Dynamic transcriptional atlas of male germ cells during porcine puberty.

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.

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.

In both male and female germ cells of mice, retinoic acid (RA) is a meiosis-inducing factor. In the present study, we used a germ cell culture system to examine the direct effects of RA on meiotic initiation in male germ cells at the stage when they normally enter mitotic arrest to determine the extent to which fetal male germ cells can respond to exogenous RA to alter their sex-specific pathway. Male germ cells between 13.5 and 15.5 days postcoitum (dpc) were isolated from Pou5fl-green fluorescent protein transgenic fetuses and cultured with or without RA for up to 6 days. In the absence of RA, male germ cells did not undergo DNA replication and did not enter meiosis in culture. However, in the presence of RA, male germ cells isolated at 13.5 dpc expressed Stra8 and initiated the meiotic process. The ratio of cells entering meiosis gradually decreased as cells were isolated progressively at later stages. By 15.5 dpc, isolated male germ cells lost their ability to respond to RA signaling. These cells remained dispersed as single cells and progressed along the male differentiation pathway, as evidenced by the establishment of male-specific methylation imprints regardless of the presence or absence of RA. We conclude that male germ cells maintain sexual bipotency until 14.5 dpc that can be reversed by the addition of RA. Once male germ cells enter mitotic arrest, however, they appear to be committed irreversibly to the male-specific differentiation pathway even in the presence of exogenously added RA.

Changes in the expression pattern and intracellular localization of Forssman glycolipid (FA) and GM1 ganglioside (GM1) in fetal mouse gonads were examined during germ cell differentiation by immunofluorescence microscopy and immunoelectron microscopy. In male germ cells from the 12th to 14th day p.c., anti-FA binding was localized in granular structures aggregated on one side of the cytoplasm and/or in the plasma membrane. On day 16 p.c., some germ cells still showed patch-like positive reactions in the plasma membrane, but by day 18 p.c., positive reactions for FA had completely disappeared. The female germ cells showed granular bindings of anti-FA scattered throughout their cytoplasm during the 13th to 16th day p.c., although the positive reactions in female germ cells on day 12 p.c. tended to be found in one side of cytoplasm and/or plasma membrane similar to those in male germ cells from 12th to 14th day p.c. On day 18 p.c., positive reactions remained in the plasma membrane of some germ cells, but these positive reactions disappeared before birth. Immunoelectron microscopic observation showed that the sites of anti-FA bindings were equivalent to the "small dense bodies" (SDB) and the Golgi lamellae both in male and female germ cells. On the other hand, GM1 was not detected in male germ cells at any time during fetal testicular development, whereas an anti-GM1 reaction was detected in the plasma membrane of female germ cells from the 16th to 18th day p.c. (oocytes in the first meiotic prophase).(ABSTRACT TRUNCATED AT 250 WORDS)

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.

Oog1 is an oocyte-specific gene whose expression is turned on in mouse oocytes at embryonic day (E) 15.5, concomitant with the time when most of the female germ cells stop proliferating and enter meiotic prophase. Here, we characterize the Oog1 promoter, and show that transgenic GFP reporter expression driven by the 2.7 kb and 3.9 kb regions upstream of the Oog1 transcription start site recapitulates the intrinsic Oog1 expression pattern. In addition, the 3.9 kb upstream region exhibits stronger transcriptional activity than does the 2.7 kb region, suggesting that regulatory functions might be conserved in the additional 1.2 kb region found within the 3.9 kb promoter. Interestingly, the longer promoter (3.9 kb) also showed strong activity in male germ cells, from late pachytene spermatocytes to elongated spermatids. This is likely due to the aberrant demethylation of two CpG sites in the proximal promoter region. One was highly methylated in the tissues in which GFP expression was suppressed, and another was completely demethylated only in Oog1pro3.9 male and female germ cells. These results suggest that aberrant demethylation of the proximal promoter region induced ectopic expression in male germ cells under the control of 3.9 kb Oog1 promoter. This is the first report indicating that sex-dependent gene expression is altered according to the length and the methylation status of the promoter region. Additionally, our results show that individual CpG sites are differentially methylated and play different roles in regulating promoter activity and gene transcription.

Gender differences in the induction of chromosomal aberrations and gene mutations in rodent germ cells

Micromanipulation techniques were used to produce reconstituted one-cell mouse embryos after the fusion of fetal male germ cells 15.5 day post coitum with enucleated secondary oocytes. At this stage of development, male fetal germ cells are arrested at G1 of mitotic interphase. Two distinct populations of germ cells, differing in size and ploidy, were isolated from the genital ridge of a mid-term fetus. Oocytes that had received male germ cells from the population of smaller (mononuclear) germ cells developed as diploid one-cell reconstituted embryos. When the same procedures were used to produce reconstituted one-cell embryos using male fetal germ cells from a population of larger (multinucleate) cells, they exhibited ploidy of either 4x, 6x or 8x at metaphase of the first cell division. Although most reconstituted embryos (90 and 96%) developed to the two-cell stage, the proportion of embryos receiving small germ cells developed to blastocysts was much higher (62%) than that receiving large germ cells (4%). These studies indicate that not all fetal germ cells are diploid before the onset of meiosis and have identified procedures to produce reconstituted embryos from fetal germ cells that do not carry genome or chromosome anomalies.

