Testosterone positively regulates vagina NO-induced relaxation: an experimental study in rats.

PURPOSE: Female sexual response involves a complex interplay between neurophysiological mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and vagina. The aim of this study was to evaluate sex steroids regulation of the relaxant pathway in vagina, using a validated animal model. METHODS: Subgroups of OVX Sprague-Dawley rats were treated with 17ß-estradiol, testosterone, or testosterone and letrozole, and compared with a group of intact animals. Masson's trichrome staining was performed for morphological evaluation of the distal vaginal wall, in vitro contractility studies investigated the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR. RESULTS: Immunohistochemical analysis showed that OVX induced epithelial and smooth muscle structural atrophy, testosterone and testo + letrozole increased the muscle bundles content and organization without affecting the epithelium while 17ß-estradiol mediated the opposite effects. In vitro contractility studies were performed on noradrenaline pre-contracted vaginal strips from each experimental group. Acetylcholine (0.001-10 µM) stimulation induced a concentration-dependent relaxation, significantly reduced by NO-synthase inhibitor L-NAME and by guanylate cyclase inhibitor ODQ. OVX resulted in a decreased responsiveness to acetylcholine, restored by testosterone, with or without letrozole, but not by 17ß-estradiol. OVX sensitivity to the NO-donor SNP was higher than in the control. Vardenafil, a PDE5 inhibitor, enhanced SNP effect in OVX + testosterone as well as in control, as supported by RNA expression analysis. CONCLUSIONS: Our study demonstrates that testosterone improves the NO-mediated smooth muscle vaginal cells relaxation confirming its role in maintaining the integrity of muscular relaxant machinery.

Safety issues in semen banks during the COVID-19 pandemic: data from a European survey.

PURPOSE: We performed a survey among European semen banks enquiring safety protocols during the COVID-19 pandemic. We report the experience from a center searching SARS-CoV-2 mRNA in semen of patients undergoing cryopreservation from May 2020 to January 2021. METHODS: A questionnaire was submitted to accredited semen banks of the European Academy of Andrology (EAA) and the Italian Society of Andrology and Sexual Medicine (SIAMS). A total of 22 centers answered to the survey. SARS-CoV-2 mRNA in semen was evaluated by RT-PCR in 111 subjects banking in the Semen Bank of Careggi University Hospital (Florence, Italy). RESULTS: No particularly drastic safety measures were adopted by the majority of the centers to prevent the risk of contamination or transmission of the virus. The most common strategy (77.3%) was the administration of an anamnestic questionnaire. About half of the centers request a negative nasopharyngeal swab (NPS) before cryopreservation. Few centers use a quarantine tank, in case of late response of NPS, and only 4 store in a dedicated tank in case of infection. SARS-CoV-2 mRNA was not found in 111 semen samples cryopreserved in the Florentine bank. CONCLUSIONS: European semen banks use different measures to handle semen samples for cryopreservation during COVID-19 pandemic. The request of NPS is advised to better manage couples undergoing ART and to protect the personnel operating in the bank/ART center. Finally, due to the areas of uncertainties of an almost unknown virus, it is absolutely recommended the use of safe devices for sample handling and storage.

Cardiometabolic risk is unraveled by color Doppler ultrasound of the clitoral and uterine arteries in women consulting for sexual symptoms.

