Pnldc1 is essential for spermiogenesis

To investigate the physiological function of mammalian PNLDC1 and its potential role in piRNA biogenesis, we generated Pnldc1-deficient mice using CRISPR-Cas9 genome editing technology (Supplementary Fig. 1 and Methods section). We designed two guide RNAs (gRNAs) to target exon1 and exon9 of Pnldc1, respectively (Supplementary Fig. 1a). We electroporated both gRNAs together with Cas9 protein into C57BL/6J zygotes and derived the first-generation (F0) Pnldc1 mutant (Pnldc1 Mut) mice with indels or deletions on both Pnldc1 alleles (Supplementary Fig. 1b–e). Genotyping PCR and reverse transcription PCR confirmed successful targeting of Pnldc1 (Supplementary Figs. 1 and 2).

We examined five male Pnldc1 Mut mice with distinct mutations on Pnldc1 alleles (we refer to as Mut-1, Mut-2, Mut-3, Mut-4, and Mut-5) and found all are viable and grow normally but exhibit smaller testes, ~70% of wild-type control by weight (Fig. 1a). Histological analysis of Pnldc1 Mut testes indicated no obvious changes in germ cell types and cellularity (Fig. 1b; Supplementary Fig. 3a). Pnldc1 Mut seminiferous tubules contained spermatogonia, spermatocytes, round spermatids, and elongating spermatids, but lacked normal spermatozoa (Fig. 1b). At stage VIII, Pnldc1 Mut elongated spermatids showed heavily condensed chromatin, but excess cytoplasm (Fig. 1b, right). Abnormally formed heads of elongated spermatids were frequently observed, sometimes surrounded by excess cytoplasm. This represents a late spermiogenic defect at the elongated spermatid stage in the testis (Fig. 1b). In Pnldc1 Mut epididymides, germ cell numbers were drastically reduced and no normal spermatozoa were present; instead, sloughed spermatids and a number of residual cytoplasm were observed (Fig. 1c). This spermiogenic defect is consistent among all Pnldc1 Mut animals examined (n = 4) (Supplementary Fig. 3). We also established a stable Pnldc1 mutant mouse line carrying an exon1–exon9 deletion (Supplementary Fig. 1f, g) from a female F0 founder. Male mice homozygous for this allele (we refer to as Pnldc1 KO) showed the same spermiogenic arrest seen in all F0 Pnldc1 Mut males (Supplementary Fig. 3b).

Fig. 1 Pnldc1 is essential for spermatogenesis. a Testicular atrophy in Pnldc1 Mut mice. Testis sizes and weights of adult wild-type (WT) and Pnldc1 Mut mice are shown. n = 5; significance determined by unpaired Student’s t-test; **p < 0.01. Error bars represent s.e.m. b Spermiogenic arrest in adult Pnldc1 Mut mice. Hematoxylin and eosin-stained testis sections from adult WT and Pnldc1 Mut (Mut-5) mice are shown. AES abnormal elongated spermatids, P pachytene spermatocytes, R round spermatids. Scale bars, 100 μm (left) and 20 μm (right). c Hematoxylin and eosin-stained epididymis sections from adult WT and Pnldc1 Mut (Mut-5) mice are shown. Scale bar, 20 μm. d Meiotic arrest in adult Tdrkh KO mice. Hematoxylin and eosin-stained testis sections from adult WT and Tdrkh KO mice are shown. Z zygotene spermatocytes. Scale bar, 20 μm. e Co-immunostaining of CRISP2 (red) and γH2AX (green) in adult WT and Pnldc1 Mut (Mut-1) testes. DNA (blue) is stained by DAPI. Spermatogenic stages are noted. Scale bar, 20 μm. f The timeline of mouse spermatogenesis with red crosses representing the arrested spermatogenic stages in Pnldc1 Mut and Tdrkh KO testes Full size image

As additional evidence of defective spermatogenesis in Pnldc1 Mut males, staining for CRISP2, a marker of cytoplasm of step 15 and 16 elongated spermatids17, showed abnormal residue body formation and delayed cytoplasm removal, confirming the lack of normally staged seminiferous tubules throughout spermatogenesis in Pnldc1 Mut testes (Fig. 1e). This spermiogenic defect contrasts with the testicular defect of Tdrkh knockout (Tdrkh KO) mice, which shows complete meiotic arrest at the zygotene spermatocyte stage15 (Fig. 1d, f). The spermatid morphological defect in Pnldc1 Mut testes also differs from reported knockouts of other piRNA biogenesis factors with spermiogenic defect, which mostly show germ cell arrest at the round spermatid stage18,19,20,21,22,23,24. Taken together, these findings demonstrate that Pnldc1 is indispensable for spermiogenesis in mice.

