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The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor-RHOX10-critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.

RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA-Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.

Histone H1 is an abundant and essential component of chromatin whose precise role in regulating gene expression is poorly understood. Here, we report that a major target of H1-mediated regulation in embryonic stem (ES) cells is the X-linked Rhox homeobox gene cluster. To address the underlying mechanism, we examined the founding member of the Rhox gene cluster-Rhox5-and found that its distal promoter (Pd) loses H1, undergoes demethylation, and is transcriptionally activated in response to loss of H1 genes in ES cells. Demethylation of the Pd is required for its transcriptional induction and we identified a single cytosine in the Pd that, when methylated, is sufficient to inhibit Pd transcription. Methylation of this single cytosine prevents the Pd from binding GA-binding protein (GABP), a transcription factor essential for Pd transcription. Thus, H1 silences Rhox5 transcription by promoting methylation of one of its promoters, a mechanism likely to extend to other H1-regulated Rhox genes, based on analysis of ES cells lacking DNA methyltransferases. The Rhox cluster genes targeted for H1-mediated transcriptional repression are also subject to another DNA methylation-regulated process: Xp imprinting. Remarkably, we found that only H1-regulated Rhox genes are imprinted, not those immune to H1-mediated repression. Together, our results indicate that the Rhox gene cluster is a major target of H1-mediated transcriptional repression in ES cells and that H1 is a candidate to have a role in Xp imprinting. [ABSTRACT FROM AUTHOR]

Poor oocyte competence contributes to infertility in humans and livestock species. The molecular characteristics of such oocytes are generally unknown. Objectives of the present studies were to identify differences in RNA transcript abundance in oocytes and early embryos associated with reduced oocyte competence and development to the blastocyst stage. Microarray experiments were conducted using RNA isolated from germinal vesicle stage oocytes collected from adult versus prepubertal animals (model of poor oocyte competence). A total of 193 genes displaying greater mRNA abundance in adult oocytes and 223 genes displaying greater mRNA abundance in prepubertal oocytes were detected. Subsequent gene ontology analysis of microarray data revealed significant overrepresentation of transcripts encoding for genes in hormone secretion classification within adult oocytes and such genes were selected for further analysis. Real-time PCR experiments revealed greater abundance of mRNA for ßA and ßB subunits of inhibin/activin and follistatin, but not the αsubunit in germinal vesicle stage oocytes collected from adult versus prepubertal animals. Cumulus cell follistatin and ßB subunit mRNA abundance were similar in samples collected from prepubertal versus adult animals. A positive association between time of first cleavage (oocyte competence) and follistatin mRNA abundance was noted. Follistatin, ßB, and αsubunit mRNAs were temporally regulated during early bovine embryogenesis and peaked at the 16-cell stage. Collectively, results demonstrate a positive association of follistatin mRNA abundance with oocyte competence in two distinct models and dynamic regulation of follistatin, ßB, and αsubunit mRNAs in early embryos after initiation of transcription from the embryonic genome. [ABSTRACT FROM AUTHOR]

Spermatogenesis in mammals is achieved by multiple players that pursue a common goal of generating mature spermatozoa. The developmental processes acting on male germ cells that culminate in the production of the functional spermatozoa are regulated at both the transcription and post-transcriptional levels. This review addresses recent progress towards understanding such regulatory mechanisms and identifies future challenges to be addressed in this field. We focus on transcription factors, chromatin-associated factors and RNA-binding proteins necessary for spermatogenesis and/or sperm maturation. Understanding the molecular mechanisms that govern spermatogenesis has enormous implications for new contraceptive approaches and treatments for infertility.

Although improvements in culture systems have greatly enhanced in vitro embryo production, success rates under the best conditions are still far from ideal. The reasons for developmental arrest of the majority of in vitro produced embryos are unclear, but likely attributable, in part, to intrinsic and extrinsic influences on the cytoplasmic and/or nuclear environment of an oocyte and/or early embryo that impede normal progression through the maternal-to-embryonic transition. The maternal-to-embryonic transition is the time period during embryonic development spanning from fertilisation until when control of early embryogenesis changes from regulation by oocyte-derived factors to regulation by products of the embryonic genome. The products of numerous maternal effect genes transcribed and stored during oogenesis mediate this transition. Marked epigenetic changes to chromatin during this window of development significantly modulate embryonic gene expression. Depletion of maternal mRNA pools is also an obligatory event during the maternal-to-embryonic transition critical to subsequent development. An increased knowledge of the fundamental mechanisms and mediators of the maternal-to-embryonic transition is foundational to understanding the regulation of oocyte quality and future breakthroughs relevant to embryo production.

Mammalian oocytes accumulate a large pool of mRNA molecules that orchestrate subsequent embryonic development. The transcriptional machinery is silent during oocyte meiotic maturation and early embryogenesis, and thereby the early decisive events in embryo development prior to initiation of transcription from the embryonic genome are directed by the translation of pre-existing maternal mRNAs. Oocytes display remarkable post-transcriptional regulatory mechanisms that control mRNA stability and translation. The regulatory mechanisms are generally negative, and target mRNAs are either subjected to degradation or repressed from undergoing translation until specifically activated. Such negative regulatory mechanisms generally are mediated by transcript deadenylation, interaction of transcripts with RNA-binding proteins in a nonspecific or sequence-specific fashion, and/or potentially via actions of microRNA and repeat- associated small interfering RNA, which degrade maternal RNA transcripts. In contrast, translational activation is initiated via cytoplasmic polyadenylation of maternal transcripts facilitated via the binding of embryo-specific poly(A)-binding proteins (ePABs). In certain instances, translational regulation (positive or negative) is dictated by the balance of positive and negative trans-acting factors that compete for specific sequence motifs present in maternal transcripts. Coordinate post-transcriptional regulation of the oocyte mRNA pool is critical for normal progression of early embryonic development.

Despite ample evidence pointing to an obligatory involvement of progesterone in ovulation, the mechanisms responsible for the ovulation promoting effects of intrafollicular progesterone are unclear. The objectives of this study were to determine if ovulation, luteinization and the gonadotropin surge-induced regulation of select extracellular matrix-degrading enzymes and their inhibitors, and mRNAs for prostaglandin (PG) biosynthesis and metabolizing enzymes are blocked following suppression of the intrafollicular increase in progesterone. Bovine preovulatory follicles were injected with the 3 beta-hydroxysteroid dehydrogenase inhibitor trilostane or diluent and collected at 0, 12, and 24 h after GnRH induction of the preovulatory LH surge. Intrafollicular trilostane administration blocked the preovulatory increase in follicular fluid progesterone resulting in concentrations similar to those observed at time 0 post-GnRH injection. The preovulatory increase in follicular fluid PGE(2) and PGF(2alpha) was reduced in trilostane-treated follicles and accompanied by upregulation of prostaglandin dehydrogenase mRNA in the granulosal and thecal cells. However, follicle rupture was not blocked by inhibition of the preovulatory rise in intrafollicular progesterone, and normal serum progesterone concentrations were observed during subsequent luteal development. Effects of trilostane administration on preovulatory changes in mRNA abundance and protein/activity in preovulatory follicles for most regulators of extracellular matrix remodeling examined were distinct from changes previously observed following the inhibition of intrafollicular prostaglandin synthesis. Results suggest that the preovulatory increase in intrafollicular progesterone may not be obligatory for bovine follicle rupture, luteinization, or regulation of prominent matrix-degrading proteinases and their inhibitors associated with ovulation.

Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development., ((c) 2005 Wiley-Liss, Inc.)