Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility

Stallmeyer B, Bühlmann C, Stakaitis R, Dicke A-K, Ghieh F, Meier L, Zoch A, MacLeod D M, Steingröver J, Okutman Ö, Fietz D, Pilatz A, Riera-Escamilla A, Xavier M J, Ruckert C, Di Persio S, Neuhaus N, Gurbuz A S, Şalvarci A, Le May N, McEleny K, Friedrich C, van der Heijden G, Wyrwoll M J, Kliesch S, Veltman J A, Krausz C, Viville S, Conrad D F, O’Carroll D, Tüttelmann F, 09.08.2024

Abstract

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.

Stallmeyer, B., Bühlmann, C., Stakaitis, R. et al. Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility. Nat Commun 15, 6637 (2024). https://doi.org/10.1038/s41467-024-50930-9

Publication: https://doi.org/10.1038/s41467-024-50930-9

Disclaimer

The publication Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility by Stallmeyer B, Bühlmann C, Stakaitis R, Dicke A-K, Ghieh F, Meier L, Zoch A, MacLeod D M, Steingröver J, Okutman Ö, Fietz D, Pilatz A, Riera-Escamilla A, Xavier M J, Ruckert C, Di Persio S, Neuhaus N, Gurbuz A S, Şalvarci A, Le May N, McEleny K, Friedrich C, van der Heijden G, Wyrwoll M J, Kliesch S, Veltman J A, Krausz C, Viville S, Conrad D F, O’Carroll D, Tüttelmann F is published under an open access license: https://creativecommons.org/licenses/by/4.0/. Permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

Curation by the MFGA team
Relevant data sets presented in the publication have been identified. If possible, annotations (title, general information, conditions, processed tissue types and processed cell types) have been added based on information from the publication. Data tables and images that provide a good overview on the publication's findings on the data set have been extracted from the publication and/or supplement. If not stated otherwise, images are depicted with title and description exactly as in the publication. Tables have been adjusted to the MFGA table format. Conducted adjustments are explained in the detailed view of the tables. However, titles and descriptions have been adopted from the publication.

Data set 1: Genetic variants in the piRNA pathway in the MERGE cohort

Exome: Exome Sequencing

Species

Species
Human

Conditions

Human phenotype ontology	Participants	Comment
HP:0000027: Azoospermia	1448	Absence of any measurable level of sperm,whereby spermatozoa cannot be observed even after centrifugation of the semen pellet.
HP:0030974: Cryptozoospermia	454	Sperm only identified after centrifugation of the ejaculate
HP:0034815: Extreme oligozoospermia	158	Sperm count < 2 million
HP:0034818: Severe oligozoospermia	67	Sperm count <10 million

Tissue Types

BRENDA tissue ontology	Maturity	Description	Species	Replicates
BTO_0000553: peripheral blood	adult	Blood circulating throughout the body.	Human
BTO_0001363: testis	average age: 34	A typically paired male reproductive gland that produces sperm and that in most mammals is contained within the scrotum at sexual maturity.	Human	2352.0

Cell Types

Cell ontology	Maturity	Description	Species	Replicates	Cells per replicate
CL_0000020: spermatogonium		An euploid male germ cell of an early stage of spermatogenesis.	Human
CL_0000017: spermatocyte		A male germ cell that develops from spermatogonia. The euploid primary spermatocytes undergo meiosis and give rise to the haploid secondary spermatocytes which in turn give rise to spermatids.	Human
CL_0000018: spermatid		A male germ cell that develops from the haploid secondary spermatocytes. Without further division, spermatids undergo structural changes and give rise to spermatozoa.	Human

Images

Figure 1: Biallelic variants identified in human piRNA biogenesis-associated genes

