Immunofluorescence and High-Resolution Microscopy Reveal New Insights in Human Globozoospermia

Globozoospermia is a rare and severe type of teratozoospermia characterized by the presence of round-headed, acrosomeless spermatozoa with cytoskeleton defects. Current data support a negative relationship between globozoospermia and intracytoplasmic sperm injection (ICSI) outcomes, revealing the need to perform exhaustive studies on this type of sperm disorder. The aim of this study was to evaluate different structural, functional and molecular sperm biomarkers in total globozoospermia with proper embryo development after ICSI. The combination of field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) allowed us to identify and correlate eight morphological patterns with both types of microscopy. Additionally, results reported a high percentage of coiled forms, with cytoplasmic retentions around the head and midpiece. By fluorescent microscopy, we detected that most of the sperm showed tubulin in the terminal piece of the flagellum and less than 1% displayed tyrosine phosphorylation in the flagellum. Moreover, we did not detect chaperone Heat shock-related 70 kDa protein 2 (HSPA2) in 85% of the cells. Overall, these findings provide new insights into globozoospermia, which could have potential implications in improving sperm selection methods for assisted reproductive techniques.This research was funded by the Human Fertility Professorship and Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186)

Detección de compuestos orgánicos mediante LIBS en rocas de interés en exploración planetaria. Aplicaciones en Astrobiología

La integración de instrumentos LIBS en misiones de exploración planetaria es una realidad desde hace ya unos años, siendo una tecnología conducente a la obtención de información multielemental en las distintas rocas y minerales existentes en la superficie de Marte. Su gran eficacia ha sido demostrada en muchos de los trabajos publicados hasta la fecha por los equipos de investigación participantes en la misión Mars Science Laboratory (MSL). Uno de los objetivos primordiales de dichas investigaciones radica en la detección de posibles bioindicadores [1], así como en la identificación y discriminación mediante LIBS de compuestos orgánicos, tarea que puede llegar a ser compleja ya que, entre otros aspectos, se trata de una técnica muy sensible a las condiciones ambientales (composición de la atmósfera y presión existente) [2] [3]. El presente trabajo ha buscado por un lado, evaluar el efecto de la atmósfera existente en el planeta rojo (rica en CO2 con 7mb de presión media) en la formación de plasmas inducidos por láser a partir de la ablación de muestras formadas por matrices inorgánicas dopadas con compuestos orgánicos seleccionados. Por otro lado, a partir de las diferentes huellas espectrales obtenidas en dichas condiciones y mediante la aplicación de técnicas quimiométricas adaptadas, se estudian las posibilidades de identificación de dichos referentes orgánicos.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Sperm Adhesion Molecule 1 (SPAM1) Distribution in Selected Human Sperm by Hyaluronic Acid Test

The failures of binding to the oocyte zona pellucida are commonly attributed to defects in the sperm recognition, adhesion, and fusion molecules. SPAM1 (sperm adhesion molecule 1) is a hyaluronidase implicated in the dispersion of the cumulus-oocyte matrix. Therefore, the aim of this study was to characterize the SPAM1 distribution in the different physiological conditions of human sperm. Specifically, we evaluated the location of the SPAM1 protein in human sperm before capacitation, at one and four hours of capacitation and after hyaluronic acid (HA) selection test by fluorescence microscopy. Sperm bound to HA were considered mature and those that crossed it immature. Our results detected three SPAM1 fluorescent patterns: label throughout the head (P1), equatorial segment with acrosomal faith label (P2), and postacrosomal label (P3). The data obtained after recovering the mature sperm by the HA selection significantly (p < 0.05) highlighted the P1 in both capacitation times, being 79.74 and 81.48% after one hour and four hours, respectively. Thus, the HA test identified that human sperm require the presence of SPAM1 throughout the sperm head (P1) to properly contact the cumulus-oocyte matrix. Overall, our results provide novel insights into the physiological basis of sperm capacitation and could contribute to the improvement of selection techniques.This research was funded by the Cátedra Human Fertility, Departamento de Biotecnología of the Universidad de Alicante (VIGROB-186) and «Proyectos de Generación de Conocimiento» within the framework of the State Program to Promote Scientific-Technical Research and its Transfer, of the State Plan for Scientific, Technical and Innovation Research 2017–2020 (PGC2018-094781-B-I00)

Molecular Chaperone HSPA2 Distribution During Hyaluronic Acid Selection in Human Sperm

