MDC1 and HSP90. Additionally, significant decreases in Oct4 expression and NANOG- and OCT4-expressing spermatogonia were found in the Jmjd1C homozygous mature testis, suggesting that JMJD1C may participate in the maintenance of spermatogonial stem cell self-renewal by up-regulating Oct4 expression. These results indicate that JMJD1C has multiple functions during spermatogenesis through interactions with different partners during the spermatogenic stages

A New Horizon in Reproductive Research with Pluripotent Stem Cells: Successful In Vitro Gametogenesis in Rodents, Its Application to Large Animals, and Future In Vitro Reconstitution of Reproductive Organs Such as “Uteroid” and “Oviductoid”

Recent success in derivation of functional gametes (oocytes and spermatozoa) from pluripotent stem cells (PSCs) of rodents has made it feasible for future application to large animals including endangered species and to ultimately humans. Here, we summarize backgrounds and recent studies on in vitro gametogenesis from rodent PSCs, and similar approaches using PSCs from large animals, including livestock, nonhuman primates (NHPs), and humans. We also describe additional developing approaches for in vitro reconstitution of reproductive organs, such as the ovary (ovarioid), testis (testisoid), and future challenges in the uterus (uteroid) and oviduct (oviductoid), all of which may be derived from PSCs. Once established, these in vitro systems may serve as a robust platform for elucidating the pathology of infertility-related disorders and ectopic pregnancy, principle of reproduction, and artificial biogenesis. Therefore, these possibilities, especially when using human cells, require consideration of ethical issues, and international agreements and guidelines need to be raised before opening “Pandora’s Box”

Abnormal histone-protamine transition found in the <i>Jmjd1C</i> ^<i>gt/gt</i> testis.

<p>Testis sections of littermate 4-month-old +/+ (a-e) (stage XI) and gt/gt (f-j) testis were immunostained with anti-acetylated histone H4K16 (a, f) or double-stained with anti-acetylated histone H4K16 (c, h) and anti-PRM1 (d, i). (b, g, e, j) represent DAPI staining of the corresponding fields. Scale bars in (a) for (a, b and f, g) and in (c) for (c-e and h-j) are 100 μm and 50 μm, respectively.</p

QRT-PCR analyses of SSC related genes between the +/+, +/gt, and gt/gt testes.

<p>Single-stranded cDNA was prepared from the +/+, +/gt and gt/gt testes of adult (2-month-old) littermate mice. Expression levels of selected genes related to spermatogonial stem cell (SSC) characteristics were compared. The values with SEM (n = 4) were the relative gene expression values in the +/+ testis (set as 1.0) after standardization with the β-actin expression level in each testis.</p

Expression kinetics of <i>Jmjd1C</i> variants and selected spermatogenic marker genes in testicular development.

<p>Single-stranded cDNA was prepared from the testes at the indicated days after birth. The expression levels of the <i>Jmjd1C</i> variants and selected genes were quantitatively analyzed with <i>Jmjd1C</i> variant- and gene-specific primer pairs. The values indicated were the relative values of each gene expression level in the adult (4-month-old) testis (set as 1.0) after standardization with the β-actin expression level in each testis. Error bars indicate the SEM (n = 4).</p

A noncoding RNA containing a SINE-B1 motif associates with meiotic metaphase chromatin and has an indispensable function during spermatogenesis.

A search for early response genes that are activated following germ cell induction from mouse embryonic stem cells in vitro led us to the isolation of a long noncoding RNA that contains a SINE (short interspersed element)-B1F motif that was named R53. In situ hybridization and northern blot analyses revealed that the R53 subfragment RNA bears a B1F motif, is processed from the primary transcript, is expressed in adult testis and is predominantly localized in meiotic metaphase chromatin during spermatogenesis. Recent studies of chromosome-associated RNAs have explored novel functions of noncoding RNAs. Specifically, chromosome-bound noncoding RNAs function not only as structural components of chromosome but also as scaffolds that recruit epigenetic modulators for transcriptional regulation, and they are dynamically rearranged during the cell cycle. However, few studies have explored meiotic chromatin; thus, R53 RNA appears to be the first long noncoding RNA to be tightly associated with the metaphase chromatin during spermatogenesis. Furthermore, R53 knockdown using a lentivirus-mediated RNAi injected into mouse testis and organ culture of the fragments revealed a remarkable reduction in postmeiotic cells and irregular up-regulation of several postmeiotic genes, which suggests the possibility that the SINE-B1-derived noncoding RNA R53 plays an indispensable role in the transcriptional regulation of key spermatogenesis genes

Apparent loss of NANOG/OCT4-expressing cells (putative SSCs) in the <i>Jmjd1C</i> ^<i>gt/gt</i> adult testis.

<p>A) Sections of littermate +/gt (a-d) and gt/gt (e-f) adult testes (4-month-old, stage XII) were stained with anti-NANOG. DAPI staining images corresponding to each immunostaining are shown in (b, d, f). Magnification of the broken line-edged frame indicated in (a) was shown in (c, d). B) Sections of littermate +/gt (a, b) and gt/gt (c, d) adult testes were stained with anti-OCT4. C) X-gal staining of the +/gt seminiferous tubules (a). Magnification of the broken line-edged frame indicated in (a) was shown in (b). Arrows indicate positive-stained cells attached to the basement membrane. Scale bars, 50 μm. D) Immunoblots of the +/+ and gt/gt testis extracts were double-stained with anti-VASA and anti-OCT4, and the resulting VASA (83 kDa) and OCT4 (52 kDa) bands were shown.</p