MP70-03 RHOXF2 AS A GUARDIAN OF THE HUMAN MALE GERMLINE STEM CELLS

Kinesins are motor proteins that transport their cargos along microtubules in an ATP-dependent manner. The testis-specific kinesin KIF17b was shown to directly regulate cAMP-response element modulator (CREM)-dependent transcription by determining the subcellular localization of the activator of CREM in testis (ACT), the testis-specific coactivator of CREM in postmeiotic male germ cells. CREM is a crucial transcriptional regulator of many important genes required for spermatid maturation, as demonstrated by the complete block of sperm development at the first steps of spermiogenesis in crem-null mice. To better understand the complex regulation of postmeiotic germ cell differentiation, we further characterized the ACT-KIF17b interaction, the function of KIF17b, and the signaling pathways governing its action. In this study, we demonstrated that the abilities of KIF17b to shuttle between the nuclear and the cytoplasmic compartments and to transport ACT are neither dependent on its motor domain nor on microtubules, thus revealing a novel microtubule-independent function for kinesins. We also showed that the cyclic AMP-dependent protein kinase A mediates the phosphorylation of KIF17b, and this modification is important for its subcellular localization. These results indicate that cyclic AMP signaling controls CREM-mediated transcription in male germ cells through modification of KIF17b function.

The Gene Encoding Murine α1,3-Galactosyltransferase Is Expressed in Female Germ Cells but Not in Male Germ Cells

The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

Male germ cell transplantation is a novel technique in which donor male stem germ cells are surgically transferred to the seminiferous tubules of a recipient testis by direct injection or via the rete testis or efferent duct. All germ cells that are destined to become stem spermatogonia are defined as male stem germ cells, including primordial germ cells from the gonadal ridges, and gonocytes and stem spermatogonia from the testis, all of which are transplantable and capable of undergoing normal spermatogenesis. Xenotransplantation of male germ cells from one species into the testis of another species, including human testicular cells in the mouse, has so far proved to be unsuccessful. However, the immunodeficient mouse testis can support rat spermatogenesis and produce apparently normal rat spermatozoa. The underlying mechanisms remain elusive. The present mini-review will focus on the importance of stem spermatogonial transplantation for testicular stem cell biology and discuss the likelihood of immune rejection after transplantation, which may limit the success of all male germ cell transplantation.

P-element-induced wimpy testis (Piwi) is a key gene involved in germ cell development in a diverse range of organisms. However, in giant clams, the function of Piwi remains unclear. In the present study, we isolated the full-length cDNA of Piwi ortholog (Tc-Piwi1) and analyzed its expression patterns in the gonads of adult and juvenile Tridacna crocea. The results of qPCR showed that the transcript of Tc-Piwi1 was mainly expressed in gonad tissue. In addition, the relative expression level of Tc-Piwi1 increased with the proliferation of male and female germ cells during the adult gonad development stage, suggesting that Tc-Piwi1 might be involved in gametogenesis. In situ hybridization revealed that Tc-Piwi1 RNA was located in female and male germ cells and strongly expressed in male germ cells in the early stage. Furthermore, immunohistochemical experiments further confirmed that Tc-Piwi1 was mainly located in primordial germ cells (PGCs), germ stem cells (GSCs), and female and male germ cells of early development, so it could be used as a marker gene of T. crocea germ cells. Whole-mount in situ hybridization suggested that Tc-Piwi1 was of maternal origin and located in two clusters of cells in the trochophore-larvae stage, implying that these cells might be putative PGCs during the embryo development. Finally, Tc-Piwi1 was used as a molecular marker to elucidate the gonadal formation, sex differentiation, and gonadal maturation process of juvenile T. crocea for the first time in the Tridacna family. Collectively, all these results revealed that Tc-Piwi1 was involved in germline formation and sex differentiation in T. crocea.

For comparative analysis of the chronology and dynamics of development and differentiation of germ cells in human gonads in antenatal and postnatal periods, we analyzed the composition of somatic cells as well as the amount, morphology, and characteristics of the state of nuclear chromatin on the basis of the ratio of mitotic stages and prophase I of meiosis in oocytes and spermatocytes. Until week 6 of antenatal development, human gonads are classified into the indifferent type; the proportion of germ cells (GCs) in the sum of somatic and germ cells in them ranged from 9.21 to 33.29%. In the analysis of histological preparations, gonads of female fetuses on week 8–9 of antenatal development were significantly differentiated as ovaries. The formation of the pool of female germ cells is ensured by the proliferation of oogonia (1.96–10.34% of mitoses) in embryogenesis and by degeneration of their nuclei. In developing testes, among the nuclei of male germ cells of antenatal and neonatal gonads, nuclei at the leptotene stage of prophase I of meiosis and at subsequent stages were not detected. In the gonads of male fetuses on weeks 6–11 of antenatal development, mitotic and degenerating germ cells and germ cells at the preleptotene stage of chromosome condensation were detected, whereas no mitotic germ cells were identified in the histological preparations of testes of male newborns and infants. An important distinction between the chronology of differentiation of gametes and chromatin transformation dynamics in the nuclei of male and female germ cells is the lack of the chromatin decondensation phase (prochromosomes) at the preleptotene stage in prophase I of meiosis in the male germ cells.