Glucose prevents the acquisition of the capacitated state in pig spermatozoa.

BACKGROUND: Mammalian spermatozoa need to undergo a process named capacitation to be able to fertilize an oocyte. During their journey in the female tract, spermatozoa obtain energy while exposed to a changing environment containing a variety of metabolic substrates. The energy requirements for sperm capacitation are species-specific. In addition, the available energy source can hinder the process of sperm capacitation and eventually the acrosome reaction. OBJECTIVES: To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process. MATERIAL AND METHODS: The effect of different metabolic substrates on sperm capacitation process was evaluated by analyzing phosphorylation in the p32 protein; the acrosome reaction and the ATP intracellular content. RESULTS: The presence of glucose in the in vitro capacitating medium diminishes, in a concentration-dependent manner, parameters associated with the capacitated status: induced acrosome exocytosis, plasma membrane destabilization, and protein tyrosine phosphorylation. Conversely, sperm incubation with pyruvate or lactate, either individually or in combination, allows the attainment of the capacitated status. Unexpectedly, pig spermatozoa incubated without any extracellular energy substrates or with a non-metabolizable substrate (l-glucose) for 4 h displayed similar sperm viability to the control and exhibited a capacitated phenotype. The capacitation-like phenotype observed in starved pig spermatozoa (absence of glucose, lactate, and pyruvate) was dependent on extracellular bicarbonate and calcium levels, and these spermatozoa exhibited lower intracellular ATP content compared to those not capacitated. Nevertheless, the intracellular content of calcium was not modified in comparison to the control. DISCUSSION AND CONCLUSIONS: Our findings suggest that the metabolic substrates used to fuel pig sperm metabolism are important in achieving the capacitated status. The results of this work could be used to refine the capacitating medium employed in pig in vitro fertilization.

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

BACKGROUND: Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. METHODS: Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. RESULTS: Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45 min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol = 120.9 ± 2.9 vs. NC = 76.91 ± 6.9 µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol = 1.15 ± 0.02 vs. NC = 0.77 ± 0.03 µm) (straightness: bicarbonate and polyvinyl-alcohol = 0.76 ± 0.01 vs. NC = 0.87 ± 0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45 min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). CONCLUSION: These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

Active surveillance for clinical complete responders after chemoradiotherapy for oesophageal squamous cell carcinoma.

BACKGROUND: Guidelines on the treatment of oesophageal squamous cell carcinoma (SCC) recommend neoadjuvant chemoradiotherapy plus surgery or definitive chemoradiotherapy. The aim of this study was to evaluate the outcome of patients with a cCR after chemoradiotherapy who underwent active surveillance. METHODS: Patients with oesophageal SCC who were treated with chemoradiotherapy between January 2016 and June 2022 were identified from an institutional database. Survival and recurrence of patients with a cCR who underwent active surveillance were compared with those of patients who underwent planned surgery. Survival was calculated according to the Kaplan-Meier method and compared between groups using the log rank test. RESULTS: The 37 patients who underwent active surveillance were older and tumours were more often located in the middle/upper-third of the oesophagus than in the surgery group of 57 patients. Median follow-up was 28.1 (i.q.r. 17.2-47.1) months for the active surveillance group and 20 (12.9-39.1) months for the surgery group. Overall survival was comparable between the two groups, with 3-year survival rates of 50 (95% c.i. 31 to 67) and 59 (40 to 73)% for the active surveillance and surgery groups respectively (P = 0.55). Three-year progression-free survival for patients who underwent active surveillance was better than in the surgery group: 70 (43 to 85) versus 58 (40 to 72)% (P = 0.02). Overall and progression-free survival was comparable between patients in the active surveillance group and 23 patients in the surgery group who had a pCR (ypT0 N0). The overall recurrence rate was comparable between the groups: 7 of 37 (19.4%) in active surveillance group versus 16 of 49 (32.6%) in surgery group (P = 0.26). Locoregional recurrence was noted more often in the active surveillance group and systemic recurrence in the surgery group. CONCLUSION: Active surveillance is feasible and safe for patients with oesophageal SCC who have a cCR after chemoradiotherapy.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Regulatory T cells play a crucial role in maintaining sperm tolerance and male fertility.

Regulatory T cells (Tregs) modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, we show that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. We found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of antisperm antibodies (ASA). In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects. Deciphering these immunoregulatory mechanisms may help to design new strategies to treat male infertility, as well as to identify potential targets for immunocontraception.

Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways.

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites.

