Human spermatogonial stem cells and their niche in male (in)fertility: novel concepts from single-cell RNA-sequencing

Abstract

The amount of single-cell RNA-sequencing (scRNA-seq) data produced in the field of human male reproduction has steadily increased. Transcriptional profiles of thousands of testicular cells have been generated covering the human neonatal, prepubertal, pubertal and adult period as well as different types of male infertility; the latter include non-obstructive azoospermia, cryptozoospermia, Klinefelter syndrome and azoospermia factor deletions. In this review, we provide an overview of transcriptional changes in different testicular subpopulations during postnatal development and in cases of male infertility. Moreover, we review novel concepts regarding the existence of spermatogonial and somatic cell subtypes as well as their crosstalk and provide corresponding marker genes to facilitate their identification. We discuss the potential clinical implications of scRNA-seq findings, the need for spatial information and the necessity to corroborate findings by exploring other levels of regulation, including at the epigenetic or protein level.

Keywords: AZF deletion; Klinefelter; Leydig cells; Sertoli cells; azoospermia; human testis development; male infertility; single-cell RNA-sequencing; spermatogenesis; spermatogonial stem cells.

Graphical Abstract

Figure 1.

Schematic representation of the single-cell RNA-sequencing studies to analyze human testicular tissues. (A) Experimental design, (B) samples used. Details are provided in Supplementary Table SI. Studies on each stage of development, and different types of infertility: 0–13 months (Guo et al., 2018, 2021; Sohni et al., 2019; Voigt et al., 2022); 2–14 years (Guo et al., 2020; Zhao et al., 2020; Voigt et al., 2022); 17–76 years (Guo et al., 2017, 2018; Neuhaus et al., 2017; Hermann et al., 2018; Wang et al., 2018; Sohni et al., 2019; Shami et al., 2020; Xia et al., 2020; Zhao et al., 2020; Alfano et al., 2021; Di Persio et al., 2021; Mahyari et al., 2021; Chen et al., 2022; Nie et al., 2022); Crypto (Di Persio et al., 2021); Idiopathic NOA (Wang et al., 2018; Zhao et al., 2020; Alfano et al., 2021; Mahyari et al., 2021; Chen et al., 2022); Yq AZFa deletions (Zhao et al., 2020); Klinefelter syndrome (Laurentino et al., 2019; Zhao et al., 2020; Mahyari et al., 2021). Crypto, cryptozoospermia; NOA, non-obstructive azoospermia; AZF, azoospermia factor.

Figure 2.

Model of the undifferentiated human spermatogonial pool dynamics in homeostasis and following an alteration of the microenvironment. (A) During tissue homeostasis, the adult pool of spermatogonia (SPG) is highly heterogeneous in terms of transcriptional and morphological profiles. The pool is highly dynamic and the cells may transfer reversibly from one transcriptional or morphological state to the other based on different signals associated with the microenvironment. By ‘microenvironment’ we refer here to all the testicular cells exchanging signals with the stem cell pool including all the testicular somatic as well as germ cells. This flexibility may allow the cells to respond to different stimuli while still maintaining their stem cell properties. (B) In case of reduced sperm production, we propose a model according to which the altered microenvironment results in an altered crosstalk with the SPG pool. Based on data obtained from men with cryptozoospermia, the SPG pool polarizes in this altered microenvironment by reducing its morphological (reduction of A_dark) and transcriptional heterogeneity and placing more spermatogonia in a ready-to react state, potentially in a futile attempt to counterbalance the limited number of sperm being produced. It remains to be elucidated whether: the observed alterations of the SPG pool are a cause or consequence of male infertility phenotypes; and SPG can re-establish the heterogeneous stem cell pool if placed back into an intact microenvironment.

Figure 3.

Schematic summary of the cellular and molecular alterations identified using single-cell RNA-sequencing of human testicular single-cell suspensions in three types of male infertility. Crypto, cryptozoospermia; NOA, non-obstructive azoospermia; AZF, azoospermia factor; SPG, spermatogonia; UTF1, undifferentiated embryonic cell transcription factor 1; PIWIL4, Piwi-like RNA-mediated gene silencing 4; CD3, cluster of differentiation 3; EGR3, early growth response 3; WNT, Wingless/Integrated; MIF, macrophage migration inhibitory factor; XIST, X-inactive specific transcript; RSPO3, R-spondin 3.