During fertilization, sperm hyaluronidase activity is essential for spermatozoa to successfully penetrate the hyaluronic acid-enriched extracellular matrix of the cumulus cells. Since molecular chaperones, as the heat shock protein A2, are typically involved in bringing hyaluronic acid receptors to the cell surface, here we evaluated the presence and spatial location of HSPA2 on human spermatozoa based on its hyaluronic acid binding capacity. This study included 16 normozoospermic sperm samples from volunteering donors. The location of HSPA2 was studied in cells before and after 1-h incubation under capacitating conditions, as well as in spermatozoa selected according to their ability of binding to hyaluronic acid. Our results showed no significant differences in HSPA2 immunofluorescent cells before and after 1 h of incubation in capacitating conditions. Nevertheless, after hyaluronic acid selection, the percentage of HSPA2-labelled cells increased significantly, indicating that the interaction with hyaluronic acid may induce the unmasking of HSPA2 epitopes. Furthermore, after swim-up and hyaluronic acid selection, spermatozoa presented a highly immunostained equatorial band with a homogeneous fluorescence throughout the acrosomal region. This distribution has been previously suggested to have important implications in male fertility. Noteworthy, a homogeneous fluorescence among the acrosomal region with a more intense labelling at the apical region was observed only in hyaluronic acid bound sperm cells, which may be associated with primary gamete recognition. Our findings suggest that the hyaluronic acid selection technique and HSPA2 biomarker should be considered candidates to complement the classic seminal analysis before recommending an appropriate assisted reproduction technique.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the Human Fertility Cathedra of the University of Alicante and R&D&I projects financed by competitive public entities (ViGrob-186, UAIND17-03, PGC2018-094781-B-100)

Arylsulfatase A Remodeling during Human Sperm In Vitro Capacitation Using Field Emission Scanning Electron Microscopy (FE-SEM)

Capacitation drives sperm biophysical and biochemical changes for sperm-oocyte interactions. It is a well-known fact that the molecular complex arylsulfatase A (ARSA), hyaluronidase sperm adhesion molecule 1 (SPAM1), and heat shock protein 2 (HSPA2) plays a significant role in sperm–zona pellucida (ZP) binding. However, the time-dependent capacitation effects on the sperm surface ARSA presence and specific topographic distributions remain to be elucidated. Here, we quantified the ARSA density and specific membrane domain locations before (US) and after in vitro capacitation (one and four hours; CS1–CS4) in human sperm using high-resolution field emission scanning electron microscopy (FE-SEM) and immunogold labeling. Our results showed a significant and progressive capacitation-mediated increase of labeled spermatozoa from the US (37%) to CS4 (100%) physiological conditions. In addition, surface mapping revealed a close relationship between the ARSA residues and their acrosomal repositioning. Compared with the ARSA surface heterogeneous distribution found in US, the CS1–4 conditions exhibited clustering on the peri-acrosomal region, showing that time-dependent capacitation also induced a ARSA residue dramatic translocation on sperm surfaces. Our findings provide novel insights into the molecular remodeling events preceding sperm-oocyte interactions.This research was funded by the Human Fertility Cathedra of the University of Alicante and R&D&I projects financed by competitive public entities (ViGrob-186, UAIND17-03 and PGC2018-094781-B-100)

Searching for Biosignatures in Mars by LIBS Molecular Signals Discernment

In the present study, LIBS analysis of a set of six selected molecules related to organic biosignatures -or their degradation compounds- have been carried out in both simulated Martian atmosphere and air in order to identify and discriminate them on the basis of their molecular emission features. This strategy can reveal insights into how different emission spectral modes react to changes in atmospheric conditions and therefore can help to detect those species which are more sensitive to changes in pressure and composition of the atmosphere. At high laser irradiance, atomization of organic compounds is essentially complete, although at sufficiently delayed integration times, the formation of new molecules by recombination processes can be noticed[1]. Molecular species characteristic of organic emissions such as C2, CN, NH, OH and CH were studied. Results can contribute to establish the optimal conditions for the observation of organic carbon species in laser-induced plasmas and the bases for the ensuing detection of organic biosignatures in analogous geological materials from Mars. Likewise, this research is aimed at providing a tool in the interpretation of LIBS data though the application of adapted data processing algorithms for the identification and discernment of suspected compounds of organic nature[2] [1] T. Delgado, L. García-Gómez, L. M. Cabalín, J. J. Laserna, Investigation on the origin of molecular emissions in laser-induced breakdown spectroscopy under Mars-like atmospheric conditions of isotope-labeled compounds of interest in astrobiology, Spectrochim. Acta Part B (2021) 179-106114. [2] T. Delgado, L. García-Gómez, L. M. Cabalín, J. J. Laserna, Detectability and discrimination of biomarker organic precursors in a low pressure CO2 atmosphere by LIBS, J. Anal. At. Spectrom. (2020) 35:1947.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Specific lectin binding sites during in vitro capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx and plasma membrane undergo outstanding modifications during fertilization. However, it is unclear how in vitro capacitation time and acrosome reaction affect the specific location of boar sperm glycoconjugates. This study aimed to identify lectin binding patterns and to describe the sequential changes during different in vitro capacitation times (1 and 4 h) and acrosome reaction in boar spermatozoa. With Aleuria aurantia agglutinin (AAA), most uncapacitated cells were labelled in the postacrosomal region. Nevertheless, after 1 h of in vitro capacitation and the acrosome reaction, most AAA binding sites were in the acrosomal region. With Concanavalin A (ConA), most sperm were labeled in the postacrosomal region before and after capacitation. After the acrosome reaction induction, this pattern changed to a highly stained acrosomal and postacrosomal regions. Peanut agglutinin (PNA) binding sites were in the acrosomal region in uncapacitated and capacitated sperm. In acrosome reacted sperm after 4h capacitation, the most frequent pattern showed remaining positive labeling in the central area of the head. With Pisum sativum agglutinin (PSA), most uncapacitated cells showed a postacrosomal region staining. Nevertheless, faint stained all over the head and highly acrosomal region labelling was observed in the major part of capacitated and acrosome reacted sperm respectively. With Wheat germ agglutinin (WGA), the most representative pattern in uncapacitated, capacitated and acrosome reacted sperm was labelled in the acrosomal region. Regarding capacitation time, the most significant changes in the most representative pattern were observed in acrosome reacted spermatozoa after 4 h of in vitro capacitation.This research was supported by AGL2015-70159-P, PGC2018-094781-B-100 and PEJ2018-002736-P (MCINN/AEI/FEDER,UE)