The "Weekday Effect" on Enhanced Recovery after Surgery Protocol for Gastrectomy.

INTRODUCTION: While enhanced recovery after surgery (ERAS) protocol demonstrated to improve outcomes after gastrectomy, some papers evidenced a detrimental effect on postoperative morbidity related to the "weekday effect." We aimed to understand whether the day of gastrectomy could affect postoperative outcomes and compliance with ERAS items. METHODS: We included all patients that underwent gastrectomy for cancer between January 2017 and September 2021. Cohort was divided considering the day of surgery: Early group (Monday-Wednesday) and Late group (Thursday-Friday). Compliance with protocol and postoperative outcomes were compared. RESULTS: Two hundred twenty-seven patients were included in Early group, while 154 were in Late group. The groups were comparable in preoperative characteristics. No significant difference in pre/intraoperative and postoperative ERAS items' compliance was apparent between Early and Late groups, with most items exceeding the 70% threshold. Median length of stay was 6.5 days and 6 days in Early and Late groups (p = 0.616), respectively. Morbidity was 50% in both groups, with severe complications that occurred in 13% of Early patients and 15% of Late patients. Ninety-day mortality was 2%, and it was similar between the two groups. CONCLUSIONS: In a center with a standardized ERAS protocol, the weekday of gastrectomy has no significant impact on the success of each ERAS item and on postoperative surgical and oncological outcomes.

Are bacteria, fungi, and archaea present in the midtrimester amniotic fluid?

OBJECTIVES: This study was conducted to determine whether bacteria, fungi, or archaea are detected in the amniotic fluid of patients who underwent midtrimester amniocentesis for clinical indications. METHODS: Amniotic fluid samples from 692 pregnancies were tested by using a combination of culture and end-point polymerase chain reaction (PCR) techniques. Intra-amniotic inflammation was defined as an interleukin-6 concentration >2,935 pg/mL. RESULTS: Microorganisms were detected in 0.3% (2/692) of cases based on cultivation, 1.73% (12/692) based on broad-range end-point PCR, and 2% (14/692) based on the combination of both methods. However, most (13/14) of these cases did not have evidence of intra-amniotic inflammation and delivered at term. Therefore, a positive culture or end-point PCR in most patients appears to have no apparent clinical significance. CONCLUSIONS: Amniotic fluid in the midtrimester of pregnancy generally does not contain bacteria, fungi, or archaea. Interpretation of amniotic fluid culture and molecular microbiologic results is aided by the assessment of the inflammatory state of the amniotic cavity. The presence of microorganisms, as determined by culture or a microbial signal in the absence of intra-amniotic inflammation, appears to be a benign condition.

Effects of preconception exposure to phthalates on mouse sperm capacitation parameters.

BACKGROUND: Phthalates have been linked to adverse male reproductive health, including poor sperm quality and embryo quality as well as a longer time to pregnancy (months of unprotected intercourse before conception occurs). The present study aimed to evaluate the effect of preconception exposure to two ubiquitous phthalate chemicals, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and their mixture on sperm function, fertilization, and embryo development in mice. MATERIALS AND METHODS: Adult male C57BL/6J mice aged 8-9 weeks were exposed to di(2-ethylhexyl) phthalate, di-n-butyl phthalate, or their mixture (di-n-butyl phthalate + di(2-ethylhexyl) phthalate) at 2.5 mg/kg/day or vehicle for 40 days (equivalent to one spermatogenic cycle) via surgically implanted osmotic pumps. Caudal epididymal spermatozoa were extracted and analyzed for motility using computer-assisted sperm analyses. Sperm phosphorylation of protein kinase A substrates and tyrosine phosphorylation, markers of early and late capacitation events, respectively, were analyzed by Western blots. In vitro fertilization was used to evaluate the sperm fertilizing capacity. RESULTS: While the study did not reveal any significant differences in sperm motility and fertilization potential, abnormal sperm morphology was observed in all phthalate exposures, particularly in the phthalate mixture group. In addition, the study revealed significant differences in sperm concentration between control and exposed groups. Moreover, protein phosphorylation of protein kinase A substrates was decreased in the di(2-ethylhexyl) phthalate and mixture exposure groups, while no significant changes in protein tyrosine phosphorylation were observed in any of the groups. Assessment of the reproductive functionality did not reveal significant effects on in vitro fertilization and early embryo development rates but showed wide variability in the phthalate mixture group. CONCLUSION: Our findings suggest that preconception phthalate exposure affects sperm numbers and phosphorylation of protein kinase A substrates involved in capacitation. Future research is warranted to examine the associations between phthalate exposure and capacitation in human spermatozoa.

