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.

Effects of Pulsed-Wave Photobiomodulation Therapy on Human Spermatozoa.

BACKGROUND AND OBJECTIVES: Previous studies reported that photobiomodulation (PBM) positively affects the mitochondrial respiratory chain in sperm, resulting in improved motility and velocity. As laser settings are not yet fully established, the present study aimed at optimizing PBM on human sperm. In addition, possible side-effects of PBM on sperm DNA fragmentation level and acrosomal integrity have been analyzed. STUDY DESIGN/MATERIALS AND METHODS: A pulsed laser-probe (wavelength 655 nm, output power 25 mW/cm², impulse duration 200 nanoseconds) was used. Native fresh liquefied semen samples underwent radiation with energy doses of 0 (control), 4, 6, and 10 J/cm². Sperm parameters were assessed at 0, 30, 60, 90, and 120 minutes after radiation using a computer-assisted sperm analysis system. Motility and velocity of sperm from asthenozoospermic patients (n = 42) and normozoospermic controls (n = 22) were measured. The amount of DNA strand breaks was analyzed using ligation-mediated quantitative polymerase chain reaction in patients with asthenozoospermia (n = 18) and normozoospermia (n = 13). Post-irradiance acrosomal integrity was investigated using flow cytometry based on CD46 protein expression (n = 7). RESULTS: Exposure to laser energy-doses of 4 and 6 J/cm² improved sperm motility and velocity in asthenozoospermic patients. PBM exhibited no significant effect on DNA fragmentation level and expression of CD46 serving as a biomarker for acrosome integrity. CONCLUSION: PBM improves sperm motility parameters by maintaining DNA and acrosome integrity and, therefore, represents a promising new tool for assisted reproductive therapy. In particular, improving sperm motility in asthenozoospermic patients by PBM in future may contribute to increasing the chance for successful intrauterine insemination. The present trial has no clinical registration number, as only in vitro studies were performed. The study was approved by the local ethics committee and performed according to the Declaration of Helsinki. Lasers Surg. Med. © 2021 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome Leads to Impaired Semen Parameters, Increased Sperm DNA Fragmentation and Unfavorable Changes of Sperm Protamine mRNA Ratio.

BACKGROUND: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. METHODS: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. RESULTS: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. CONCLUSIONS: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.

The Role of the LINC Complex in Sperm Development and Function.

The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex is localized within the nuclear envelope and consists of SUN (Sad1/UNc84 homology domain-containing) proteins located in the inner nuclear membrane and KASH (Klarsicht/Anc1/Syne1 homology domain-containing) proteins located in the outer nuclear membrane, hence linking nuclear with cytoplasmic structures. While the nucleoplasm-facing side acts as a key player for correct pairing of homolog chromosomes and rapid chromosome movements during meiosis, the cytoplasm-facing side plays a pivotal role for sperm head development and proper acrosome formation during spermiogenesis. A further complex present in spermatozoa is involved in head-to-tail coupling. An intact LINC complex is crucial for the production of fertile sperm, as mutations in genes encoding for complex proteins are known to be associated with male subfertility in both mice and men. The present review provides a comprehensive overview on our current knowledge of LINC complex subtypes present in germ cells and its central role for male reproduction. Future studies on distinct LINC complex components are an absolute requirement to improve the diagnosis of idiopathic male factor infertility and the outcome of assisted reproduction.

Unexplained recurrent miscarriages are associated with an aberrant sperm protamine mRNA content.