a Localization of variants in schematic of MOV10L1, PLD6, GPAT2, PNLDC1, MAEL, DDX4, and HENMT1 structure with protein domains colored and newly identified biallelic variants (red, bold for homozygous) as well as previously described homozygous variants (black) indicated. Pairs of compound heterozygous variants are indicated by identical symbols (*,#) in superscript. Helicase domains (green): DEAD/DEAH, Helicase_C, DNA2/NAM7; CAF1 chromatin assembly factor 1 domain (yellow); GPAT/DHAPAT acetyltransferase and methyltransferase domains (blue). b Periodic acid-Schiff (PAS) staining of representative testicular tissue of variant carriers demonstrating SCO in M2803 [PLD6, p.(His157Thrfs*102)], M2173 [PLD6, p.(Met1?)] and M454 [GPAT2, p.(His377Arg)/(Arg49His)] and presence of haploid germ cells (round/elongated spermatids) in M1125 [PNLDC1, p.(Tyr353Cys)], M3079 [HENMT1, p.(Ile134Leu)], M928 [DDX4, p.(Ala511Val)], and M2435 [MAEL, p.(Arg267*)/ p.(Cys283_Ala303del)]. Representative tubules showing the most advanced stage of spermatogenesis observed in three independent sections are shown. c Immunohistochemical (IHC) staining for GPAT2 in testicular tissue with full spermatogenesis (control) and GPAT2 variant carriers with meiotic arrest, [M690, p.(Arg627Trp), [M2556, p.(Glu386Valfs*16)]. In control tissue, GPAT2 is expressed in perinuclear structures in spermatocytes and this staining pattern is absent in M690 and M2556. Representative tubules showing the staining pattern observed in independent sections (control: N = 3, proband: N = 2) are shown. d IHC for MAEL in testicular tissue with full spermatogenesis (control) and M2435 with compound heterozygous presence of two MAEL LoF variants p.(Arg267*)/p.(Cys283_Ala303del). In control tissue, MAEL is expressed in perinuclear structures in spermatocytes and distinct condensed structures in round spermatids and this staining pattern is absent in the variant carrier. Representative tubules showing the staining pattern observed in independent sections (control: N = 3, proband: N = 2) are shown. Scale bar = 50 µm. SC Sertoli cell, SPC spermatocyte, RS round spermatid, ES elongated spermatid.

Source: Stallmeyer, B., Bühlmann, C., Stakaitis, R. et al. Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility. Nat Commun 15, 6637 (2024). https://doi.org/10.1038/s41467-024-50930-9

Licensed under: https://creativecommons.org/licenses/by/4.0/

Figure 2: Functional impact of disturbed piRNA biogenesis

a Immunohistochemical staining demonstrating diminished expression of PIWIL1 in variant carriers M2243 [GTSF1, p.(Arg74Lysfs*4)], M1648 [TDRD1, p.(Ser296Tyr)], M690 [GPAT2, p.(Arg627Trp)], M2556, [GPAT2, p.(Glu386Valfs*16)] and M3079 [HENMT1, p.(Ile134Leu)]. Representative tubules showing the staining pattern observed in independent sections (control: N = 3, proband: N = 2) are shown. b Schematic depicting impact of loss of piRNA biogenesis protein function on expression of additional piRNA factors. Circles represent piRNA protein and inhibiting effects of loss-of-protein functions on the expression of further piRNA proteins are indicated. c Effect of biallelic variants in genes of the piRNA pathway on biogenesis of pachytene piRNAs. RNA isolated from snap frozen testicular tissue of M2006 [PIWIL1 p.(Arg230*)], M1648 [TDRD1 p.(Ser296Tyr)], M2317 [TDRD12 p.(Arg811Gln)] and M2595 [TDRD12 p.(Leu1053Phefs*4)] revealed a major loss of pachytene piRNAs with expected lengths of 26–31 bases when compared with controls with complete spermatogenesis (ctrl1-3; P = 0.000007). Shapiro-Wilk test was used to estimate the normality of the data. Since Shapiro-Wilk test indicated abnormal data distribution in both control and case groups, two-sided Mann-Whitney U test was used for comparing the expression changes of piRNAs with different length (26–31 nt) between both groups. Source data are provided as a Source Data file. d Immunohistochemical staining for LINE1 transposon specific protein LINE1 ORF1p in testicular tissue. LINE1 ORF1p was not detected in testicular tissue of controls with full spermatogenesis and PIWIL1, GTSF1, and TDRD1 variant carriers. In contrast, all three TDRD12 variant carriers, both GPAT2 variant carriers, and all three FKBP6 variant carriers revealed a concordant distinct and specific LINE1 ORF1p staining in spermatogonia. A similar effect was also seen in testicular tissue of MAEL, HENMT1, and TDRD9 variant carriers. Representative tubules showing the staining pattern observed in independent sections (control: N = 3, proband: N = 2) are shown. Scale bar = 50 µm. SG spermatogonia. b Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0

Source: Stallmeyer, B., Bühlmann, C., Stakaitis, R. et al. Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility. Nat Commun 15, 6637 (2024). https://doi.org/10.1038/s41467-024-50930-9

Licensed under: https://creativecommons.org/licenses/by/4.0/