Abstract
Spermatogenesis is a highly regulated process during which haploid sperm cells are generated. Although autophagy is involved in the spermatogenesis process, the molecular pathways and regulations of autophagy in germ cell development remain elusive. Here, we showed that Ppp1r36, a regulatory subunit of protein phosphatase 1, is expressed during gonadal development, mainly in testes during spermatogenesis. Autophagy protein LC3 (microtubule associated protein 1 light chain 3), especially its active form LC3-II, had a similar expression pattern to Ppp1r36. Moreover, LC3-II level and puncta analysis showed that autophagy is up-regulated around 21 dpp (day postpartum) in postnatal testis, indicating a potential role of autophagy during the first wave of spermatogenesis. We demonstrated that Ppp1r36 promotes autophagosome formation upon starvation induction. Further autophagy flux analysis using a tandem fluorescent indicator, mCherry-GFP-LC3, confirmed that Ppp1r36 participated in autophagy. We further determined that Ppp1r36 is associated with Atg16L1 (autophagy related 16-like 1) in autophagy of starvation induction. Thus, our results uncover a potential role of the regulatory subunit Ppp1r36 of protein phosphatase 1 in enhancing autophagy during spermatogenesis.
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Introduction
In mammals, the primordial germ cells migrate into the genital ridge, which then differentiate into gonads during embryogenesis. Spermatogenesis is a complexly regulated process during which haploid sperm cells are generated from spermatocytes through meiosis in the seminiferous tubules in male testis1. The haploid round spermatids first appear around postnatal day 18 and further undergo a series of dramatic morphological transformations and structural changes, such as the nuclei condensed and cytoplasm discharged, as well as the formations of flagella and acrosome. Finally, elongated sperm cells are generated in the seminiferous tubules in approximately 35 days and a new round of spermatogenesis is initiated about every 12 days2,3,4,5.
Spermatogenesis is a developmental process, which is involved in both mitosis and meiosis. Nearly half of total coding genes are expressed in mouse testis6, which are localized on both sex-chromosomes and autosomes. The Y-linked genes are particularly involved in spermatogenesis. For example, Azf (Azoospermia factor) mutations can cause sertoli cell-only syndrome and spermatogenic arrest7, Daz (Deleted in azoospermia) mutations lead to oligozoospermia and azoospermia8,9, and Zfy2 is responsible for sperm formation10. Some X-linked genes are also required for spermatogenesis in mammals. For example, the Dax1-deficiency in mice results in spermatogenic failure11. Many autosomal genes are essential to spermatogenesis, of which Dmrt1 is a key transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells12,13. Endocrine hormones regulate coordinately spermatogenesis, such as follicle stimulating hormone and luteinizing erythropoietin5. It is suggested that the GnRH (gonadotropin-releasing hormone) pulse generator is a master ON/OFF switch of the hypothalamus-pituitary-gonad reproductive axis5. In addition, small non-coding RNA pathways are involved in regulations of spermatogenesis in mammals. Ablation of Dicer, which is required for microRNAs biogenesis in Sertoli cells, leads to a complete absence of spermatozoa and progressive testicular degeneration14. In contrast to miRNAs, piRNAs are enriched in germ line, which play their roles through their interactive proteins including Mili and Miwi. Their interactions are important in inhibition of transposable elements, translation regulation and RNA degradation during spermatogenesis15,16,17.
Accumulating evidence shows the importance of interplay between subcellular activities and germ cell development. Dynamic mitochondrial fusion is essential for spermatogenesis. Germ cell-specific gene Gasz contains a mitochondrial localization signal, which interacts with Mfn1 to promote mitofusion18, indicating a role of mitofusion during germ cell development. Autophagy, as an important degradation and recycling pathway, can transport the intracellular components such as the excess or dysfunctional proteins to the lysosomes for degradation to maintain metabolism homeostasis19. Autophagy-related proteins (Atg) are highly conserved from yeast to mammals. Knockout of Atg7 in germ cells resulted in irregular or nearly round-headed spermatozoa in mice, which is similar to human globozoospermia20, highlighting an importance of autophagy in germ cell development. However, roles and underlying molecular mechanisms of autophagy during spermatogenesis remain elusive.