Female sexual dysfunction (FSD) may be a mirror of a poor cardiometabolic state. In a small pilot study enrolling 71 women with FSD, we previously demonstrated that clitoral Pulsatility Index (PI) evaluated by using color Doppler ultrasound (CDU), reflecting vascular resistance, was associated with cardiometabolic risk factors. Data on uterine CDU in this context are lacking. First, to confirm previously reported data on the direct association between clitoral PI and cardiometabolic risk factors on a larger study population of women consulting for sexual symptoms; second, to investigate eventual similar correlations between cardiometabolic risk factors and CDU parameters of the uterine artery. We also ascertained whether uterine artery PI, similarly to what had previously been observed for clitoral artery PI, was directly related to body image uneasiness and psychopathological symptoms, assessed by validated questionnaires. N = 230 women consulting our clinic for sexual symptoms were examined with clitoral CDU and blood sampling and were asked to fill out the Female Sexual Function Index, the Middlesex Hospital Questionnaire (MHQ) and the Body Uneasiness Test (BUT). In a subgroup of women (n = 164), we also performed transvaginal CDU with measurement of uterine artery parameters. At multivariate analysis, we found a direct association between clitoral PI and body mass index (BMI) (p = 0.004), waist circumference (WC) (p = 0.004), triglycerides (p = 0.006), insulin (p = 0.029) and HOMA-IR (p = 0.009). Furthermore, a correlation between obesity and Metabolic Syndrome (MetS) and a higher clitoral PI was observed (p = 0.003 and p = 0.012, respectively). Clitoral PI was also correlated with MHQ-S (p = 0.010), a scale exploring somatized anxiety symptoms, and BUT-B Positive Symptom Distress Index (p = 0.010), a measure of body image concerns. Similarly, when investigating the uterine artery, we were able to demonstrate an association between its PI and BMI (p < 0.0001), WC (p = 0.001), insulin (p = 0.006), glycated haemoglobin (p = < 0.0001), and HOMA-IR (p = 0.009). Women diagnosed with obesity and MetS showed significantly higher uterine PI values vs. those without obesity or MetS (p = 0.001 and p = 0.004, respectively). Finally, uterine PI was associated with BUT-A Global Severity Index (p < 0.0001) and with several other BUT-A subdomains. Vascular resistance of clitoral and uterine arteries is associated with cardiometabolic risk factors and body image concerns in women consulting for sexual symptoms. If further confirmed in different populations, our data could suggest CDU, a common examination method, as a useful tool for an identification-and possible correction-of cardiometabolic risk factors.

Semen impairment and occurrence of SARS-CoV-2 virus in semen after recovery from COVID-19.

STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection? SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen. WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants. STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method. MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2. LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study. WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

LH supplementation of ovarian stimulation protocols influences follicular fluid steroid composition contributing to the improvement of ovarian response in poor responder women.

In this prospective study, we evaluated the steroid levels in 111 follicular fluids (FF) collected from 13 women stimulated with FSH monotherapy and 205 FF collected from 28 women stimulated with FSH + LH because of a previous history of hypo-responsiveness to FSH. Steroid levels were measured by HPLC/MS-MS and related to ovarian stimulation protocol, oocyte maturity, fertilization and quality of blastocysts, after individually tracking the fate of all retrieved oocytes. 17-Hydroxy-Progesterone, Androstenedione, Estradiol and Estrone were significantly higher in the FSH + LH protocol. Progesterone, 17-Hydroxy-Progesterone and Estradiol were more expressed in FF yielding a mature oocyte (p < 0.01) in the FSH + LH protocol. FF Progesterone concentration was correlated with the rate of normal fertilization in the FSH protocol. None of the FF steroids measured were associated with blastocyst quality and achievement of pregnancy. Our results indicate that LH supplementation in hypo-responsive women modifies ovarian steroid production, mimicking physiological production better and likely contributing to an improved ovarian response. Employing a correct methodological procedure to evaluate the relationship between FF steroid hormones and assisted reproduction outcomes, our study reveals that some steroids in single follicles may be helpful in predicting oocyte maturity and fertilization.

Acute effects on human sperm exposed in vitro to cadmium chloride and diisobutyl phthalate.

Epidemiological studies reported a negative relationship between concentrations of heavy metals and phthalates in seminal fluid and semen quality, likely compromising male fertility potential. The aim of this study was to investigate the in vitro effects of cadmium chloride (CdCl2), a common heavy metal, and diisobutyl phthalate (DIBP), a common phthalate ester, on human sperm functions necessary for fertilization. After in vitro incubation of spermatozoa with 10 µM CdCl2 or 100 and 200 µM DIBP for 24 h, a significant decrease of sperm progressive and hyperactivated motility was observed. The exposure to each of the two toxic agents also induced spontaneous sperm acrosome reaction and blunted the physiological response to progesterone. Both agents induced an increase of caspase activity suggesting triggering of an apoptotic pathway. Our results suggest that acute exposure of spermatozoa to these pollutants may impair sperm ability to reach and fertilize the oocyte.

Chromatin Protamination and Catsper Expression in Spermatozoa Predict Clinical Outcomes after Assisted Reproduction Programs.