Lectin spatial immunolocalization during in vitro capacitation in Tursiops truncatus spermatozoa

Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.Cátedra Human Fertility of University of Alicante and VIGROB-186

Pancreatic and psoas abscesses as a late complication of intravesical administration of bacillus Calmette-Guerin for bladder cancer: a case report and review of the literature

<p>Abstract</p> <p>Introduction</p> <p>Bacillus Calmette-Guerin (BCG) is a live attenuated strain of <it>Mycobacterium bovis</it> that has been used to treat urothelial carcinoma since 1976, and has been reported to eradicate disease in more than 70% of patients with <it>in situ</it> and stage I disease. To the best of our knowledge, we report the first case of disseminated bacillus Calmette-Guerin infection causing multiple abscesses affecting the pancreatic head and right psoas muscle, diagnosed 5 years after intravesical treatment with bacillus Calmette-Guerin therapy for bladder cancer.</p> <p>Case presentation</p> <p>An 83-year-old Caucasian man was hospitalized with a 2-month history of back pain, anorexia, generalized weakness and a 47-pound weight loss. He had previously undergone two transurethral resections for high-grade transitional cell carcinoma of the bladder and had received 12 intravesical bacillus Calmette-Guerin instillations without any complications. He complained of abdominal pain in his right flank. A computed tomography scan of the abdomen showed multiple abscesses affecting the pancreatic head and right psoas muscle. Growth of <it>Mycobacterium bovis</it> was determined in cultures of the purulent material obtained by surgical drainage of the abscesses.</p> <p>Conclusions</p> <p>This case illustrates the fact that although intravesical administration of bacillus Calmette-Guerin is generally considered to be safe, it is not exempt from complications and these could appear immediately after treatment or as a delayed complication many years later.</p

Detection of indigenous organic matter in rocks from the interpretation of carbon molecular forms in the laser-induced plasma.

Oil shale, a sedimentary rock containing organic matter and a variety of inorganic minerals including carbonates and kerogens, serves as a significant source of organic material on Earth [1]. Kerogen, the most abundant form of organic matter, differs in chemical composition based on the microorganisms that contributed to its formation [2]. Laser-induced breakdown spectroscopy (LIBS) is a powerful analytical technique used on the Mars rover, allowing elemental characterization of Martian rocks, soils, and sediments. This study presents the first-ever detection of natural organic matter in oil shale using LIBS under simulated Martian conditions. Through an analysis of emitting species including CN and C2, LIBS successfully identifies the presence of organic compounds in this sedimentary rock. The ability to detect and characterize natural organic matter in oil shale, known for its potential to suggest the existence of ancient life, holds significant relevance in astrobiology. Furthermore, this information contributes to the identification of biosignatures and aids in the development of planetary exploration strategies. Oil shale samples were analyzed using LIBS under simulated Martian conditions after being crushed, pressed into pellets, and subjected to pyrolysis to remove organic matter. The analysis revealed significant changes in the infrared spectra, confirming the absence of aliphatic hydrocarbons after pyrolysis [3]. The LIBS results demonstrated the presence of molecular species associated with hydrocarbons, such as CN and C2, through distinct spectral emissions. The absence of these emissions in the pyrolyzed sample further supported the detection of organic matter originating from kerogen.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech