Human Sperm Head Vacuoles Are Related to Nuclear-Envelope Invaginations.

Nuclear vacuoles are specific structures present on the head of the human sperm of fertile and non-fertile men. Human sperm head vacuoles have been previously studied using motile sperm organelle morphology examination (MSOME) and their origin related to abnormal morphology, abnormal chromatin condensation and DNA fragmentation. However, other studies argued that human sperm vacuoles are physiological structures and consequently, to date, the nature and origin of the nuclear vacuoles remains to be elucidated. Here, we aim to define the incidence, position, morphology and molecular content of the human sperm vacuoles using transmission electron microscopy (TEM) and immunocytochemistry techniques. The results showed that ~50% of the analyzed human sperm cells (n = 1908; 17 normozoospermic human donors) contained vacuoles mainly located (80%) in the tip head region. A significant positive correlation was found between the sperm vacuole and nucleus areas. Furthermore, it was confirmed that nuclear vacuoles were invaginations of the nuclear envelope from the perinuclear theca and containing cytoskeletal proteins and cytoplasmic enzyme, discarding a nuclear or acrosomal origin. According to our findings, these human sperm head vacuoles are cellular structures originating from nuclear invaginations and contain perinuclear theca (PT) components, allowing us to define a new term of 'nuclear invaginations' rather than 'nuclear vacuoles'.

Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation.

Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50-99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation.

Carbohydrate analysis of the zona pellucida and cortical granules of human oocytes by means of ultrastructural cytochemistry.

BACKGROUND: The zona pellucida (ZP), the mammalian oocyte coat, consists of a restricted number of highly glycosylated proteins. In vitro sperm binding studies suggest a higher binding affinity for the outer region of the ZP compared to its inner region in different species. However, the reason for this difference in binding distribution remains unresolved. Many studies suggest that the carbohydrate sequences linked to ZP glycoproteins act as ligands for sperm binding to this matrix. METHODS: Lectins and antibodies that recognize different carbohydrates were employed to perform an ultrastructural analysis of human ZP and cortical granule glycosylation. RESULTS: This study reveals variable glycosylation of the human ZP throughout its thickness, with pronounced differences between the most external and internal regions of this matrix. The binding studies also indicate that ZP glycoproteins express some carbohydrate sequences not previously detected in other species. Finally, cytochemical analysis of human cortical granules suggests similarities in glycosylation to ZP glycoproteins but not to cortical granules from other mammalian species. CONCLUSION: A heterogeneous carbohydrate composition was observed in the thickness of the human ZP that could be responsible for the different sperm binding affinity detected between the outer and inner regions of the ZP.