STUDY QUESTION: Are unexplained recurrent miscarriages associated with abnormal protamine-1 and protamine-2 mRNA levels in spermatozoa? SUMMARY ANSWER: Both protamine-1 and protamine-2 mRNA levels as well as the protamine-1 to protamine-2 mRNA ratio in spermatozoa from men whose female partners experienced two or more consecutive miscarriages were significantly different compared to those from both healthy control men and subfertile couples undergoing IVF/ICSI. WHAT IS KNOWN ALREADY: Aberrant sperm protamine ratios are known to be associated with male-factor infertility. Data from this study suggest that the protamine mRNA ratio may additionally affect early embryo development. STUDY DESIGN, SIZE, DURATION: The study population was recruited from men whose female partners presented with two or more consecutive unexplained miscarriages in a consultation for recurrent pregnancy loss between 2014 and 2016. At the research laboratory of the Urological Clinic of the University Giessen, spermatozoa from cases and controls were subjected to reverse transcription quantitative PCR (RTqPCR) using specific primer pairs for protamine-1 and protamine-2. PARTICIPANTS/MATERIALS, SETTING, METHODS: Protamine-1 and protamine-2 mRNA levels were analysed in semen samples from 25 men whose female partners experienced at least two consecutive idiopathic miscarriages before the 20th week of gestation. The couples were recruited during consultation at the Fertility Center of the LMU Munich, Germany, and at the Clinical Division of Gynecologic Endocrinology and Reproductive Medicine of the Medical University of Vienna, Austria. Results were compared with those from 32 healthy donors (WHO, 2010) recruited at the Department of Urology, Pediatric Urology and Andrology, Giessen, Germany, and 107 men whose partners participated in an IVF/ICSI program at the Fertility Center of the LMU Munich, Germany. MAIN RESULTS AND THE ROLE OF CHANCE: Protamine-1 and protamine-2 mRNA levels as well as the protamine mRNA ratio and all routine semen parameters revealed significant differences between recurrent miscarriage couples and healthy volunteers (P < 0.01). When comparing recurrent miscarriage couples with couples undergoing IVF/ICSI, Ct-values of protamine-1 and protamine-2 mRNAs were significantly higher and the protamine mRNA ratio was significantly lower in RM couples (P < 0.01). When comparing protamine mRNA levels and the protamine mRNA ratio with routine semen parameters, a significant negative correlation was evident between progressive motility and the protamine-2 mRNA level (P = 0.015), as well as between non-progressive motility and the protamine mRNA ratio (P = 0.023). LIMITATIONS REASONS FOR CAUTION: Although our data demonstrate significant abnormalities in RM, larger sample sizes will be needed to confirm our results. Larger sample sizes should also balance the fact that we had to focus mainly on median protamine mRNA levels. Finally, men in the healthy control group were younger in age than those in the case group, which might have introduced some bias, at least concerning the classic semen parameters. Moreover, only protamine mRNA instead of protein levels could be measured. WIDER IMPLICATIONS OF THE FINDINGS: Although the exact mechanism remains to be elucidated, our data suggest that protamine mRNA levels in spermatozoa are not only important for successful fertilization, but also for proper development of the early embryo. STUDY FUNDING/COMPETING INTEREST(S): Grant from the University Clinic Giessen and Marburg (UKGM 29/2015GI). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Impairment of IGF2 gene expression in prostate cancer is triggered by epigenetic dysregulation of IGF2-DMR0 and its interaction with KLF4.