Protein phosphatase 1 (PP1) is one of the main members of the serine/threonine protein phosphatase family which are ubiquitously expressed in eukaryotic cells21. It regulates a variety of cellular functions through interaction of its catalytic subunit with different established or putative regulatory subunits21,22,23. PP1 has multifaceted cellular functions in many physiological and pathological processes such as metabolism, immune response, apoptosis, mitosis, meiosis, protein synthesis and cytoskeletal reorganization23,24,25,26. A recent study shows that PPP1 inhibited autophagy in cardiomyocytes through Atg16L1 dephosphorylation, indicating a role of Atg16L1 phosphorylation in autophagy in cardiomyocytes27. Particularly, deletion of the Ppp1cc in germ cells resulted in oligo-terato-asthenozoospermia and male infertility in mice28. Ppp1cc mutant males had predominantly round spermatids and only occasional mature sperms visible in the epididymis, eventually led to male sterility, which is similar to a human condition known as nonobstructive azoospermia29. Recently, other PP1 isoforms including PPP1CB, PPP4C and PPP6C have also identified in human sperm30. In addition, sperm motility is initiated by inhibiting PP1 by Wnt signaling31. These studies indicate that protein phosphorylation is relevant to sperm physiology. However, regulations of protein phosphatase 1, especially with regard to the importance and underlying molecular mechanisms in germ cell development, remain largely unknown.
In the present study, we identify a regulatory subunit of protein phosphatase 1, Ppp1r36, in mouse gonads and show that Ppp1r36 and LC3 are associated with spermatogenesis in mice. Furthermore, we demonstrate that Ppp1r36 promotes autophagy upon starvation induction, probably through its interaction with Atg16L1. Thus, these results uncover a potential role for the regulatory subunit Ppp1r36 of protein phosphatase 1 in enhancing autophagy during spermatogenesis.
Results
Ppp1r36 is expressed in testes during spermatogenesis
Ppp1r36 is an evolutionarily conserved gene in vertebrates (Suppl. Fig. 1). To explore a potential role of Ppp1r36 gene in spermatogenesis, we first investigated its expression patterns in mice. In adult tissues, Ppp1r36 was highly expressed in testis compared with other tissues (Fig. 1a). During testis development in postnatal mice, Ppp1r36 expression increased to a high level at 21 dpp and then had a stable level until adulthood (Fig. 1b,c).
Further immunofluorescence analysis showed that Ppp1r36 protein can be detected in testis at 21 dpp. With the progress of development, Ppp1r36 presented higher expression level until adulthood (Fig. 1d). Ppp1r36 positive signals were observed in spermatids and spermatozoa in testis, as well as in oocytes and granulosa cells in ovary (Fig.1e). In spermatozoa from epididymis of adult male mice, Ppp1r36 protein was mainly distributed in the acrosome (Fig. 1f). These data suggested that Ppp1r36 is associated with spermatogenesis in mice.
LC3 is expressed in testes during spermatogenesis
A previous study showed a role of autophagy protein Atg7 in acrosome biogenesis in mice20, indicating that autophagy is important for spermatogenesis. We thus detected autophagy marker LC3 in mouse testes at different developmental stages. Western blot analysis showed that LC3 reached a high level of expression at 18–21 dpp. Notably, LC3-II (active LC3 lipidation form) had a highest abundance at the 18–21 dpp compared to other stages (Fig. 2a), indicating occurrence of autophagy during the first wave of spermatogenesis.
Immunofluenrence analysis further confirmed the observation (Fig. 2b). LC3 can be detected in testes from 18 dpp to adult, mainly in the cytoplasm of spermatocytes, spermatids and spermatozoa. Some LC3 positive puncta were observed at 21 dpp (Fig. 2b). These results suggested a potential role of autophagy during spermatogenesis.
Ppp1r36 over-expression promotes autophagy upon starvation induction
To explore whether Ppp1r36 participates in autophagy, Ppp1r36 was over-expressed in HEK293T cells and then the cells were treated in starvation medium to induce autophagy. Western blot analysis showed that LC3-II level was higher when Ppp1r36 transfected upon starvation induction for 0.5–1 h compared to controls (Fig. 3a). We then confirmed the results by gradient transfection of Ppp1r36 (Fig. 3b).
Fluorescence analysis in mouse germ cell GC-1 showed that the number of Red-LC3 puncta was significantly increased in Ppp1r36 over-expression upon starvation induction compared to the cells without Ppp1r36 transfection (Fig. 3c). In addition, BAF (Bafilomycin A1, a blocker of the formation of autolysosome) treatment also markedly increased the puncta numbers in comparison with controls. These results indicated that Ppp1r36 promotes autophagosome formation upon starvation induction.
Autophagy flux associated with Ppp1r36
To investigate autophagy process involved in Ppp1r36, we tested autophagy flux by analysis of a tandem fluorescent indicator, mCherry-GFP-LC3. Since green fluorescence of the fusion protein is very sensitive to the acidic environment of lysosomes and quickly quenched in autolysosomes, just red fluorescence could be detected in the autolysosomes32,33. Fluorescence analysis using the indicator system in GC-1 cells showed that Ppp1r36 over-expression promoted autophagy flux upon starvation induction, while BAF treatment inhibited the process (Fig. 4a). In addition, Ppp1r36-Red could co-aggregate with GFP-LC3 puncta in GC-1 cells upon starvation induction (Fig. 4b). These results further confirmed that Ppp1r36 promotes autophagy.
Ppp1r36 interacts with Atg16L1
To further illustrate the molecular mechanism of Ppp1r36-associated autophagy, we tested levels of autophagy related proteins in autophagosome formation. Western blot analysis showed that endogenous Atg16L1 protein level increased with an increasing amount of Ppp1r36, while protein levels of Atg6 and Atg12-Atg5 did not change (Fig. 5a). Co-immunoprecipitation analysis indicated an interaction of Ppp1r36 with Atg16L1 (Fig. 5b). Further fluorescence analysis showed that Ppp1r36 and Atg16L1 puncta were co-aggregated in GC-1 cells (Fig. 5c). These results indicated that Ppp1r36 was associated with Atg16L1.
Discussion
Both kinases and phosphatases cooperatively control protein activities through phosphorylation/dephosphorylation. Ppp1c is ubiquitously expressed in nearly all tissues. Cellular and tissue specificity of dephosphorylation by Ppp1c are dependent on its regulatory subunits. Hence, search for the interactive proteins that regulate Ppp1c activity is essential to understand signaling pathways of autophagy regulation, especially in germ cell development. In the present study, we identify a regulatory subunit of protein phosphatase 1, Ppp1r36, in mouse gonads.
The study presents several major findings: (i) Ppp1r36 is expressed during gonadal development, especially in testes during spermatogenesis. (ii) Interestingly its expression pattern is consistent with autophagy key protein LC3. (iii) Moreover, autophagy is up-regulated around 21 dpp in postnatal testis, revealed by LC3-II level and puncta, an active form of LC3, indicating a potential role of autophagy during the first wave of spermatogenesis. (iv) To explore a possible association of Ppp1r36 with autophagy, we show that Ppp1r36 promotes autophagosome formation upon starvation induction. (v) Further autophagy flux analysis using a tandem fluorescent indicator, mCherry-GFP-LC3, confirms that Ppp1r36 promotes autophagy. (vi) Lastly, we show that Ppp1r36 can interact with Atg16L1. It has been shown that Atg16L1 interacted with Atg12-Atg5 to promote LC3 lipidation by acting as an E3-like ubiquitin ligase34,35. Thus, our results uncover a potential role for the regulatory subunit Ppp1r36 of protein phosphatase 1 in enhancing autophagy during spermatogenesis.
To date, nearly 40 genes for serine⁄threonine phosphatases have been identified36. Many interaction proteins of PP1 are detected, for example, 77 proteins in human testis and 7 proteins in human sperm that bind PP1 have been identified36. This is consistent with the fact that cellular and tissue specificity of dephosphorylation by PP1 are dependent on its regulatory subunits in various tissues. Ppp1r36 has been identified as an interactive protein of PP1 by a global proteome analysis37. We show that as a regulatory subunit, Ppp1r36 probably exerts its role in spermatogenesis through autophagy. The process is probably dependent on its interaction with Atg16L1. A recent study indicated that PPP1 inhibited autophagy in cardiomyocytes through Atg16L1 dephosphorylation, suggesting a role of Atg16L1 phosphorylation for autophagy induction in cardiomyocytes27.