Pnldc1 is required for LINE1 retrotransposon silencing

The piRNA pathway plays pivotal roles in transposon silencing and germline genome protection1, 5, 9, 10, 25. LINE1, the most studied active retrotransposon in the mouse genome, is activated in knockout mice of almost all piRNA biogenesis factors26, 27. We examined the expression of LINE1 messenger RNA (mRNA) in adult Pnldc1 Mut testes. In situ hybridization with a probe against LINE1 open reading frame1 (ORF1) mRNA revealed a significantly elevated LINE1 mRNA expression in Pnldc1 Mut spermatocytes (Fig. 2a). To examine the LINE1 protein levels, we generated LINE1 ORF1 specific antisera (see Methods section). Western blot analysis confirmed the elevated LINE1 protein level in Pnldc1 Mut testes (Supplementary Fig. 4). Consistent with in situ hybridization results, LINE1 ORF1 protein expression dramatically increased in Pnldc1 Mut pachytene spermatocytes (Fig. 2b). LINE1 ORF1 protein could be first detected in Pnldc1 Mut zygotene spermatocytes and peaked at the mid-pachytene stage. LINE1 ORF1 was not detected in Pnldc1 Mut spermatogonia, preleptotene/leptotene spermatocytes or round spermatids (Fig. 2c). As a positive control, LINE1 ORF1 was upregulated in Tdrkh −/− testes15; the positive cells were leptotene or zygotene spermatocytes because of the early meiotic arrest (Supplementary Fig. 5). These data indicate that PNLDC1 is essential for transposon repression in mammalian male germ cells.

Fig. 2 Retrotransposon LINE1 derepression in Pnldc1 mutant spermatocytes. a In situ hybridization of LINE1 Orf1 mRNA in adult WT and Pnldc1 Mut (Mut-1) testes. LINE1 Orf1 mRNA was upregulated in spermatocytes in Pnldc1 Mut testes but was undetectable in WT testes. Scale bar, 50 μm. b Immunostaining was performed using LINE1 ORF1 antibody on adult testis sections from WT and Pnldc1 Mut (Mut-1) mice. LINE1 ORF1 was upregulated in spermatocytes in Pnldc1 Mut testes but was undetectable in WT testes. Scale bar, 20 μm. c Immunostaining was performed using LINE1 ORF1 antibody and γH2AX antibody on adult testis sections from WT and Pnldc1 Mut (Mut-1) mice. Different cell types were distinguished according to γH2AX staining and DAPI staining. LINE1 ORF1 was expressed from zygotene spermatocytes to mid-pachytene spermatocytes in Pnldc1 Mut testes. Scale bar, 5 μm Full size image

Mislocalization of piRNA pathway factors in Pnldc1 Mut testes

We next examined the expression levels and localization patterns of several piRNA pathway factors in Pnldc1 Mut testes. Western blotting showed similar expression levels for TDRKH, MILI, GASZ, and MVH between mutant and control testes. MIWI protein level was decreased in Pnldc1 Mut testes (Fig. 3a). Although the expression level of TDRKH was similar to that of wild type (Fig. 3a), its localization was polarized to large perinuclear granules in Pnldc1 Mut pachytene spermatocytes (Fig. 3b), which may correspond to mitochondrial clusters observed in other piRNA biogenesis factor knockouts22, 28, 29. MILI, MIWI, and GASZ staining also showed the same polarized pattern in Pnldc1 Mut testes (Fig. 3b). We confirmed that the large granules consisted of clustered mitochondria by staining with a mitochondrial marker AIF (Fig. 3c). MVH, which is also essential for piRNA biogenesis but has a wider distribution throughout the cytoplasm, showed a normal distribution in Pnldc1 Muttestes (Fig. 3b). Because piRNA biogenesis factors are normally localized to the intermitochondrial cement of pachytene spermatocytes where piRNA biogenesis is believed to occur, the polarization of TDRKH, MILI, GASZ, and MIWI in a mitochondria congregation zone in Pnldc1 Mut testes suggests PNLDC1 deficiency may disrupt piRNA biogenesis.