BACKGROUND: Human cancer cells often exhibit impaired IGF2 expression and the underlying mechanisms are multifaceted and complex. Besides the well-known imprinting control region IGF2/H19-ICR, the involvement of a differentially methylated region in the promoter P0 of IGF2 gene (IGF2-DMR0) has been suggested. Here, we evaluate several mechanisms potentially leading to up- and/or down-regulation of IGF2 expression in prostate cancer and present a novel role of Kruppel-like factor 4 (KLF4) as a transcriptional regulator of IGF2 binding in IGF2-DMR0. METHODS: Putative binding sites for transcription factors were identified in IGF2-DMR0 using JASPAR CORE database. Gene expressions were analyzed by RT-qPCR in prostate carcinoma and adjacent benign prostate hyperplasia samples obtained by radical prostatectomy (86 RP-PCa and 47 RP-BPH) and BPH obtained by transurethral prostate resection (13 TUR-BPH). Pyrosequencing and qMSP were used for DNA methylation studies in IGF2-DMR0, IGF2/H19-ICR and Glutathione-S-transferase-P1 (GSTP1) promoter. Loss of imprinting (LOI) was analyzed by RFLP. Copy number variation (CNV) test was performed using qBiomarker CNV PCR Assay. KLF4-binding and histone-modifications were analyzed by ChIP-qPCR in prostate cancer cell lines exhibiting differentially methylated IGF2-DMR0 (LNCaP hypomethylated and DU145 hypermethylated). KLF4 protein was analyzed by western blot. Statistical associations of gene expression to methylation, IGF2 LOI and CNV were calculated by Mann-Whitney-U-test. Correlations between gene expression and methylation levels were evaluated by Spearman's-Rank-Correlation-test. RESULTS: We found a significant reduction of IGF2 expression in the majority of RP-PCa and RP-BPH in comparison to TUR-BPH. Analyzing potential molecular reasons, we found in RP-PCa and RP-BPH in comparison to TUR-BPH a significant hypomethylation of IGF2-DMR0, which coincided with hypermethylation of GSTP1-promoter, a prominent marker of prostate tumors. In contrast, IGF2 LOI and CNV did not associate significantly with up- and/or down-regulation of IGF2 expression in prostate tumors. By analyzing IGF2-DMR0, we detected a consensus sequence for KLF4 with a z-score of 7.6. Interestingly, we found that KLF4 binds to hypomethylated (17%) IGF2-DMR0 enriched with H3K9me3 and H3K27me3 (LNCaP), but does not bind under hypermethylated (85%) and H3K4me3-enriched conditions (DU145). KLF4 expression was detected in TUR-BPH as well as in RP-BPH and RP-PCa and showed a highly significant correlation to IGF2 expression. CONCLUSIONS: Our study demonstrated that in human prostate cancer the impairment of IGF2 expression is accompanied by hypomethylation of IGF2-DMR0. We revealed that KLF4 is a putative transcriptional regulator of IGF2, which binds in IGF2-DMR0 in dependence of the prevailing epigenetic state in this region. Herewith we provide complementary new insights into IGF2 dysregulation mechanisms as a critical process in prostate tumorigenesis.

TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility.

STUDY QUESTION: Are ten-eleven-translocation (TET) 1-3 family enzymes involved in human spermatogenesis and do they impact male fertility? SUMMARY ANSWER: TET1, TET2 and TET3 are successively expressed at different stages of human spermatogenesis, and their expression levels associate with male fertility. WHAT IS KNOWN ALREADY: Spermatogenesis is a complex cell differentiation process accompanied by a drastic epigenetic remodeling. TET1-3 dioxygenases are essential for active DNA demethylation in the paternal pronucleus and in embryonic stem cells. STUDY DESIGN, SIZE, DURATION: Expression of TET1-3 mRNAs and proteinss and 5-hydroxymethylcytosine (5-hmC) proteins were analyzed in human testis tissues from men with obstructive azoospermia and exhibiting histologically normal spermatogenesis. Ejaculated spermatozoa from normozoospermic healthy volunteers, the 'controls' (TET1: n = 58; TET2-3: n = 63), and subfertile men who participated with their female partners in an ICSI-program, the 'patients' (TET1: n = 66; TET2-3: n = 64), were analyzed concerning the stored TET1-3 mRNAs, and the values were correlated to semen parameters and ICSI-outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis sections were used for in situ hybridization (ISH) and immunohistochemical (IHC) studies to determine TET1-3 mRNA and protein expression, and for immunofluorescence (IF) detection of 5-hmC. Sperm samples from controls were analyzed by western blot, immunocytochemistry (ICC) and RT-PCR concerning the presence of non-degraded TET1-3 protein and mRNA. Sperm samples from controls and patients were used for quantitative TET1-3 mRNA analyses (reverse transcription-polymerase chain reaction) and for comparative statistical evaluations under consideration of semen parameters and ICSI-outcome (pregnancy). MAIN RESULTS AND THE ROLE OF CHANCE: During human spermatogenesis TET1-3 proteins are successively expressed: TET2 is expressed in the cytoplasm of late pachytene spermatocytes of Stage V, TET1 starts to be expressed in the nuclei of Step 1 round spermatids at Stage I, and TET3 starts to be expressed in the nuclei of Step 3 round spermatids at Stage III. Five-hmC appears only in Step 5 elongated spermatids. All three TETs are still detectable at the mRNA and protein level in sperm cells in considerable amounts. Control men generally exhibited higher levels of TET1-3 in sperm. TET1- and TET3-mRNA levels in sperm were significantly negatively correlated with age (P = 0.0025 and P = 0.0343) and positively correlated with progressive sperm motility (P = 0.0007 and P = 0.018). All TETs showed a significant association with sperm concentration (P < 0.03). Patients diagnosed with oligozoospermia and/or asthenozoospermia (TET1: n = 35; TET2-3: n = 32) showed significantly reduced TET1-3 in sperm in comparison to controls (P = 0.003, P = 0.041 and P = 0.028), but not compared with normozoospermic patients. Levels of TET3 in sperm was significantly associated with high-fertilization rates (P = 0.009). Concerning ICSI-outcome, the lowest levels of TET1-3 mRNAs in sperm were found in the non-pregnant group. Increased TET2 in sperm was significantly associated with pregnancy (P = 0.006). LIMITATIONS, REASONS FOR CAUTION: Our results concerning the association of the mRNA level of TETs in ejaculated sperm cells to different fertility parameters are descriptive. Further studies clarifying the reasons for decreased TET1-3 levels in subfertile men and their effect on their sperm methylome are essential. WIDER IMPLICATIONS OF THE FINDINGS: The study gives a substantial indication that in human spermiogenesis, an active DNA demethylation process occurs with an involvement of TET enzymes, and that the level of TET1-3 expression is pivotal for male fertility. STUDY FUNDING: Research grant from the German Research Foundation (DFG) to U.S. (SCHA1531/1-1 and SCHA1531/2-1). COMPETING INTERESTS: None.

Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

Expression and role of leptin under hypoxic conditions in human testis: organotypic in vitro culture experiment and clinical study on patients with varicocele.

PURPOSE: We investigated the expression and the exact role of leptin under hypoxic conditions in the human testis. MATERIALS AND METHODS: Five testes from patients treated with orchiectomy for prostate cancer were used to construct an in vitro hypoxic culture system for human testicular tissue. Immunohistochemistry was performed to analyze leptin protein expression. Leptin, leptin receptor and HIF-1α mRNA were examined by quantitative reverse transcriptase-polymerase chain reaction. Serum and seminal plasma leptin, gonadal hormones and semen parameters were evaluated in 10 healthy donors and 42 infertile patients with varicocele before and after surgery. RESULTS: The viability of in vitro cultured testicular tissue was well maintained within 48 hours based on the results of morphological analysis, cell number and cell specific mRNAs. Immunohistochemistry demonstrated that leptin was mainly expressed in seminiferous tubules. Interestingly the optical density of leptin, leptin mRNA and HIF-1α mRNA was significantly increased under hypoxia. Leptin mRNA and HIF-1α mRNA correlated positively (Rs = 0.843, p <0.01). In the clinical study the concentration of seminal plasma leptin before varicocelectomy was markedly higher in patients (mean ± SD 3.01 ± 1.23 ng/l, p <0.01). It was highest in the grade 3 group (mean 3.95 ± 1.37 ng/l, p <0.01) and significantly decreased in the 6-month postoperative group (2.35 ± 0.78, p <0.05). Furthermore, negative correlations were observed between seminal plasma leptin and the sperm concentration (Rs = -0.187, p <0.05), and progressive motility (Rs = -0.234, p <0.05). CONCLUSIONS: Leptin expression was induced under hypoxia in the human testis, probably via the HIF-1α related response pathway. Seminal plasma leptin closely correlated with varicocele related spermatogenesis dysfunction. It might effectively reflect the testicular hypoxic environment.

Sperm protamine mRNA ratio and DNA fragmentation index represent reliable clinical biomarkers for men with varicocele after microsurgical varicocele ligation.