Ppp1r36 is not only expressed in the developing testis during the first wave of spermatogenesis, but also presents in the acrosome of mature spermatozoa, indicating a role of Ppp1r36 in sperm activity, probably through autophagy. Previous study using gene chip (Affymetrix) exhibited a differential expression pattern of Ppp1r36 in XX and XY SSCs (spermatogonial stem cells) with a higher level in XY than XX gonad of E12.538. Accumulating evidences show that spermatogenesis process is involved in autophagy. Specific knockout of Atg7 in mouse germ cells resulted in irregular or nearly round-headed spermatozoa, which is similar to human globozoospermia, thus the knockout mice were infertile because of the defection of acrosome biogenesis20. Heat stress can induce autophagy in addition to apoptosis in mouse germ cells39. In deed, autophagy plays a role in the survival of spermatozoa during conservation in refrigeration40. Autophagy marker LC3-II and autophagosomes were also detected in primary cultures of spermatocytes isolated from male rats41. Recent study showed that Ol-epg5 (ectopic P-granules autophagy protein 5 homolog, a new critical component of the autophagy pathway) knockout in medaka fish resulted in an impaired autophagic flux42. Taken together, these data suggest that autophagy is probably involved in several processes of spermatogenesis from the first wave of spermatogenesis to mature of spermatozoa. Nevertheless, the molecular mechanisms of autophagy in spermatogenesis still need further investigation.
Methods
Ethics statement
All animal experiments and methods were performed in accordance with the relevant approved guidelines and regulations, as well as under the approval of the Ethics Committee of Wuhan University.
Antibodies and reagents
Primary antibodies: Anti-Atg5 (Cat# AP1812b) was purchased from Abgent, San Diego, USA. Anti-Atg6 (Cat# PD017) was from MBL, Nagoya, Japan. Anti-Atg16L1 (Cat# 8089) was purchased from Cell Signaling Technology, Pickering, Canada. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Cat# CW0100) was from CWBIO, Beijing, China. Anti-Actin (Cat# 14395-1-AP) was from Proteintech Group, Chicago, USA. Anti-Flag antibody (Cat# F3165) and monoclonal anti-LC3 (Cat# SAB4200361) were from Sigma-Aldrich, St Louis, USA. Anti-Myc (Cat# 11667149001) and anti-GFP (Cat# 11814460001) were from Roche Applied Science, Indianapolis, USA. Anti-Ppp1r36 was prepared by Beijing Huada Protein Innovation, Beijing, China.
Secondary antibodies: Goat anti-mouse IgG (H + L), horseradish peroxidase conjugated antibody (Cat# 31430) and goat anti-rabbit IgG (H + L), horseradish peroxidase conjugated antibody (Cat# 31460) were from Pierce Company, Rockford, USA. FITC-conjugated immunopure goat anti-rabbit IgG (H + L) (Cat# ZF-0311) was purchased from Feiyi Technology, Wuhan, China. Cy3-conjugated affinipure goat anti-rabbit IgG (H + L) (Cat# SA00009-2) was from Proteintech Group, Chicago, USA.
Cloning of Ppp1r36 and RT-PCR
Total RNAs from mouse tissues were isolated using the Trizol Reagent (Cat# 15596-026, Invitrogen, Carlsbad, USA). We synthesized cDNA by reverse transcription from the total RNAs with MMLV reverse transcriptase (Cat# M1701, Promega, Madison, USA). Full-length Ppp1r36 (NM_001163103.1) was cloned into pRK-Flag using Sal1 and Not1 (Fermentas, Lithuania) to generate Flag-Ppp1r36 and into pDsRed-N1 using XhoI and BamHI (Fermentas) to Ppp1r36-Red. Primers were as follows:
Ppp1r36-Sal1: 5′-acgcgtcgaccatggtcaagagtgaggccatgttcacc-3′
Ppp1r36-Not1: 5′-aaggaaaaaagcggccgctttagggcaggtgcttggcgaag-3′
Ppp1r36-Xho1: 5′-ccgctcgagatggtcaagagtgaggccatgttc-3′
Ppp1r36-BamH1: 5′-cgcggatccgctttagggcaggtgcttggcg-3′
The underlined letters indicated restriction endonuclease sites.
