STAGETOOL, a Novel Automated Approach for Mouse Testis Histological Analysis.

Spermatogenesis is a complex differentiation process that takes place in the seminiferous tubules. A specific organization of spermatogenic cells within the seminiferous epithelium enables a synchronous progress of germ cells at certain steps of differentiation on the spermatogenic pathway. This can be observed in testis cross-sections where seminiferous tubules can be classified into distinct stages of constant cellular composition (12 stages in the mouse).

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells.

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development.

Isolation of chromatoid bodies from mouse testis as a rich source of short RNAs.

RNA-protein (RNP) complexes and granules are powerful composites of merged functions and unique properties. The importance of RNPs in carrying out complex tasks in RNA processing and regulation is being increasingly revealed. One of the biggest RNP granules is the chromatoid body (CB) that is believed to orchestrate the RNA posttranscriptional regulation in haploid male germ cells.

An atlas of chromatoid body components.

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis.

Epigenetic regulation of male germ cell differentiation.

Male germ cell differentiation is a complex developmental program that produces highly specialized mature spermatozoa capable of independent movement and fertilization of an egg. Germ cells are unique in their capability to generate new organisms, and extra caution has to be taken to secure the correct inheritance of genetic and epigenetic information. Male germ cells are epigenetically distinct from somatic cells and they undergo several important epigenetic transitions.

The RNA binding protein SAM68 transiently localizes in the chromatoid body of male germ cells and influences expression of select microRNAs.

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes.

Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects.

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements.

Dicer is required for haploid male germ cell differentiation in mice.

Background: The RNase III endonuclease Dicer is an important regulator of gene expression that processes microRNAs (miRNAs) and small interfering RNAs (siRNAs). The best-characterized function of miRNAs is gene repression at the post-transcriptional level through the pairing with mRNAs of protein-encoding genes. Small RNAs can also act at the transcriptional level by controlling the epigenetic status of chromatin.

Chromatoid body and small RNAs in male germ cells.

The chromatoid body (CB) is a germ granule in the cytoplasm of postmeiotic haploid round spermatids that is loaded with RNA and RNA-binding proteins. Following the discovery of small non-coding RNA-mediated gene regulation and the identification of PIWI-interacting RNAs (piRNAs) that have crucial roles in germ line development, the function of the CB has slowly begun to be revealed. Male germ cells utilise small RNAs to control the complex and specialised process of sperm production.

Accumulation of piRNAs in the chromatoid bodies purified by a novel isolation protocol.

Haploid male germ cells are featured by an intriguing cytoplasmic cloud-like structure that has been named as chromatoid body (CB) on the basis of its staining properties and appearance under a microscope. Notwithstanding its early discovery in the late 19th century, the function of the CB is still largely obscure. Emerging evidence suggests a role for the CB and other similar RNA-containing granules, such as germ plasm in lower organism and processing bodies in somatic cells, in the control and organization of RNA processing and/or storage.