PURPOSE: We investigated whether the sperm protamine-1/2 mRNA ratio and DNA fragmentation index are reliable biomarkers in patients with clinical varicocele. MATERIALS AND METHODS: We performed a prospective study in 42 subfertile patients with left clinical varicocele and 10 normozoospermic healthy donors with proven fertility. All patients and female partners were seen 3 and 6 months after varicocelectomy for fertility examination. Real-time quantitative reverse transcriptase-polymerase chain reaction and SCSA® were performed to analyze the sperm protamine-1/2 mRNA ratio and DNA fragmentation index. RESULTS: The protamine-1/2 mRNA ratio and DNA fragmentation index were significantly higher in the preoperative group than in the control group (p <0.01). After varicocelectomy in the pregnant group the protamine-1/2 mRNA ratio recovered to the normal value and the DNA fragmentation index was significantly lower than in the preoperative group (p <0.01). In the nonpregnant group the protamine-1/2 mRNA ratio and DNA fragmentation index did not differ (p >0.05). However, significant differences were present preoperatively according to varicocele severity in the protamine-1/2 mRNA ratio and DNA fragmentation index (p <0.05 and <0.01, respectively) with the highest values in the grade 3 group. Nevertheless, postoperatively no difference in varicocele severity was noted (p >0.05). The protamine-1/2 mRNA ratio was strongly related to preoperative and postoperative sperm concentration (Rs -0.238, p <0.01), progressive motility (Rs -0.327, p <0.01), total motility (Rs -0.206, p <0.05) and DNA fragmentation index (Rs 0.293, p <0.01) except for normal morphology (Rs -0.064, p >0.05). CONCLUSIONS: The sperm protamine-1/2 mRNA ratio and DNA fragmentation index can effectively be used to evaluate male fertility. Male infertility due to varicocele may be associated with protamine deficiency and sperm DNA damage. The post-varicocelectomy protamine-1/2 mRNA ratio and DNA fragmentation index are associated with the post-varicocelectomy pregnancy rate.

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.

The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART programme.

STUDY QUESTION: Could the protamine-1 to protamine-2 mRNA ratio serve as a biomarker to estimate the fertilizing capacity of sperm from men taking part in an IVF/ICSI programme? SUMMARY ANSWER: The protamine mRNA ratio clearly discriminates between fertile and subfertile men and sperm with a normal protamine mRNA ratio exhibit a higher fertilizing capacity in IVF/ICSI. WHAT IS KNOWN ALREADY: Aberrant sperm protamine ratios are associated with male factor infertility and mRNA ratio is comparable with protein ratio (due to transcriptional stop in elongating spermatids). STUDY DESIGN, SIZE, DURATION: The study population was drawn from subfertile men, whose female partners participated in IVF or ICSI programmes between September 2010 and February 2012. Normozoospermic healthy volunteers served as controls. Sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine-1 and protamine-2. Relative protamine-1 and protamine-2 mRNA levels were analysed with the Mann-Whitney U-test (two-tailed). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative RT-PCR for protamines 1 and 2 has been performed in ejaculates from 32 normozoospermic volunteers (control, University Clinic Giessen, Germany) and 306 patients, whose female partners took part in an IVF (n = 76; University Clinic Hamburg, Germany and Shanghai Jiaotong University, China) or an ICSI (n = 230; University Clinic Munich, Germany and Kinderwunschzentrum Wiesbaden, Germany) programme. MAIN RESULTS AND THE ROLE OF CHANCE: The sperm protamine mRNA ratio in normozoospermic men (0.98 ± 0.3) differed significantly from that of ICSI patients (Munich 0.81 ± 0.1; Wiesbaden 0.78 ± 0.2; P < 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0 ± 0.07; Shanghai 1.0 ± 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc > 80%; P = 0.017) and ICSI (65.1% of patients with fc > 70%; P = 0.028). LIMITATIONS, REASONS FOR CAUTION: The protamine mRNA ratio in an individual sperm cell used for ICSI may be different from the overall value obtained from a semen aliquot. WIDER IMPLICATIONS OF THE FINDINGS: Data are in line with current literature and suggest the protamine mRNA ratio as a diagnostic marker to estimate the fertilizing capacity of sperm. STUDY FUNDING: The German Research Foundation (DFG) to K.S., W.W. and A.P. (STE 892/9-2), as well as to A.S. and H.C.O. (SP721/1-3). COMPETING INTEREST(S): None.