Robotic versus open Ivor-Lewis esophagectomy: A more accurate lymph node dissection without burdening the leak rate.

BACKGROUND: Robotic-assisted minimally invasive esophagectomy (RAMIE) combines the beneficial effects of minimally invasive surgery on postoperative complications, especially on pulmonary ones, with the safety of the anastomosis performed in open surgery. Moreover, RAMIE could allow a more accurate lymphadenectomy. METHODS: We reviewed our database to identify all patients with adenocarcinoma of the esophagus treated by Ivor-Lewis esophagectomy in the period January 2014 to June 2022. Patients were divided according to the thoracic approach into RAMIE and open esophagectomy (OE) groups. We compared the groups for early surgical outcomes, 90-day mortality as well as R0 rate, and the number of lymph nodes harvested. RESULTS: We identified 47 patients in RAMIE and 159 patients in the OE group. Baseline characteristics were comparable. Operative time was significantly longer for RAMIE procedures (p < 0.01); however, we did not observe the difference in overall (RAMIE 55.5% vs. OE 61%, p = 0.76) and severe complications rate (RAMIE 17% vs. OE 22.6%, p = 0.4). The anastomotic leak rate was 2.1% after RAMIE and 6.9% after OE (p = 0.56). We did not report the difference in 90-day mortality (RAMIE 2.1% vs. OE 1.9%, p = 0.65). In the RAMIE group, we observed a significantly higher number of thoracic lymph nodes harvested, with a median of 10 lymph nodes in the RAMIE group versus 8 in the OE group (p < 0.01). CONCLUSIONS: In our experience, RAMIE has morbimortality rates comparable to OE. Moreover, it allows a more accurate thoracic lymphadenectomy which results in a higher thoracic lymph nodes retrieval rate.

Prenatal Diagnosis and Fetopsy Validation of Complete Atrioventricular Septal Defects Using the Fetal Intelligent Navigation Echocardiography Method.

(1) Background: Artificial Intelligence (AI) is a modern tool with numerous applications in the medical field. The case series reported here aimed to investigate the diagnostic performance of the fetal intelligent navigation echocardiography (FINE) method applied for the first time in the prenatal identification of atrioventricular septal defects (AVSD). This congenital heart disease (CHD) is associated with extracardiac anomalies and chromosomal abnormalities. Therefore, an early diagnosis is essential to advise parents and make adequate treatment decisions. (2) Methods: Four fetuses diagnosed with AVSD via two-dimensional (2D) ultrasound examination in the second trimester were enrolled. In all cases, the parents chose to terminate the pregnancy. Since the diagnosis of AVSD with 2D ultrasound may be missed, one or more four-dimensional (4D) spatiotemporal image correlation (STIC) volume datasets were obtained from a four-chamber view. The manual navigation enabled by the software is time-consuming and highly operator-dependent. (3) Results: FINE was applied to these volumes and nine standard fetal echocardiographic views were generated and optimized automatically, using the assistance of the virtual intelligent sonographer (VIS). Here, 100% of the four-chamber views, and after the VISA System application the five-chamber views, of the diagnostic plane showed the atrioventricular septal defect and a common AV valve. The autopsies of the fetuses confirmed the ultrasound results. (4) Conclusions: By applying intelligent navigation technology to the STIC volume datasets, 100% of the AVSD diagnoses were detected.

Treatment of EGJ Cancer within or outside Clinical Trials: Does the Setting Matter? A Monocentric Prospective Observational Study.

INTRODUCTION: RCTs support neoadjuvant chemoradiotherapy (nCRT) followed by surgery in locally advanced esophago-gastric junction (LA-EGJ) adenocarcinoma. However, RCTs are performed in highly controlled settings with limited representativeness of real-life patients (RLS). The aim of the study was to compare the outcomes in RLS and clinical trial settings. METHODS: The outcomes of RLS, which comprised 125 patients consequently treated for LA-EGJ adenocarcinoma between 2012 and 2017, were compared with the phase II trial (PIIS), performed on 65 patients from 2003 to 2011. RESULTS: About half of RLS (51.2%) were treated with nCRT according to VR protocol, 20.8% with standard CRT according to CROSS/Al-Sarraf, 20% with chemotherapy (CT) alone. pCR was 36.8%, 28.6%, and 9.1% after VR protocol, standard CRT, and CT, respectively (p = 0.082), while 3-year overall survival (OS) was 58.6% (95% CI 43.2-71.1%), 32.8% (14.6-52.4%), and 44.8% (21.3-65.9%), respectively (p = 0.030). With respect to PIIS, RLS had a higher proportion of cN+ (94% vs. 54%; p < 0.001) and a lower proportion of pCR after CT/CRT (23% vs. 39%; p = 0.041). Three-year OS was slightly higher, although not significantly, in PIIS (58.9%, 45.1-70.2%) than RLS (47.9%, 37.4-57.7%) and nearly identical to 3-year OS in RLS treated with VR protocol. CONCLUSION: Real-life patients with EGJ adenocarcinoma have more advanced cancer at baseline, lower pathologic response to neoadjuvant treatment than patients enrolled in clinical trials, but similar survival.