Fig. 3 Pnldc1 mutation causes mitochondria disorder. a Expression of TDRKH, MILI, MIWI, GASZ, and MVH in WT and Pnldc1 Mut (Mut-1) testes revealed by western blotting. β-actin is a loading control. b Aggregation of TDRKH, MILI, MIWI, and GASZ in Pnldc1 Mut spermatocytes. Immunostaining was performed using indicated antibodies on adult testis sections from WT and Pnldc1 Mut (Mut-1) mice. DNA (blue) is stained with DAPI. Protein aggregations are indicated by arrows. Scale bar, 10 μm. c Conglomeration of mitochondria in Pnldc1 Mut testes. Immunostaining of WT and Pnldc1 Mut (Mut-1) testis sections with an antibody against AIF, a mitochondrial marker. DNA (blue) is stained with DAPI. Conglomeration of mitochondria is indicated by arrows. Scale bar, 10 μm Full size image

Pachytene piRNA biogenesis is impaired in Pnldc1 Mut testes

To explore a role of PNLDC1 in piRNA biogenesis, we examined the size and abundance of small RNA populations from adult wild-type and Pnldc1 Mut testes. Radiolabeling of total RNA showed a normal wild-type piRNA population at around 30 nt in length (Fig. 4a). Strikingly, this population was absent in Pnldc1 Mut testes; instead, an abnormally longer small RNA population of 30–40 nt in length was observed (Fig. 4a). The absence of normal piRNA population and the presence of longer small RNA population are consistent among all Pnldc1 Mut animals examined (Supplementary Fig. 6). To test whether this population of longer small RNAs is piRNAs associated with PIWI proteins, we immunoprecipitated MILI and MIWI, and labeled the associated RNAs. Small RNAs with extended length were observed in Pnldc1 Mut MILI and MIWI immunoprecipitates, indicating that these small RNAs are most likely aberrantly processed piRNA intermediates (pre-piRNAs) (Fig. 4b, c). Next, we sequenced small RNA libraries constructed from total RNA, MILI-bound RNAs, or MIWI-bound RNAs from adult wild-type and Pnldc1 Mut testes. Sequencing results revealed that the amount of total piRNAs was dramatically decreased in Pnldc1 Mut testes as normalized by the microRNA reads (Fig. 4d). In addition, the small RNA population in Pnldc1 Mut testes showed extended lengths of 24–44 nt, as compared to 24–32 nt in the wild type (Fig. 4d). The peak of MILI-bound piRNAs (MILI-piRNAs) likewise shifted from 27 nt in the wild type to 32 nt in Pnldc1 Mut samples (Fig. 4e), and the peak of MIWI-bound piRNAs (MIWI-piRNAs) shifted from 30 to 34 nt (Fig. 4f). These data indicate that PNLDC1 is required for production of normal mature piRNAs and may facilitate the proper processing of pre-piRNAs.

Fig. 4 Increased piRNA sizes and reduced normal piRNAs in adult Pnldc1 Mut testes. a piRNA extension in Pnldc1 Mut mice. Total RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testes were end-labeled with [32P]-ATP, and detected by 15% TBE urea gel and autoradiography. Square bracket indicates extended piRNAs. b MILI-piRNA extension in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MILI RNPs and were end-labeled with [32P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. c MIWI-piRNA extension and reduction in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MIWI RNPs and were end-labeled with [32P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MIWI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. d The length distribution of small RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testicular small RNA libraries. Data were normalized by microRNA reads (21–23 nt). e The length distribution of MILI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MILI-piRNA libraries. f The length distribution of MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MIWI-piRNA libraries Full size image

