Protamine 2 Deficiency Results In Septin 12 Abnormalities.

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 -/- sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2 -/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in Prm2 -/- testes. Furthermore, the Prm2 -/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone- protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.

Overexpression of Human sFLT1 in the Spongiotrophoblast Is Sufficient to Induce Placental Dysfunction and Fetal Growth Restriction in Transgenic Mice.

Preeclampsia (PE) is characterized by maternal hypertension and placental dysfunction, often leading to fetal growth restriction (FGR). It is associated with an overexpression of the anti-angiogenic sFLT1 protein, which originates from the placenta and serves as a clinical biomarker to predict PE. To analyze the impact of sFLT1 on placental function and fetal growth, we generated transgenic mice with placenta-specific human sFLT1 (hsFLT1) overexpression. Immunohistochemical, morphometrical, and molecular analyses of the placentas on 14.5 dpc and 18.5 dpc were performed with a focus on angiogenesis, nutrient transport, and inflammation. Additionally, fetal development upon placental hsFLT1 overexpression was investigated. Dams exhibited a mild increase in serum hsFLT1 levels upon placental hsFLT1 expression and revealed growth restriction of the fetuses in a sex-specific manner. Male FGR fetuses expressed higher amounts of placental hsFLT1 mRNA compared to females. FGR placentas displayed an altered morphology, hallmarked by an increase in the spongiotrophoblast layer and changes in labyrinthine vascularization. Further, FGR placentas showed a significant reduction in placental glycogen storage and nutrient transporter expression. Moreover, signs of hypoxia and inflammation were observed in FGR placentas. The transgenic spongiotrophoblast-specific hsFLT1 mouse line demonstrates that low hsFLT1 serum levels are sufficient to induce significant alterations in fetal and placental development in a sex-specific manner.

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human.

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Male humans, mice and other animals produce sex cells known as sperm that seek out and fertilize egg cells from females. Sperm have a very distinctive shape with a head and a long tail that enables them to swim towards an egg. At the front of the sperm's head is a pointed structure known as the acrosome that helps the sperm to burrow into an egg cell. A structure known as the cytoskeleton is responsible for forming and maintaining the shape of acrosomes and other parts of cells. Two proteins, known as Cylicin 1 and Cylicin 2, are unique to the cytoskeleton of sperm, but their roles remain unclear. To investigate the role of the Cylicins during spermiogenesis, Schneider, Kovacevic et al. used an approach called CRISPR/Cas9-mediated gene-editing to generate mutant mice that were unable to produce either Cylicin 1 or Cylicin 2, or both proteins. The experiments found that healthy female mice were less likely to become pregnant when they mated with mutant males that lacked Cylicin 1 compared with males that had the protein. When they did become pregnant, the females had smaller litters of babies. Mutant male mice lacking Cylicin 2 or both Cylicin proteins (so-called "double" mutants), were infertile and mating with healthy female mice did not lead to any pregnancies. Further experiments found that the sperm of such mice had smaller heads than normal sperm, defective acrosomes, and curled tails that wrapped around the head. Schneider, Kovacevic et al. also examined the sperm of a human patient who had inherited genetic variants in the genes encoding both Cylicin proteins. Similar to the double mutant mice, the patient was infertile, and his sperm also had defective acrosomes and curled tails. These findings indicate that Cylicins are required to make the acrosome as sperm cells mature and help maintain the structure of the cytoskeleton of sperm. Further studies of Cylicins and other sperm proteins in mice may help us to understand some of the factors that contribute to male infertility in humans.

Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis.

Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and is highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7b-null allele (Actl7b-/-) were infertile, whereas heterozygous males (Actl7b+/-) were fertile. Severe spermatid defects, such as detached acrosomes, disrupted membranes and flagella malformations start to appear after spermiogenesis step 9 in Actl7b-/- mice, finally resulting in spermatogenic arrest. Abnormal spermatids were degraded and levels of autophagy markers were increased. Co-immunoprecipitation with mass spectrometry experiments identified an interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establish that mutations in ACTL7B are directly related to male infertility, pressing for additional research in humans.