Intra-amniotic inflammation in the mid-trimester of pregnancy is a risk factor for neuropsychological disorders in childhood.

OBJECTIVES: Intra-amniotic inflammation is a subclinical condition frequently caused by either microbial invasion of the amniotic cavity or sterile inflammatory stimuli, e.g., alarmins. An accumulating body of evidence supports a role for maternal immune activation in the genesis of fetal neuroinflammation and the occurrence of neurodevelopmental disorders such as cerebral palsy, schizophrenia, and autism. The objective of this study was to determine whether fetal exposure to mid-trimester intra-amniotic inflammation is associated with neurodevelopmental disorders in children eight to 12 years of age. METHODS: This is a retrospective case-control study comprising 20 children with evidence of prenatal exposure to intra-amniotic inflammation in the mid-trimester and 20 controls matched for gestational age at amniocentesis and at delivery. Amniotic fluid samples were tested for concentrations of interleukin-6 and C-X-C motif chemokine ligand 10, for bacteria by culture and molecular microbiologic methods as well as by polymerase chain reaction for eight viruses. Neuropsychological testing of children, performed by two experienced psychologists, assessed cognitive and behavioral domains. Neuropsychological dysfunction was defined as the presence of an abnormal score (<2 standard deviations) on at least two cognitive tasks. RESULTS: Neuropsychological dysfunction was present in 45% (9/20) of children exposed to intra-amniotic inflammation but in only 10% (2/20) of those in the control group (p=0.03). The relative risk (RR) of neuropsychological dysfunction conferred by amniotic fluid inflammation remained significant after adjusting for gestational age at delivery [aRR=4.5 (1.07-16.7)]. Of the 11 children diagnosed with neuropsychological dysfunction, nine were delivered at term and eight of them had mothers with intra-amniotic inflammation. Children exposed to intra-amniotic inflammation were found to have abnormalities in neuropsychological tasks evaluating complex skills, e.g., auditory attention, executive functions, and social skills, whereas the domains of reasoning, language, and memory were not affected in the cases and controls. CONCLUSIONS: Asymptomatic sterile intra-amniotic inflammation in the mid-trimester of pregnancy, followed by a term birth, can still confer to the offspring a substantial risk for neurodevelopmental disorders in childhood. Early recognition and treatment of maternal immune activation in pregnancy may be a strategy for the prevention of subsequent neurodevelopmental disorders in offspring.

C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Sperm Energy Restriction and Recovery (SER) Alters Epigenetic Marks during the First Cell Cycle of Development in Mice.

The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first cell cycle indicated that progression through the pronuclear stages of SER-generated embryos is advanced in comparison with control-generated embryos. These findings prompted further analysis of potential effects of SER on pronuclear chromatin dynamics, focusing on the key H3K4me3 and H3K27ac histone modifications. Nearly all the SER-generated embryos displayed H3K4me3 in the male pronuclei at 12 h post-insemination (HPI), while a subset of the control-generated embryos did not. Additionally, SER-generated embryos displayed a more homogenous intensity of H3K27ac at 8 and 12 HPI compared to control embryos. These changes in histone modifications during the first cell cycle were accompanied by differences in gene expression at the two-cell stage; both of these changes in early embryos could potentially play a role in the improved developmental outcomes of these embryos later in development. Our results indicate that sperm incubation conditions have an impact on early embryo development and can be useful for the improvement of assisted reproductive technology outcomes.

Human Sperm Remain Motile After a Temporary Energy Restriction but do Not Undergo Capacitation-Related Events.