Epigenetic down regulation of RASSF10 and its possible clinical implication in prostate carcinoma.

BACKGROUND: Ras association domain family (RASSF) comprises several tumor suppressor genes, which are often epigenetically inactivated in human tumors. Here, we aim to analyze the relevance of the recently identified member RASSF10 in prostate carcinogenesis. METHODS: RASSF10 promoter methylation and mRNA expression were investigated by bisulfite-pyrosequencing and qRT-PCR, respectively, in prostate carcinoma (PCa) cell lines (LNCaP, 22Rv1, DU-145) and in 83 primary PCa and 53 primary benign prostatic hyperplasia (BPH) tissues obtained after radical prostatectomy. Histological localization of RASSF10 was done by in situ hybridization. To prove the epigenetic nature of RASSF10 down regulation, PCa cell lines were treated with 5-aza-2-deoxycytidine and trichostatin A. Potential function of RASSF10 was analyzed in LNCaP by colony formation and apoptosis assays. RESULTS: RASSF10 mRNA was localized to cells of the basal layer of the prostatic gland. Absence (LNCaP) and decrease (22Rv1, DU-145) of RASSF10 expression was associated with promoter methylation and could be restored by demethylating agents. A link between RASSF10 mRNA reduction and promoter methylation was also detected in primary prostate tissues (P = 0.006), where PCa showed more frequently reduced RASSF10 levels when compared with BPH (33.7% vs. 13.2%, P = 0.009). RASSF10 methylation could be further associated with advanced tumor stage and advanced age (P-values < 0.05). Our preliminary functional assays revealed the ability of RASSF10 to inhibit colony formation (P = 0.018) and to increase apoptosis (P = 0.035). CONCLUSIONS: This is the first study, which demonstrates the frequent epigenetic inactivation of RASSF10 in PCa and its implication in clinical symptoms of PCa.

14-3-3 beta in the healthy and diseased male reproductive system.

BACKGROUND: Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been implicated in several diseases including tumourigenesis. Here we investigated the expression of the 14-3-3 beta isoform in healthy testicular tissues of humans, rats and mice as well as in tissues of Sertoli-cell-only (SCO) syndrome, intratubular germ cell neoplasia (IGCN) and classical seminoma. METHODS: Samples of healthy and diseased testes from humans, rats and mice were analysed by immunohistochemistry. For PCR, human testis cell lysates were used. Immunoblot analyses of rats and humans healthy testes were performed. Duolink proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were carried out to investigate interactions between 14-3-3 beta and vimentin in human, rat and mouse testes. RESULTS: In healthy testes and SCO syndrome, strong 14-3-3 beta-positive cells could be identified as Sertoli cells. Furthermore, 14-3-3 beta proteins were detected in cells of the peritubular stroma. In samples of IGCN and classical seminoma, the malignant transformed cells stained positive for 14-3-3 beta antigen. Immunoblot analyses revealed the presence of 14-3-3 beta in healthy testicular tissues. 14-3-3 beta mRNA transcripts were detected in cell lysates of healthy human testes. Interaction of 14-3-3 beta with the intermediate filament vimentin was revealed by Duolink PLA and Co-IP. Co-IP experiments identified tubulin as another 14-3-3 beta binding partner. CONCLUSIONS: Our data suggest that 14-3-3 beta expression is essential for normal spermatogenesis by interacting with vimentin in Sertoli cells. Additionally, 14-3-3 beta expression in malignant transformed cells in IGCN and classical seminoma may lead to tumourigenesis and cell survival.

Effect of Khat (Catha edulis Forsk) extract on testicular maturation in pre-pubertal and pubertal rats: A morphological and biochemical study.