SPAG17 mediates nuclear translocation of protamines during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Genome-scale CRISPR screen reveals neddylation to contribute to cisplatin resistance of testicular germ cell tumours.

BACKGROUND: Type II testicular germ cell tumours (TGCT) are the most prevalent tumours in young men. Patients suffering from cisplatin-resistant TGCTs are facing very poor prognosis demanding novel therapeutic options. Neddylation is a known posttranslational modification mediating many important biological processes, including tumorigenesis. Overactivation of the neddylation pathway promotes carcinogenesis and tumour progression in various entities by inducing proteasomal degradation of tumour suppressors (e.g., p21, p27). METHODS: We used a genome-scale CRISPR/Cas9 activation screen to identify cisplatin resistance factors. TGCT cell lines were treated with the neddylation inhibitor (MLN4924)/cisplatin/combination and investigated for changes in viability (XTT assay), apoptosis/cell cycle (flow cytometry) as well as in the transcriptome (3'mRNA sequencing). RESULTS: NAE1 overexpression was detected in cisplatin-resistant colonies from the CRISPR screen. Inhibition of neddylation using MLN4924 increased cisplatin cytotoxicity in TGCT cell lines and sensitised cisplatin-resistant cells towards cisplatin. Apoptosis, G2/M-phase cell cycle arrest, γH2A.X/P27 accumulation and mesoderm/endoderm differentiation were observed in TGCT cells, while fibroblast cells were unaffected. CONCLUSIONS: We identified overactivation of neddylation as a factor for cisplatin resistance in TGCTs and highlighted the additive effect of NAE1 inhibition by MLN4924 in combination with cisplatin as a novel treatment option for TGCTs.

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells.

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB's impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

PFN4 is required for manchette development and acrosome biogenesis during mouse spermiogenesis.

Profilin 4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established; however, ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Furthermore, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed that the proteins ARF3, SPECC1L and FKBP1, which are involved in Golgi membrane trafficking and PI3K/AKT pathway, are more abundant in Pfn4-/- testes. Levels of PI3K, AKT and mTOR were elevated, whereas AMPK level was reduced, consistent with inhibition of autophagy. This seems to result in blockage of autophagic flux, which could explain the failure in acrosome formation. In vitro fertilization demonstrated that PFN4-deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment for PFN4-related human infertility.

Protamines: lessons learned from mouse models.

In brief: Protamines package and shield the paternal DNA in the sperm nucleus and have been studied in many mouse models over decades. This review recapitulates and updates our knowledge about protamines and reveals a surprising complexity in protamine function and their interactions with other sperm nuclear proteins. Abstract: The packaging and safeguarding of paternal DNA in the sperm cell nucleus is a critical feature of proper sperm function. Histones cannot mediate the necessary hypercondensation and shielding of chromatin required for motility and transit through the reproductive tracts. Paternal chromatin is therefore reorganized and ultimately packaged by protamines. In most mammalian species, one protamine is present in mature sperm (PRM1). In rodents and primates among others, however, mature sperm contain a second protamine (PRM2). Unlike PRM1, PRM2 is cleaved at its N-terminal end. Although protamines have been studied for decades due to their role in chromatin hypercondensation and involvement in male infertility, key aspects of their function are still unclear. This review updates and integrates our knowledge of protamines and their function based on lessons learned from mouse models and starts to answer open questions. The combined insights from recent work reveal that indeed both protamines are crucial for the production of functional sperm and indicate that the two protamines perform distinct functions beyond simple DNA compaction. Loss of one allele of PRM1 leads to subfertility whereas heterozygous loss of PRM2 does not. Unprocessed PRM2 seems to play a distinct role related to the eviction of intermediate DNA-bound proteins and the incorporation of both protamines into chromatin. For PRM1, on the other hand, heterozygous loss leads to strongly reduced sperm motility as the main phenotype, indicating that PRM1 might be important for processes ensuring correct motility, apart from DNA compaction.

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice.

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice.

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/- mice were subfertile, whereas Prm1-/- mice were infertile. Prm1-/- and Prm2-/- sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/- sperm displayed only moderate DNA damage. The majority of Prm1+/- sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/- sperm. However, sperm from Prm1+/- and Prm1-/- mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

COVID-19 Infection Induce miR-371a-3p Upregulation Resulting in Influence on Male Fertility.