To acquire fertilization competence, mammalian sperm must undergo several biochemical and physiological modifications known as capacitation. Despite its relevance, the metabolic pathways that regulate the capacitation-related events, including the development of hyperactivated motility, are still poorly described. Previous studies from our group have shown that temporary energy restriction in mouse sperm enhanced hyperactivation, in vitro fertilization, early embryo development and pregnancy rates after embryo transfer, and it improved intracytoplasmic sperm injection results in the bovine model. However, the effects of starvation and energy recovery protocols on human sperm function have not yet been established. In the present work, human sperm were incubated for different periods of time in medium containing glucose, pyruvate and lactate (NUTR) or devoid of nutrients for the starving condition (STRV). Sperm maintained in STRV displayed reduced percentages of motility and kinematic parameters compared to cells incubated in NUTR medium. Moreover, they did not undergo hyperactivation and showed reduced levels of ATP, cAMP and protein tyrosine phosphorylation. Similar to our results with mouse sperm, starvation induced increased intracellular Ca2+ concentrations. Starved human sperm were capable to continue moving for more than 27 h, but the incubation with a mitochondrial uncoupler or inhibitors of oxidative phosphorylation led to a complete motility loss. When exogenous nutrients were added back (sperm energy recovery (SER) treatment), hyperactivated motility was rescued and there was a rise in sperm ATP and cAMP levels in 1 min, with a decrease in intracellular Ca2+ concentration and no changes in sperm protein tyrosine phosphorylation. The finding that human sperm can remain motile for several hours under starvation due to mitochondrial use of endogenous metabolites implies that other metabolic pathways may play a role in sperm energy production. In addition, full recovery of motility and other capacitation parameters of human sperm after SER suggests that this treatment might be used to modulate human sperm fertilizing ability in vitro.

TSSK3, a novel target for male contraception, is required for spermiogenesis.

We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.

Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation.

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

Isolated Dissection of the Ductus Arteriosus Associated with Sudden Unexpected Intrauterine Death.

We report five cases of sudden intrauterine death due to premature closure of the ductus arteriosus. In four cases, this was caused by dissecting the hematoma of the ductus arteriosus with intimal flap and obliteration of the lumen. In one case, the ductus arteriosus was aneurysmatic, with lumen occlusion caused by thrombus stratification. No drug therapy or free medication consumption were reported during pregnancy. The time of stillbirth ranged between 26 and 33 gestational weeks. We performed TUNEL analysis for apoptosis quantification. The dissecting features were intimal tears with flap formation in four of the cases, just above the origin of the ductus arteriosus from the pulmonary artery. The dissecting hematoma of the ductus arteriosus extended downward to the descending aorta and backward to the aortic arch with involvement of the left carotid and left subclavian arteries. TUNEL analysis showed a high number of apoptotic smooth muscle cells in the media in two cases. Abnormal ductal remodeling with absence of subintimal cushions, lacunar spaces rich in glycosaminoglycans (cystic medial necrosis), and smooth muscle cell apoptosis were the pathological substrates accounting for failure of remodeling process and dissection.

Gamma Delta TCR and the WC1 Co-Receptor Interactions in Response to Leptospira Using Imaging Flow Cytometry and STORM.

The WC1 cell surface family of molecules function as hybrid gamma delta (γδ) TCR co-receptors, augmenting cellular responses when cross-linked with the TCR, and as pattern recognition receptors, binding pathogens. It is known that following activation, key tyrosines are phosphorylated in the intracytoplasmic domains of WC1 molecules and that the cells fail to respond when WC1 is knocked down or, as shown here, when physically separated from the TCR. Based on these results we hypothesized that the colocalization of WC1 and TCR will occur following cellular activation thereby allowing signaling to ensue. We evaluated the spatio-temporal dynamics of their interaction using imaging flow cytometry and stochastic optical reconstruction microscopy. We found that in quiescent γδ T cells both WC1 and TCR existed in separate and spatially stable protein domains (protein islands) but after activation using Leptospira, our model system, that they concatenated. The association between WC1 and TCR was close enough for fluorescence resonance energy transfer. Prior to concatenating with the WC1 co-receptor, γδ T cells had clustering of TCR-CD3 complexes and exclusion of CD45. γδ T cells may individually express more than one variant of the WC1 family of molecules and we found that individual WC1 variants are clustered in separate protein islands in quiescent cells. However, the islands containing different variants merged following cell activation and before merging with the TCR islands. While WC1 was previously shown to bind Leptospira in solution, here we showed that Leptospira bound WC1 proteins on the surface of γδ T cells and that this could be blocked by anti-WC1 antibodies. In conclusion, γδ TCR, WC1 and Leptospira interact directly on the γδ T cell surface, further supporting the role of WC1 in γδ T cell pathogen recognition and cellular activation.