The present study aimed at analysing the effect of Khat plant extract on rat testicular development. Thirty-two weaned male albino rats (4 weeks old) were divided into four groups consisting of eight animals each. While control animals received normal saline, rats of groups I, II and III received 100, 200 and 300 mg Khat extract per kg body weight dissolved in distilled water by oral gavage daily for 8 weeks, respectively. Blood samples were collected in separate heparinized tubes by cardiac puncture from each rat and processed for measuring plasma levels of reproductive hormones LH, FSH, testosterone and prolactin. Five-µm sections were stained with haematoxylin and eosin and examined by light microscope. Some sections were immunostained for protamine-1 representing a biomarker for intact sperm differentiation. The present study clearly demonstrated that Khat extract has a pronounced effect on testicular maturation of developing albino rats at both the morphological and functional levels. Khat-treated groups revealed a significantly low serum testosterone level and severe impairment of spermatogenesis when compared with control animals. The current findings also verified, for the first time, that the final stages of sperm maturation (spermiogenesis) were strongly impaired after administration of Khat extract to experimental rats particularly at a higher dose (300 mg/kg body weight). This was proved by the very weak, if any, expression of protamine-1 in the maturing spermatids in Khat-treated rats.

PTPIP51-a myeloid lineage specific protein interacts with PTP1B in neutrophil granulocytes.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) was identified as an in vitro interacting partner of protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP). The full-length form of PTPIP51 encompasses 470aas and has a molecular weight of 52kDa. The physiological function is poorly understood but an involvement in differentiation processes and apoptosis has been suggested. Preliminary observations suggested differences in PTPIP51 expression in blood cells. To analyze a possible involvement of PTPIP51 in hematopoietic processes, we studied its expression in samples of peripheral venous blood (PVB), umbilical cord blood (UCB) and human bone marrow (HBM). In both, PVB and UCB PTPIP51 expression was restricted to neutrophil granulocytes. In HBM samples, besides in mature neutrophil ganulocytes PTPIP51 protein and mRNA was present in myeloid precursor cells of neutrophils. The expression of PTPIP51 in neutrophil granulocytes was corroborated by immunoblot analysis exhibiting different molecular weight forms of PTPIP51 protein. Anti-peptide antibodies, identifying specific regions of the PTPIP51 protein (C-terminus, N-terminus and aas114-129) revealed a distinct isoform expression pattern in neutrophil granulocytes of different sources. In PVB and UCB neutrophil granulocytes reacted positive for all three peptide antibodies. In contrast, neutrophils of HBM express solely an N-terminal variant of PTPIP51 protein, lacking the C-terminal and aas114-129 sequence. Immunocytochemical results displayed a strict co-localization of PTPIP51 and PTP1B in PVB and UCB. The interaction of both proteins was verified by a proximity ligation assay. Neither proliferating cells, as identified by PCNA immunostaining, nor apoptotic cells, labeled by TUNEL assay, displayed an immunoreactivity for PTPIP51 in HBM. In fact, PTPIP51 expression was restricted to myeloid precursor cells undergoing differentiation. In blood cells therefore, PTPIP51 expression is restricted to differentiating and mature neutrophil granulocytes.

The interaction of modified histones with the bromodomain testis-specific (BRDT) gene and its mRNA level in sperm of fertile donors and subfertile men.

As histone modifications have been suggested to be involved in the regulation of gene expression after fertilisation, the present study aimed to analyze the interaction between the bromodomain testis-specific (BRDT) gene and differentially modified histones in human spermatozoa. The BRDT transcript level was studied to identify possible correlations between epigenetic changes, mRNA level and subfertility associated with impaired sperm chromatin condensation. Chromatin immunoprecipitation (ChIP) was performed with ejaculates from fertile and subfertile men using antibodies against specifically acetylated and methylated histone H3. Immunoprecipitated DNA was analysed by real-time quantitative PCR with primer pairs for BRDT. The BRDT mRNA level was screened by real-time RT-PCR. ChIP assay revealed co-localisation of acetylated and methylated histones within promoter and exon regions of the BRDT gene in fertile men. Interestingly, reduced binding of investigated modified histone modifications was observed in the BRDT promoter of subfertile patients. Different mRNA levels of BRDT have been detected in a group of infertile patients, as well as in fertile men. Enrichment of methylated histones within the BRDT promoter of fertile sperm suggests that this epigenetic mark may cause repression of BRDT after fertilisation, and may be changed in infertile patients. Our data suggest that reduced histone methylation in the promoter of BRDT may be associated with increased transcript levels in subfertile patients.