Identification of parameters predicting assisted reproductive technologies (ARTs) success is a major goal of research in reproduction. Quality of gametes is essential to achieve good quality embryos and increase the success of ARTs. We evaluated two sperm parameters, chromatin maturity and expression of the sperm specific calcium channel CATSPER, in relation to ART outcomes in 206 couples undergoing ARTs. Chromatin maturity was evaluated by Chromomycin A3 (CMA3) for protamination and Aniline Blue (AB) for histone persistence and CATSPER expression by a flow cytometric method. CMA3 positivity and CATSPER expression significantly predicted the attainment of good quality embryos with an OR of 6.6 and 14.3 respectively, whereas AB staining was correlated with fertilization rate. In the subgroup of couples with women ≤35 years, CATSPER also predicted achievement of clinical pregnancy (OR = 4.4). Including CMA3, CATSPER and other parameters affecting ART outcomes (female age, female factor and number of MII oocytes), a model that resulted able to predict good embryo quality with high accuracy was developed. CMA3 staining and CATSPER expression may be considered two applicable tools to predict ART success and useful for couple counseling. This is the first study demonstrating a role of CATSPER expression in embryo development after ARTs programs.

Quantification of CatSper1 expression in human spermatozoa and relation to functional parameters.

STUDY QUESTION: Is CatSper1 expression in human spermatozoa related to semen parameter values and sperm functions? SUMMARY ANSWER: CatSper1 expression is positively related to progressive and hyperactivated (HA) motility, [Ca(2+)]i responsiveness to progesterone but not the acrosome reaction (AR). WHAT IS KNOWN ALREADY: The role of cationic channel of sperm (CatSper) in sperm functions is clear in animal models but less defined in human sperm cells. Current knowledge is mostly based on low specificity CatSper inhibitors showing agonistic and toxic effects on human spermatozoa and is thus of little help in clarifying the role of the CatSper channel in human sperm functions. STUDY DESIGN, SIZE, DURATION: CatSper1 protein expression was evaluated in 115 men undergoing semen analysis for couple infertility. CatSper1 expression was related to routine semen parameters, motility kinematic parameters and basal and progesterone-stimulated [Ca(2+)]i and the AR. PARTICIPANTS/MATERIALS, SETTING, METHODS: CatSper1 expression was evaluated (n = 85 normozoospermic, n = 30 asthenozoospermic patients) by immunofluorescence coupled to flow cytometry leading to quantitative measurement of the percentage of ejaculated sperm cells expressing the protein. Semen analysis was evaluated according to World Health Organization guidelines. Kinematic parameters were evaluated by a computer-aided sperm analysis system. [Ca(2+)]i was measured by a spectrofluorimetric method in fura-2-loaded spermatozoa. The AR was evaluated in live sperm cells by fluorescent-labeled lectin. MAIN RESULTS AND THE ROLE OF CHANCE: CatSper1 protein expression in spermatozoa was reduced in asthenozoospermic men (mean ± SD: 53.0 ± 15.5%, n = 30 versus 67.9 ± 17.1% in normozoospermic, n = 85, P < 0.01) and was significantly correlated with progressive (r = 0.36, P < 0.001), total (r = 0.35, P < 0.001) and HA (r = 0.41, P < 0.005) motility. In addition to a higher percentage of spermatozoa not expressing CatSper1, asthenozoospermic men showed a large number of spermatozoa with immunofluorescent signal localized outside the principal piece compared with those in normozoospermia. A significant positive correlation was found between CatSper1 protein expression and the increase of [Ca(2+)]i in response to progesterone (r = 0.36, P < 0.05, n = 40) but not with basal [Ca(2+)]i. No correlation was found with the AR, either basal or in response to progesterone. LIMITATIONS, REASONS FOR CAUTION: The study is partly descriptive. Furthermore, we cannot rule out the possibility that some round cells remain after a single round of 40% density gradient centrifugation or that this step may have removed some defective or slow swimming sperm, and therefore this preparation may not be representative of the entire sperm sample. Although our data suggest that CatSper1 may be a useful marker for infertility, and a possible contraceptive target, any clinical application is limited without further research. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate an association of CatSper1 expression with human sperm progressive and HA motility and provide preliminary evidence that lack of expression or mislocalization of CatSper1 in spermatozoa may be involved in the pathogenesis of asthenozoospermia. However, mechanistic studies are needed to confirm that the correlations between CatSper1 expression and sperm functions are causative. STUDY FUNDING/COMPETING INTERESTS: Supported by grants from Ministry of University and Scientific Research (PRIN project to E.B. and FIRB project to S.M.) and by Regione Toscana (to G.F.). L.T. was recipient of a grant from Accademia dei Lincei (Rome, Italy). The authors have no conflicts of interest to declare.