PNLDC1 is required for pachytene pre-piRNA 3′ end trimming

To examine the characteristics of extended piRNA species in Pnldc1 Mut mice, we mapped 24–48 nt reads from both wild-type and Pnldc1 Mut small RNA libraries to the mouse genome. Similar to wild-type piRNAs, the longer piRNAs in Pnldc1 Mut mice were primarily mapped to piRNA clusters (~80%) (Fig. 5a). We observed the same results for MILI-piRNAs and MIWI-piRNAs, indicating that the longer small RNAs in Pnldc1 Mut are from the correct genomic source and therefore are true pre-piRNAs (Fig. 5a). We next measured the composition of the first nucleotide of Pnldc1 Mut pre-piRNAs. piRNAs from both the 24–32 nt reads and 33–40 nt reads of the Pnldc1 Mut library had a strong U bias at the first nucleotide position (Fig. 5b). Examination of MILI-piRNAs and MIWI-piRNAs from Pnldc1 Mut testes revealed the same strong U bias at the first nucleotide (Fig. 5b). These data indicate that the formation of the Pnldc1 Mut piRNA 5′ end was normal, suggesting that the extended length of Pnldc1 Mut piRNAs likely resulted from defective 3′ end trimming. To test this hypothesis, we extracted the 24–32 nt reads from wild-type total piRNA library and 33–40 nt reads from Pnldc1 Mut total piRNA library, and performed 5′ end match analysis26. We obtained 3,827,727 unique wild-type library reads from the 24–32 nt population and 2,104,602 unique Pnldc1 Mut library reads from the 33–40 nt population. After measuring the overlap between these two populations, we identified 1,604,365 unique reads of 33–40 nt piRNAs from the Pnldc1 Mut library that could perfectly match to at least one 24–32 nt read from the wild-type library. Within these matching pairs, 70% could perfectly match at the 5′ end, leaving an extended 3′ end tail (Fig. 5c). We further mapped Pnldc1 Mut pre-piRNAs to the most highly expressed piRNA clusters and compared piRNA 5′ end and 3′ end positions between wild-type and Pnldc1 Mut piRNAs. The 5′ end positions had similar start nucleotides between wild-type and Pnldc1 Mut, while the 3′ end positions showed an obvious extended variety (Fig. 5d). These results indicate that accumulated Pnldc1 Mut pre-piRNAs are the bona fide piRNA precursors with untrimmed 3′ end extensions. Therefore, PNLDC1 is required for pre-piRNA 3′ end trimming in mice. Consistent with recently demonstrated in vitro 3′–5′ exonuclease activity13, 30, PNLDC1 is likely the trimmer for mammalian adult piRNA 3′ end maturation.

Fig. 5 piRNA 3′ end extension in adult Pnldc1 Mut testes. a Genomic annotation of total piRNA, MILI-piRNAs, and MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1) testes. Sequence reads (24–48 nt) from small RNA libraries were aligned to mouse sequence sets in the following order: piRNA clusters, coding RNA, non-coding RNA, repeats, and intronic sequences (see Methods for details). “Other” represents sequence reads that did not mapped to the above five sequence sets. The percentage of mapped reads is shown. b Nucleotide distributions at the first position in total piRNA, MILI-piRNAs, and MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1) testes. The 24–40 nt reads from small RNA libraries were used. c Extended piRNA 3′ ends in Pnldc1 Mut testes. The 33–40 nt reads from Pnldc1 Mut (Mut-1 and Mut-2) total piRNA library were mapped to 24–32 nt reads from WT total piRNA library. The number of 33–40 nt Pnldc1 Mut piRNA reads that perfectly match at least one WT piRNA was calculated. The percentage of matching pairs that have identical 5′ ends or identical 3′ ends against all matched pairs are shown. n = 2. Error bars represent s.e.m. d Two examples of 3′ end extension in Pnldc1 Mut (Mut-1) piRNAs. Alignments between 33–48 nt reads from Pnldc1 Mut total piRNA library and 24–32 nt reads from WT total piRNA library within a selected region from two representative piRNA clusters. The genomic locations of these two piRNA clusters are shown at the bottom Full size image

Pachytene piRNA biogenesis is phased in mice

In flies and mouse pre-pachytene stages, Zucchini/MitoPLD-mediated cleavage acts both in the 3′ end formation of upstream piRNA and in the 5′ end formation of downstream piRNA, consecutively generating “phased” piRNAs31, 32. It is unknown whether the production of meiotic pachytene piRNAs, the most abundant piRNA population in mammalian germ cells, is phased due to lack of animal models. The accumulation of pachytene piRNA intermediates in Pnldc1 Mut mice allowed us to explore the pachytene piRNA precursor cleavage mechanism prior to the piRNA trimming step. We analyzed untrimmed pachytene pre-piRNAs in Pnldc1 Mut mice to detect the existence of phased piRNA biogenesis in adult testes using methods described31, 32. We mapped the 24–48 nt reads from wild-type and Pnldc1 Mut small RNA libraries to the most highly expressed pachytene piRNA clusters and analyzed the 5′ end and 3′ end sequences from mapped piRNAs. A strong 5′ end U bias appeared in both wild-type and Pnldc1 Mut piRNAs, confirming the normal 5′ end formation in Pnldc1 Mut piRNA intermediates (Fig. 6a). As expected, at the 10th nt position, there was no obvious adenine (A) residue bias, indicating that the secondary piRNA biogenesis (ping-pong) pathway was not aberrantly activated in Pnldc1 Mut mice. Analysis of the piRNA 3′ end downstream sequences mapped to piRNA clusters revealed that U residues are enriched at the first position immediately downstream of Pnldc1 Mut piRNA 3′ end (+1 position) (Fig. 6a; Supplementary Fig. 7). This specifies a signature of phased piRNA processing. The 3′ end +1 U signature is likely generated by MitoPLD cleavage, which is known to produce phased pre-pachytene piRNAs by generating simultaneously the 3′ end of current piRNA and the 5′ end of immediate next piRNA. We also observed this +1 position U enrichment in Pnldc1 Mut MILI-piRNAs and MIWI-piRNAs (Fig. 6b; Supplementary Figs. 8 and 9). To confirm the phased pachytene piRNA biogenesis in adult testes, we performed 3′–5′ coupling analysis as described31, 32. We mapped piRNA reads to the 10 most highly expressed piRNA clusters and extracted the top 1000 most abundant distinct piRNAs of each cluster as references. We then measured the distance from the 3′ end of each reference piRNA (position 0) to the 5′ end of all the mapped downstream piRNAs within the window of −10 to +50 nt surrounding reference piRNA 3′ end. In wild-type mice, piRNA 5′ ends distribute randomly around the upstream piRNA 3′ ends (Fig. 6c). In contrast, when we analyzed the untrimmed Pnldc1 Mut pre-piRNAs, the neighboring piRNA 5′ ends were enriched at positions immediately downstream of the piRNA 3′ ends (+1), indicating that a single cleavage event produces the 3′ end of one piRNA and the 5′ end of the next downstream piRNA (Fig. 6c; Supplementary Fig. 10). Together, these data demonstrate that phased piRNA biogenesis contributes to mammalian pachytene piRNA production.