In December 2019, the first case of COVID-19 was reported and since then several groups have already published that the virus can be present in the testis. To study the influence of SARS-CoV-2 which cause a dysregulation of the androgen receptor (AR) level, thereby leading to fertility problems and inducing germ cell testicular changes in patients after the infection. Formalin-Fixed-Paraffin-Embedded (FFPE) testicular samples from patients who died with or as a result of COVID-19 (n = 32) with controls (n = 6), inflammatory changes (n = 9), seminoma with/without metastasis (n = 11) compared with healthy biopsy samples (n = 3) were analyzed and compared via qRT-PCR for the expression of miR-371a-3p. An immunohistochemical analysis (IHC) and ELISA were performed in order to highlight the miR-371a-3p targeting the AR. Serum samples of patients with mild or severe COVID-19 symptoms (n = 34) were analyzed for miR-371a-3p expression. In 70% of the analyzed postmortem testicular tissue samples, a significant upregulation of the miR-371a-3p was detected, and 75% of the samples showed a reduced spermatogenesis. In serum samples, the upregulation of the miR-371a-3p was also detectable. The upregulation of the miR-371a-3p is responsible for the downregulation of the AR in SARS-CoV-2-positive patients, resulting in decreased spermatogenesis. Since the dysregulation of the AR is associated with infertility, further studies have to confirm if the identified dysregulation is regressive after a declining infection.

Transcriptional CDK Inhibitors as Potential Treatment Option for Testicular Germ Cell Tumors.

Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15% of patients with metastatic non-seminomas show cisplatin resistance and a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in the treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. An XTT viability assay revealed a strong cytotoxic impact of CDK7/12/13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild-type cell lines (2102EP, NCCIT, TCam2) and the cisplatin-resistant cell lines (2102EP-R, NCCIT-R). The CDK7 inhibitor YKL-5-124 showed a strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines, leaving the MPAF control cell line mostly unaffected. FACS-based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell-line-specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination, CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity.

Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels.

Low expression levels of the E3 ubiquitin­protein ligase Parkin (PARK2) are exhibited in several cancer entities, including clear cell renal cell carcinoma (ccRCC), and are associated with poor prognosis; however, PARK2 can also function as a tumor suppressor gene. The aim of the present study was to thoroughly investigate the effects of PARK2 overexpression in ccRCC cell lines and to determine its effects on malignancy by conducting functional assays such as cell cycle analysis, apoptosis analysis, migration and invasion assays. Furthermore, liquid chromatography­mass spectrometry was used to decipher potential targets of PARK2 that may influence the behavior of ccRCC tumor cells. In addition, ccRCC tumor tissues from a patient cohort were examined in tissue microarrays to find correlations between different clinical parameters. In the present study, it was demonstrated that the induction of PARK2 resulted in a less aggressive phenotype, as indicated by lower migration and invasion in ccRCC cell lines. Mass spectrometry revealed decreased levels of 29 proteins in cells with PARK2 overexpression, including CDC28 protein kinase regulatory subunit 2 (CKS2), which is highly expressed in numerous types of cancer. The link between the function of PARK2 as an E3 ubiquitin ligase and the low expression levels of CKS2 was investigated by mutating the catalytic domain of the PARK2 gene, and it was found that the effect of decreased migration was abolished in 786­O and RCC­MH ccRCC cell lines. CKS2 silencing decreased migratory ability of the cells. Furthermore, it was revealed that high CKS2 levels are associated with high tumor grading in patient samples and lower patient survival. In conclusion, the results from the present study indicated that PARK2 may signal via CKS2 to affect tumor behavior. In consequence, CKS2 may be a biomarker in ccRCC and may also serve as potential target for ccRCC therapy.

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

Loss of Profilin3 Impairs Spermiogenesis by Affecting Acrosome Biogenesis, Autophagy, Manchette Development and Mitochondrial Organization.

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome-manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3-/- males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3-/- sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3-/- sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3-/- testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3-ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3-/- testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs.

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-sequencing reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5-E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility.

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.