SUMO1 in human sperm: new targets, role in motility and morphology and relationship with DNA damage.

In studies carried out previously, we demonstrated that small ubiquitin-like modifier 1 (SUMO1) is associated with poor sperm motility when evaluated with a protocol that reveals mostly SUMO1-ylated live sperm. Recently, with another protocol, it has been demonstrated that SUMO is expressed in most sperm and is related to poor morphology and motility, suggesting that sumoylation may have multiple roles depending on its localisation and targets. We show herein, by confocal microscopy and co-immunoprecipitation, that dynamin-related protein 1 (DRP1), Ran GTPase-activating protein 1 (RanGAP1) and Topoisomerase IIα, SUMO1 targets in somatic and/or germ cells, are SUMO1-ylated in mature human spermatozoa. DRP1 co-localises with SUMO1 in the mid-piece, whereas RanGAP1 and Topoisomerase IIα in the post-acrosomal region of the head. Both SUMO1 expression and co-localisation with the three proteins were significantly higher in morphologically abnormal sperm, suggesting that sumoylation represents a marker of defective sperm. DRP1 sumoylation at the mid-piece level was higher in the sperm of asthenospermic men. As in somatic cells, DRP1 sumoylation is associated with mitochondrial alterations, this protein may represent the link between SUMO and poor motility. As SUMO pathways are involved in responses to DNA damage, another aim of our study was to investigate the relationship between sumoylation and sperm DNA fragmentation (SDF). By flow cytometry, we demonstrated that SUMO1-ylation and SDF are correlated (r=0.4, P<0.02, n=37) and most sumoylated sperm shows DNA damage in co-localisation analysis. When SDF was induced by stressful conditions (freezing and thawing and oxidative stress), SUMO1-ylation increased. Following freezing and thawing, SUMO1-Topoisomerase IIα co-localisation and co-immunoprecipitation increased, suggesting an involvement in the formation/repair of DNA breakage.

Characterization and sorting of flow cytometric populations in human semen.

Human semen is a complex biological matrix. It contains mature spermatozoa, immature germ cells, residual apoptotic bodies and, in some cases, epithelial cells and leucocytes. Hence, one of the challenges in applying flow cytometry in spermatology is the correct recognition of spermatozoa and their separation from signals of other semen cells/elements. In this study, we show that semen spermatozoa are included in a well-defined, flame-shaped FSC/SSC region (FR), by demonstrating that the count of the spermatozoa contained in such region overlaps that obtained by microscopy in the same samples. In FR, nuclear staining of semen samples reveals three different populations: unstained, brighter and dimmer. Unstained elements were previously characterized as apoptotic bodies of testis origin and the brighter elements represent the majority of semen spermatozoa, whereas the composition and the origin of the population with a lower nuclear staining is less clear, albeit we have previously shown that all the elements constituting it are positive for TUNEL. In this study, we sorted all the elements contained in FR region and demonstrated that the dimmer elements are spermatozoa. To further characterize dimmer spermatozoa, we evaluated apoptotic caspases and chromatin immaturity, the latter detected by aniline blue (AB) and chromomycin A (CMA3) staining. We found that caspases were much more expressed in the dimmer spermatozoa (71.4 ± 18.8%) than in the brighter (46.7 ± 15.1%), whereas similar amounts of spermatozoa with chromatin immaturity were found in both populations (brighter, AB: 48.2 ± 19.5%; CMA3: 48.5 ± 20.4% and dimmer, AB: 43.4 ± 19.8%; CMA3: 36.1 ± 18.0%). Hence, the role of apoptosis in generating dimmer spermatozoa and their DNA fragmentation appears clear, whereas the involvement of defects during the chromatin packaging remains elusive.

Development of a specific method to evaluate 8-hydroxy, 2-deoxyguanosine in sperm nuclei: relationship with semen quality in a cohort of 94 subjects.

Oxidative stress (OS) is involved in many disoders including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most sperm functions are impaired in the case of OS. In addition unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, probably due to the different methods and/or targets used to reveal the OS. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei they yielded different measures, and only with the immunofluorescence method was the labelling specific for sperm 8-OHdG. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-OHdG content in semen samples from 94 subfertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-OHdG (mean±s.d., 11.4±6.9%) were related to sperm count (Pearson's correlation coefficient (r)=-0.27, P=0.04 (ANOVA and student's t-test)), motility (progressive: r=-0.22, P=0.04; non-progressive: r=0.25, P=0.01), and normal morphology (r=-0.27, P=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-OHdG at single-cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on male fertility with respect to routine semen analysis.