For RT-PCR analysis, cDNA synthesis was as described above. Ppp1r36 and Actin were amplified with an annealing temperature of 60 °C for 36 cycles (Ppp1r36) and 25 cycles (Actin) respectively. Primers for the amplification were:
Ppp1r36-1: 5′-agaaaggcaagaaagggaa-3′
Ppp1r36-2: 5′-cgtggtgaaggagcagat-3′
Actin-1: 5′-actgtgcccatctacgaggg-3′
Actin-2: 5′-gtggtggtgaagctgtagcc-3′.
Immunofluorescence analysis
Cells and tissue sections were fixed in pre-cooled 100% methanol at −20 °C or 4% paraformaldehyde at room temperature for 30 min respectively, 0.1% Triton X-100/PBS permeabilized for 10 min and then blocked in 5% bovine serum albumin/PBS for 30 min at 37 °C. The samples were incubated with anti-Ppp1r36 polyclonal antibodies in 5% bovine serum albumin/PBS overnight at 4 °C. Then the samples were incubated with FITC-conjugated affinipure goat anti-rabbit IgG for 1 h at room temperature. Nuclear staining was carried out with DAPI Staining Solution (Cat# C1002, Beyotime Institute of Biotechnology, Jiangsu Province, China) for 10 min in the dark. Images were captured with a confocal fluorescence microscope (Olympus, FV1000, Tokyo, Japan).
Cell culture, treatment and transfection
HEK293T and GC-1 cells were cultured in DMEM (Cat# SH30022.01B, HyClone, Beijing, China) with 10% FBS (fetal bovine serum, Cat# SV30087.02, HyClone). The Cells were transfected in 12/24-well plates using Lipofectamine 2000 (Cat# 11668027, Invitrogen) according to the routine protocol. For starvation treatment, the cells were cultured in the medium HBSS (Cat# SH30030.02B, HyClone). For BAF treatment, Bafilomycin A1 (Cat# B1793, Sigma-Aldrich) was added in the medium for 3 hrs before harvest.
Western blot analysis
HEK293T cells transfected with expression vectors were lysed in RIPA lysates (50 mM Tris pH7.4, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, 1% Protease inhibitors). Cocktail (Roche) was used as protease inhibitor. After centrifuged for 10 min at 4 °C, the supernatant was used for Western blot analyses. Western blots were performed according to routine protocols. The protein extracts from HEK293T cells or mouse tissues were separated using 15% SDS-PAGE and transferred onto a 0.45 μm PVDF membrane (Cat# NK0414, Roche Diagnostics, Indianapolis, USA). The membranes were blocked with 5% non-fat dried milk in TBST (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% Tween-20) and incubated with the antibodies overnight at 4 °C, followed by the horseradish peroxidase-conjugated secondary antibody. The protein bands were visualized by incubating membranes with the ECL Plus detecting reagents (Cat# WBKLS0500, Millipore, Billerica, USA).
Co-immunoprecipitation assays
To analyze protein interactions, co-immunoprecipitation assays were performed in HEK293T cells. The cells were lysed in RIPA lysates as above. The cell lysates were incubated with the anti-Flag antibody and Protein G Agarose (Roche) overnight at 4 °C. The resins were collected by centrifugation at 4 °C and then washed four times with RIPA lysates. Bound proteins were eluted using loading buffer (3% SDS, 1.5% mercaptoethanol, 8% glycerol, 0.01% Coomassie blue G-250, 150 mM Tris-HCl pH7.0) and separated using 15% SDS-PAGE, followed by immunoblotting with anti-Atg16L1 or anti-Myc antibody.
Additional Information
How to cite this article: Zhang, Q. et al. The germline-enriched Ppp1r36 promotes autophagy. Sci. Rep. 6, 24609; doi: 10.1038/srep24609 (2016).
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Acknowledgements
We would specifically thank Dr. Mingzhou Chen for plasmid mCherry-GFP-LC3. This work was supported by the National Natural Science Foundation of China, the National Key Basic Research project, National Key Technologies R&D Program and Hubei Province Science and Technology project.
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Conceived and designed the experiments: R.Z. and H.C. Performed the experiments: Q.Z., M.G., Y.Z. and Y.S. Analyzed the data: Q.Z. Wrote the paper: Q.Z. and R.Z.
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Zhang, Q., Gao, M., Zhang, Y. et al. The germline-enriched Ppp1r36 promotes autophagy. Sci Rep 6, 24609 (2016). https://doi.org/10.1038/srep24609
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DOI: https://doi.org/10.1038/srep24609
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