In the testis, interstitial macrophages are thought to be derived from the yolk sac during fetal development, and later replaced by bone marrow-derived macrophages. By contrast, the peritubular macrophages have been reported to emerge first in the postnatal testis and solely represent descendants of bone marrow-derived monocytes. Here, we define new monocyte and macrophage types in the fetal and postnatal testis using high-dimensional single-cell analyses. Our results show that interstitial macrophages have a dominant contribution from fetal liver-derived precursors, while peritubular macrophages are generated already at birth from embryonic precursors. We find that bone marrow-derived monocytes do not substantially contribute to the replenishment of the testicular macrophage pool even after systemic macrophage depletion. The presence of macrophages prenatally, but not postnatally, is necessary for normal spermatogenesis. Our multifaceted data thus challenge the current paradigms in testicular macrophage biology by delineating their differentiation, homeostasis and functions.

SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

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Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.

SummaryAccurate analysis and quantification of different testicular cell populations are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities of applying flow cytometry in studies of male reproduction. Here, we report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and γH2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat testis ontogenesis.

Maintenance of the differentiated state and cell cycle exit in adult Sertoli cells depends on tumor suppressor retinoblastoma protein (RB, also known as RB1). We have previously shown that RB interacts with transcription factor E2F3 in the mouse testis. Here, we investigated how E2f3 contributes to adult Sertoli cell proliferation in a mouse model of Sertoli cell-specific knockout of Rb by crossing these mice with an E2f3 knockout mouse line. In the presence of intact RB, E2f3 was redundant in Sertoli cells. However, in the absence of RB, E2f3 is a key driver for cell cycle re-entry and loss of function in adult Sertoli cells. Knockout of E2f3 in Sertoli cells rescued the breakdown of Sertoli cell function associated with Rb loss, prevented proliferation of adult Sertoli cells and restored fertility of the mice. In summary, our results show that RB-mediated repression of E2F3 is critical for the maintenance of cell cycle exit and terminal differentiation in adult mouse Sertoli cells.

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The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17β) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17β-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17β) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.