Semen apoptotic M540 body levels correlate with testis abnormalities: a study in a cohort of infertile subjects.

STUDY QUESTION: What are the associations between semen apoptotic M540 bodies and other parameters of semen quality and sonographic alterations of the male genital tract in a cohort of infertile subjects? SUMMARY ANSWER: In infertile subjects, semen M450 bodies are highly correlated with ultrasound and clinical signs of testis abnormalities but not with alterations of other parts of the male genital tract, suggesting a testicular origin of M540 bodies. WHAT IS KNOWN ALREADY: We have reported the presence in semen of round anucleate elements, named 'M540 bodies', resembling apoptotic bodies as they contain several apoptotic markers. STUDY DESIGN AND SIZE: A consecutive series of 130 males with couple infertility were evaluated, during the same day session, for clinical, scrotal and transrectal color-Doppler ultrasound characteristics, and hormonal and semen parameters, including interleukin 8 (sIL-8) and M540 body levels. PARTICIPANTS/MATERIALS, SETTING METHODS: Semen parameters were analyzed by WHO recommended procedures. CDU was performed using the ultrasonographic console Hitachi H21. sIL-8 and serum hormones were evaluated by ELISA methods. MAIN RESULTS AND THE ROLE OF CHANCE: The average percentage value of M540 bodies was 24.6 ± 18.3. After adjusting for possible confounders (age, waist, calculated free testosterone and smoking habit), M450 body levels negatively correlated with sperm number/ejaculate, progressive motility, normal morphology and sIL-8 levels (adj.r = -0.455, P < 0.0001; adj.r = -0.464, P < 0.0001; adj.r = -0.430, P < 0.001; adj.r = -0.236, P < 0.05, respectively). In a subset of patients with a history of cryptorchidism (n = 8), M540 bodies were higher than in non-cryptorchid men (40.5 ± 14.8 versus 23.6 ± 18.2%; P < 0.02). A negative correlation was found between M540 and ultrasound testis volume (adj.r = -0.241, P < 0.05), whereas a positive association was found with testis inhomogeneity [HR = 1.06 (1.02-1.09); P = 0.002], hypoechogenicity [HR = 1.05 (1.01-1.08); P < 0.02] and FSH levels (adj.r = 0.309, P < 0.01). No relationships were found with CDU characteristic of the prostate, seminal vesicles, epididymis and vas deferens. In a multivariate model, testis inhomogeneity and history of cryptorchidism were independently associated with M540 body levels (adj.r = 0.355, P < 0.01 and adj.r = 0.223, P < 0.05, respectively). Receiver operating characteristic analysis demonstrated that at the threshold of 27%, M540 bodies discriminate subjects with testis inhomogeneity with a sensitivity of 72% and specificity of 73%. LIMITATIONS, REASONS FOR CAUTION: The increased M540 body semen levels in men with a history of cryptorchidism should be confirmed in a larger number of patients. WIDER IMPLICATIONS OF THE FINDINGS: M540 bodies may be considered a semen marker of altered testis function and thus their evaluation may be helpful in the diagnosis of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Ministry of University and Scientific Research (Prin project to E.B. and FIRB project to S.M.) and Regione Toscana (to G.F.).

Sumo1-ylation of human spermatozoa and its relationship with semen quality.

Sumoylation is a post-translational modification involved in the regulation of several cell functions. Recent studies suggest its involvement in spermatogenesis, but occurrence and function of SUMO (small ubiquitin-like modifier) in mature spermatozoa remain unknown. We report the occurrence of several SUMO1-conjugated proteins, in a range of 20-85 kDa, in ejaculated spermatozoa. By cytofluorimetric analysis, we evaluated the percentage of SUMO1-positive spermatozoa in 58 subjects undergoing semen analysis in our laboratory and correlated the obtained values with semen parameters. We found that the percentage of SUMO1-positive spermatozoa was inversely correlated with total (r = -0.35, p < 0.01) and progressive motility (r = -0.29, p < 0.05). Such correlations become stricter when only asthenospermic subjects were included in the analysis (r = -0.58, p = 0.01 for progressive motility, n = 17) and were lost in non-asthenospermic subjects. By immunofluorescence and immunoconfocal fluorescence, we demonstrated that SUMO1 is mainly located in the nucleus and, occasionally, in the midpiece of spermatozoa. Immunoelectron microscopy as well as a long permeabilization protocol demonstrated a massive localization of SUMO-1 in the nucleus. By using a fluorescent probe to distinguish dead/live cells, we show that SUMO1 is mainly present in live spermatozoa. In conclusion, sumoylation of human spermatozoa may be involved in the regulation of motility.