Comparison of ART outcomes in men with altered mRNA protamine 1/protamine 2 ratio undergoing intracytoplasmic sperm injection with ejaculated and testicular spermatozoa.

Assisted reproductive technologies involving the use of spermatozoa and eggs for in vitro fertilization (IVF) have come as the solution for many infertile couples to become parents. However, in some cases, the use of ejaculated spermatozoa delivers poor IVF performance. Some studies have suggested the use of testicular spermatozoa in severe male infertility cases, but no guidelines regarding their utilization are currently available. In the present study, we found the mRNA protamine 1/protamine 2 (P1/P2) ratio to be a valuable biomarker of poor sperm function that could be used as a diagnostic key for the identification of cases that would benefit from the use of testicular spermatozoa. A total of 23 couples undergoing egg donation cycles with at least one previous cycle failure were studied. All couples underwent two consecutive intracytoplasmic sperm injection (ICSI) cycles with either ejaculated or testicular spermatozoa (TESA). The sperm mRNA P1/P2 ratio, fertilization rate, blastocyst rate, and pregnancy and live birth rate were compared. Results showed improved ICSI and clinical outcomes in cycles with testicular spermatozoa in men with altered mRNA P1/P2 ratios. TESA cycles presented significantly higher rates of fertilization (mean ± standard deviation: 76.1% ± 15.1% vs 65.5% ± 18.8%), blastocyst formation (55.0% ± 20.3% vs 30.8% ± 23.8%), and good morphological quality blastocyst (28.9% ± 22.9% vs 13.5% ± 17.9%) and also improvements on pregnancy (60.9% vs 0%) and healthy birth rates (56.5% vs 0%) than EJACULATE cycles. The results described here suggest that in patients with previous IVF/ICSI failures and aberrant mRNA protamine ratios, the use of testicular spermatozoa may be a good alternative to improve clinical outcomes.

Protamine-2 Deficiency Initiates a Reactive Oxygen Species (ROS)-Mediated Destruction Cascade during Epididymal Sperm Maturation in Mice.

Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Protamine-2 (Prm2)-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm's antioxidant capacity, indicated by downregulation of key proteins like Superoxide dismutase type 1 (SOD1) and Peroxiredoxin 5 (PRDX5), ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination.

PTPIP51 mRNA and protein expression in tissue microarrays and promoter methylation of benign prostate hyperplasia and prostate carcinoma.

BACKGROUND: Protein tyrosine phosphatase interacting protein 51 (PTPIP51) shows a tissue-specific expression pattern and is associated with cellular differentiation and apoptosis in several mammalian tissues. Overexpression of the full-length protein enhances apoptosis. It is also expressed in various carcinomas. In this study the expression of PTPIP51 and its in vitro interaction partners was investigated in human benign prostate hyperplasia (BPH) and in prostate carcinoma (PCa). METHODS: Tissue microarrays of human BPH and PCa were analyzed by immunohistochemistry. For polymerase chain reaction (PCR), cryo samples of BPH and PCa were used. Bisulfite DNA treatment, followed by sequencing of PCR products was performed in order to analyze CpGs methylation within the promoter region of the PTPIP51 gene. RESULTS: PTPIP51 mRNA and protein expression was detected in prostatic epithelia of BPH and in tumor cells of PCa, respectively, and within smooth muscle cells of the stromal compartment. A stronger expression was present in nerve fibers, particularly in PCa, in immune cells and in smooth muscle and endothelial cells of vessels of BPH and PCa. On mRNA levels, a slightly elevated expression of PTPIP51 was observed in the PCa group as tested by real-time quantitative PCR analyses. Methylation experiments revealed that at least 70% of methylated CpGs in the CpG island of the PTPIP51 gene promoter region were identified in BPH samples. In contrast, a loss of methylation has been found in the PCa group. CONCLUSION: The promoter methylation status of PTPIP51 seems to influence the expression of PTPIP51, which was seen as elevated in the PCa.