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). For a detailed analysis of spermatogenesis, these stages have to be individually observed from testis cross-sections. However, the recognition of stages requires special training and expertise. Furthermore, the manual scoring is laborious considering the high number of tubule cross-sections that have to be analyzed. To facilitate the analysis of spermatogenesis, we have developed a convolutional deep neural network-based approach named "STAGETOOL." STAGETOOL analyses histological images of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI)-stained mouse testis cross-sections at ×400 magnification, and very accurately classifies tubule cross-sections into 5 stage classes and cells into 9 categories. STAGETOOL classification accuracy for stage classes of seminiferous tubules of a whole-testis cross-section is 99.1%. For cellular level analysis the F1 score for 9 seminiferous epithelial cell types ranges from 0.80 to 0.98. Furthermore, we show that STAGETOOL can be applied for the analysis of knockout mouse models with spermatogenic defects, as well as for automated profiling of protein expression patterns. STAGETOOL is the first fluorescent labeling-based automated method for mouse testis histological analysis that enables both stage and cell-type recognition. While STAGETOOL qualitatively parallels an experienced human histologist, it outperforms humans time-wise, therefore representing a major advancement in male reproductive biology research.

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. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

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. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

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. Here, we describe the CB isolation procedure, from mouse testis. After cross-linking and lysing the cells, the CBs are enriched by slow-speed centrifugation and immunoprecipitated using anti-MVH/DDX4 antibody. The method yields pure fractions of CBs, and it is robust, reproducible and does not require special equipment or abundant starting material. The CB is packed with large amounts of RNA, especially small RNAs. Isolation of the CBs provides a tool to enrich these RNA species.

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. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.

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. In primordial germ cells (PGCs), epigenome is reprogrammed by genome-wide resetting of epigenetic marks, including the sex-specific imprinting of certain genes. Postnatal spermatogenesis is characterized by drastic chromatin rearrangements during meiotic recombination, sex chromosome silencing, and compaction of sperm nuclei, which is accomplished by replacing near to all histones by sperm-specific protamines. Small RNAs, including microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and PIWI-interacting RNAs (piRNAs) are also involved in the control of male gamete production. The activities of small RNAs in male germ cells are diverse, and include miRNA- and endo-siRNA-mediated posttranscriptional mRNA regulation and piRNA-driven transposon silencing and the control of DNA methylation in PGCs. In this chapter, we give a brief review on the epigenetic processes that govern chromatin organization and germline-specific gene expression in differentiating male germ cells.

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. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68(-/-) germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis.

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. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice. PRINCIPAL FINDINGS: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

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. Dicer and other mediators of small RNA pathways are present in mouse male germ cells, and several miRNAs and endogenous siRNAs are expressed in the testis, suggesting that Dicer-dependent small RNAs are involved in the control of the precisely timed and highly organised process of spermatogenesis. PRINCIPAL FINDINGS: Being interested in the Dicer-mediated functions during spermatogenesis, we have analysed here a male germ cell-specific Dicer1 knockout mouse model, in which the deletion of Dicer1 takes place during early postnatal development in spermatogonia. We found that Dicer1 knockout testes were reduced in size and spermatogenesis within the seminiferous tubules was disrupted. Dicer1 knockout epididymides contained very low number of mature sperm with pronounced morphological abnormalities. Spermatogonial differentiation appeared unaffected. However, the number of haploid cells was decreased in knockout testes, and an increased number of apoptotic spermatocytes was observed. The most prominent defects were found during late haploid differentiation, and Dicer was demonstrated to be critical for the normal organization of chromatin and nuclear shaping of elongating spermatids. CONCLUSIONS/SIGNIFICANCE: We demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility.

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. Several microRNAs have been identified during spermatogenesis. In addition, a high number of piRNAs are present both in embryonic and postnatal male germ cells, with their expression being impressively induced in late meiotic cells and haploid round spermatids. At postmeiotic stage of germ cell differentiation, the CB accumulates piRNAs and proteins of piRNA machinery, as well as several other proteins involved in distinct RNA regulation pathways. All existing evidence suggests a role for the CB in mRNA regulation and small RNA-mediated gene control, but the mechanisms remain uncharacterised. In this review, we summarise the current knowledge of the CB and its association with small RNA pathways.

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. Despite the increasing scientific demand, the lack of CB purification protocols has still been the main obstacle in the functional characterization of this structure. We have successfully isolated CBs from mouse testis by a novel immunoaffinity purification procedure and validated by several different methods that pure CB fractions are obtained. Analysis of the CB RNA content reveals enrichment of PIWI-interacting RNAs (piRNAs), further emphasizing the role of CB as the RNA processing body.