[Fragmentation and/or decondensation of spermatic DNA: which consequences?]. / Fragmentation et/ou décondensation de l'ADN spermatique: quelles conséquences?

Absence of DNA fragmentation and/or decondensation is a marker of sperm quality and is related to outcome of assisted reproductive techniques: new tests have been set up to determine fragmentation rate.

Androgen-responsive and -unresponsive prostate cancer cell lines respond differently to stimuli inducing neuroendocrine differentiation.

The treatment of advanced prostate cancer (CaP) with androgen deprivation therapy inevitably renders the tumours castration resistant and incurable. Under these conditions, neuroendocrine differentiation (NED) of CaP cells occurs and neuropeptides released by neuroendocrine cells facilitate tumour progression. Pharmacological strategies aiming to prevent or delay NED during androgen ablation could, therefore, increase the effectiveness of the therapy. Mechanisms and pathways inducing NED in CaP are poorly understood and data are often discordant. In the present study, we used several CaP cell lines (androgen-responsive: LNCaP, PC3-AR, 22RV1 and -irresponsive: DU145 and PC3) to evaluate NED after androgen deprivation or treatment with epidermal growth factor (EGF). NED was determined by neuron-specific enolase and chromogranin A expression and by the occurrence of morphological changes in the cells. Androgen-deprivation conditions induced NED in LNCaP and PC3-AR, but not in 22Rv1, PC3 and DU145 cells. LNCaP and PC3-AR cells also became resistant to thapsigargin-induced apoptosis. In all the AR-positive cell lines, androgen deprivation caused a decrease in androgen receptor expression indicating that it is downregulated irrespective of NED induction. Treatment with EGF induced NED in DU145 cells and the EGF receptor inhibitor gefinitib prevented the process. On the contrary, no effect of EGF was demonstrated in LNCaP or 22Rv1 cells. CaP cell lines did not respond univocally to treatments inducing NED, suggesting that studies on this topic should be performed in a wide spectrum of cell models which can be more indicative of the tumour variability in vivo.

Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters.

BACKGROUND: Sperm DNA fragmentation is a possible predictive parameter for male fertility status. The occurrence of M540 bodies in semen of subfertile subjects affects flow cytometric investigations in sperm. We set up a new method to evaluate DNA fragmentation excluding M540 bodies. METHODS: DNA fragmentation was evaluated by flow cytometry in semen of 75 subjects both by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL, traditional method) and by double staining with TUNEL and propidium iodide (PI, new method). RESULTS: The use of the new method revealed that TUNEL underestimates sperm DNA fragmentation in flow cytometry and showed two sperm populations stained with low (PI(dim)) and high (PI(br)) avidity for PI. The PI(dim) population is entirely composed of DNA fragmented sperm and its incidence shows highly significant negative correlations with morphology, motility, sperm count and concentration (respectively, r = -0.51, -0.52, -0.46 and -0.32, n = 75). DNA fragmentation in the PI(br) sperm population is independent from semen quality. CONCLUSIONS: The correlations between sperm DNA breakage and semen quality previously reported are mainly driven by the occurrence of the PI(dim) population. DNA fragmented sperm in this population are more likely to have poorer morphology, reduced motility and thus a reduced chance to fertilize an oocyte than DNA damaged sperm in PI(br) population. Distinguishing between the two types of sperm DNA fragmentation appears to be important in clinical investigations.

Characterization of M540 bodies in human semen: evidence that they are apoptotic bodies.