Fig. 6 Pachytene piRNA biogenesis is phased in mice. a U bias at position +1 downstream of piRNA 3′ ends in Pnldc1 Mut total piRNA. The 24–48 nt reads from WT and Pnldc1 Mut (Mut-1) total piRNA libraries were mapped to one representative pachytene piRNA cluster (2-qE1-35981.1, the most abundantly expressed). Sequence logos showing nucleotide composition at mapped piRNA 5′ ends, 3′ ends, and downstream regions of 3′ ends were generated. Gray shading marks the piRNA region. b U bias at position +1 downstream of piRNA 3′ end in Pnldc1 Mut MILI-piRNAs and MIWI-piRNAs. The 24–48 nt reads from WT and Pnldc1 Mut (Mut-1) MILI- and MIWI-piRNA libraries were mapped to one representative pachytene piRNA cluster (2-qE1-35981.1). Sequence logos showing nucleotide composition in the vicinity of mapped piRNA 3′ ends were generated. Gray shading marks the piRNA region. c Untrimmed Pnldc1 Mut piRNAs exhibit coupling of piRNA 3′ ends with subsequent piRNA 5′ ends. The 24–48 nt reads from WT and Pnldc1 Mut (Mut-1) total piRNA libraries were mapped to one representative pachytene piRNA cluster (2-qE1-35981.1). Top 1000 distinctively mapped piRNAs were extracted as references to perform 3′–5′ coupling analysis. The frequency of piRNA 5′ ends around referenced piRNA 3′ ends was calculated. Z-scores at position +1 (Z1) are shown Full size image

PNLDC1 is required for pre-pachytene piRNA trimming

To investigate whether PNLDC1 plays a role in pre-pachytene piRNA biogenesis, we analyzed neonatal (P0) control Pnldc1 heterozygous (HET) and Pnldc1 homozygous (KO) mutant mice carrying an exon 1–exon 9 deletion. MILI localization was not altered in Pnldc1 KO testes (Fig. 7a). However, MIWI2 in Pnldc1 KO germ cells was mislocalized partially to cytoplasm as compared to control (Fig. 7b). This contrasts with the MIWI2 localization pattern in Tdrkh KO testes in which vast majority of MIWI2 was mislocalized to cytoplasm (Fig. 7c). The mislocalization of MIWI2 in Pnldc1 KO mice suggests defective piRNA biogenesis and function. We immunoprecipitated MILI from Pnldc1 HET and KO testes and examined piRNA length distribution (Fig. 7d). Pnldc1 KO piRNAs bound to MILI showed an extended length (~33 nt) as compared to control (~27 nt), indicating a piRNA trimming defect. Mapping of extended Pnldc1 KO piRNAs to a representative pre-pachytene piRNA cluster revealed a 3′ end extension indicative of defect in pre-piRNA 3′ end trimming (Fig. 7e). To examine the effect of defective piRNA trimming on LINE1 expression in neonatal Pnldc1 KO germ cells, we performed immunostaining of LINE1. Interestingly, no obviously LINE1 upregulation was observed (n = 4) (Supplementary Fig. 11). This indicates that the piRNA pathway in PNLDC1-deficient neonatal male germ cells is still at least functional for LINE1 silencing despite defective piRNA trimming and partial MIWI2 mislocalization.