Our group has recently identified, in human semen, round bodies of different size and density which were termed M540 bodies due to their staining with the fluorochrome merocyanine 540. Here, we investigate the hypothesis that such structures represent apoptotic bodies. To this aim, by both fluorescence-activated cell sorting (FACS) and fluorescence microscopy, we examined the occurrence of apoptotic markers such as caspase activity, Fas, p53 and Bcl-x in M540 bodies. In addition, we evaluated their ultrastructure by transmission electron microscopy. We found that M540 bodies express all the investigated markers, strongly supporting our hypothesis. We also found that M540 bodies contain fragmented DNA, another evidence of their apoptotic derivation. We investigated also the presence of M540 bodies in the different categories of patients. With respect to normozoospermic subjects, a higher content of M540 bodies was found in oligoasthenoteratozoospermic and asthenoteratozoospermic, but not in asthenozoospermic and teratozoospermic men. Interestingly, these subjects are those whose semen shows the highest levels of apoptotic signs. The variable occurrence of apoptotic bodies in semen may thus be considered a sign of abortive apoptosis in male reproductive organs. Of interest, since M540 bodies exhibit a similar size and density to sperm, they represent a confounding factor in FACS studies on ejaculated sperm.

Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate.

Mammalian testicular spermatozoa are immotile, thus, to reach the oocyte, they need to acquire swimming ability under the control of different factors acting during the sperm transit through the epididymis and the female genital tract. Although bicarbonate is known to physiologically increase motility by stimulating soluble adenylate cyclase (sAC) activity of mammalian spermatozoa, no extensive studies in human sperm have been performed yet to elucidate the additional molecular mechanisms involved. In this light, we investigated the effect of in vitro addition of bicarbonate to human spermatozoa on the main intracellular signaling pathways involved in regulation of motility, namely, intracellular cAMP production and protein tyrosine phosphorylation. Bicarbonate effects were compared with those of the phosphatidyl-inositol-3 kinase inhibitor, LY294002, previously demonstrated to be a pharmacological stimulus for sperm motility. Bicarbonate addition to spermatozoa results in a significant increase in sperm motility as well as in several hyperactivation parameters. This stimulatory effect of bicarbonate and LY294002 is mediated by an increase in cAMP production and tyrosine phosphorylation of the A kinase anchoring protein, AKAP3. The specificity of bicarbonate effects was confirmed by inhibition with 4,4'-di-isothiocyanostilbene-2,2'-disulfonic acid. We remark that, in human spermatozoa, bicarbonate acts primarily through activation of sAC to stimulate tyrosine phosphorylation of AKAP3 and sperm motility because both effects are blunted by the sAC inhibitor 2OH-estradiol. In conclusion, our data provide the first evidence that bicarbonate stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, finally leading to an increased recruitment of PKA to AKAP3.

The androgen receptor associates with the epidermal growth factor receptor in androgen-sensitive prostate cancer cells.

Many recent evidences indicate that androgen-sensitive prostate cancer cells have a lower malignant phenotype that is in particular characterized by a reduced migration and invasion. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with the synthetic androgen R1881 further reduced invasion of the cells without, however, modifying alpha6beta4 expression on the cell surface, suggesting an interference with the invasion process in response to EGF. We investigated whether the presence of the AR could affect EGF receptor (EGFR)-mediated signaling in response to EGF by evaluating autotransphosphorylation of the receptor as well as activation of downstream signalling pathways. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. An interaction between EGFR and AR has been demonstrated by immunoconfocal and co-immunoprecipitation analysis in PC3-AR cells, suggesting a possible interference of AR on EGFR signalling by interaction of the two proteins. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signalling in response to EGF leading to invasion through a mechanism involving an interaction between AR and EGFR.

Signaling mechanisms that mediate invasion in prostate cancer cells.

Recent evidence indicates that androgen-sensitive prostate cancer cells have a less malignant phenotype characterized by reduced migration and invasion. We investigated whether the presence of the androgen receptor could affect EGFR-mediated signaling by evaluating autotransphosphorylation of the receptor as well as activation of the downstream signaling pathway PI3K/AKT. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. Our results suggest that the expression of androgen receptors by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signaling leading to invasion in response to EGF. We used the selective tyrosine kinase inhibitor of the EGFR gefitinib (also known as Iressa or ZD1839) to further investigate the role of EGFR in the invasion and growth of PC cells. We demonstrate that in the androgen-insensitive cell lines PC3 and DU145 this compound was able to decrease in vitro invasion of Matrigel by inhibiting EGFR autotransphosphorylation and subsequent PI3K activation. Gefitinib may be useful in the treatment of androgen-independent prostate cancer to limit not only the proliferation but also the invasion of these tumors.