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Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: 20 May 2024 | Viewed by 6335

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Dr. Manuel Álvarez-Rodríguez

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Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
Interests: animal reproduction and related biotechnologies; -omics in gametes and reproductive tissues; cryopreservation and in vitro procedures
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Dr. Jaime Catalan

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Department of Medicine and Animal Surgery, Autonomous University of Barcelona, Barcelona, Spain
Interests: equids reproduction; reproductive biology; cryopreservation, semen evaluation, semen preservation; sperm fertility; sperm biology, oxidative stress, seminal plasma
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Dear Colleagues,

The complex process of sperm development and maturation involves multiple interconnected steps that begin in the testis and result in fertility acquisition inside the male genital tract as well as the female genital tract. The sperm then must cope with the active immune system and particular homeostasis of the tract. A special segment, the sperm reservoir, stores the sperm cells until they are released and move toward the fertilization site.

This Special Issue aims to cover the crucial molecular events and mechanisms in sperm development. The scope includes research on sperm cell physiology as well as on components in the seminal plasma and/or in the female allowing for the realization of the full fertilization of sperm, including extracellular vesicles. Thus, in vitro and in vitro studies aiming at unraveling new molecular determinants of the acquisition of the full functionality in sperm are welcome.

Dr. Manuel Álvarez-Rodríguez
Dr. Jaime Catalan
Guest Editors

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Keywords

sperm physiology
seminal plasma
extracellular vesicles
reproduction
spermatogenesis
capacitation
fertility

Related Special Issue

Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization in International Journal of Molecular Sciences (10 articles)

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Research

23 pages, 1770 KiB

Open AccessArticle

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression

by Rebeka Podgrajsek, Luka Bolha, Tjasa Pungert, Joze Pizem, Katerina Jazbec, Elvira Malicev and Martin Stimpfel

Int. J. Mol. Sci. 2024, 25(8), 4157; https://doi.org/10.3390/ijms25084157 - 09 Apr 2024

Viewed by 553

Abstract

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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23 pages, 6722 KiB

Open AccessArticle

SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization

by Nathaly Cormier, Asha E. Worsham, Kinsey A. Rich and Daniel M. Hardy

Int. J. Mol. Sci. 2024, 25(7), 3652; https://doi.org/10.3390/ijms25073652 - 25 Mar 2024

Viewed by 487

Abstract

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a–Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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17 pages, 3348 KiB

Open AccessArticle

Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro

by Areej Jorban, Eitan Lunenfeld and Mahmoud Huleihel

Int. J. Mol. Sci. 2024, 25(4), 2160; https://doi.org/10.3390/ijms25042160 - 10 Feb 2024

Viewed by 832

Abstract

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO₂ incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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11 pages, 2543 KiB

Open AccessArticle

Overdue Calcium Oscillation Causes Polyspermy but Possibly Permits Normal Development in Mouse Eggs

by Mio Fukuoka, Woojin Kang, Daiki Katano, Sae Horiike, Mami Miyado, Mamoru Tanaka, Kenji Miyado and Mitsutoshi Yamada

Int. J. Mol. Sci. 2024, 25(1), 285; https://doi.org/10.3390/ijms25010285 - 24 Dec 2023

Viewed by 738

Abstract

In some non-mammalian eggs, the fusion of one egg and multiple sperm (polyspermy) induces a robust rise in intracellular calcium ion (Ca²⁺) concentration due to a shortage of inducers carried by a single sperm. Instead, one of the sperm nuclei is selected inside the egg for normal embryogenesis. Polyspermy also occurs during the in vitro fertilization of human eggs; however, the fate of such eggs is still under debate. Hence, the relationship between polyspermy and repetitive Ca²⁺ increases (Ca²⁺ oscillation) in mammals remains unknown. To address this issue, we used mouse sperm lacking extramitochondrial citrate synthase (eCS), which functions as a Ca²⁺ oscillation inducer; its lack causes retarded Ca²⁺ oscillation initiation (eCs-KO sperm). Elevated sperm concentrations normalize Ca²⁺ oscillation initiation. As expected, eCS deficiency enhanced polyspermy in both zona pellucida (ZP)-free and ZP-intact eggs despite producing the next generation of eCs-KO males. In conclusion, similarly to non-mammalian eggs, mouse eggs may develop normally under polyspermy conditions caused by problematic Ca²⁺ oscillation. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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16 pages, 1147 KiB

Open AccessArticle

Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems

by Clara Agudo-Rios, Ana Sanchez-Rodriguez, Ingrid I. D. Idrovo, Juan Ángel Laborda-Gomariz, Ana J. Soler, Maria E. Teves and Eduardo R. S. Roldan

Int. J. Mol. Sci. 2023, 24(21), 15954; https://doi.org/10.3390/ijms242115954 - 03 Nov 2023

Viewed by 1135

Abstract

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode’s medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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17 pages, 1911 KiB

Open AccessArticle

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples

by András Balló, Péter Czétány, Kinga Székvári Busznyákné, László Márk, Nóra Mike, Attila Török, Árpád Szántó and Gábor Máté

Int. J. Mol. Sci. 2023, 24(15), 11981; https://doi.org/10.3390/ijms241511981 - 26 Jul 2023

Cited by 1 | Viewed by 934

Abstract

There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10–12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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12 pages, 2497 KiB

Open AccessArticle

Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR

by Erwin Muñoz, Macarena Castro, Luis Aguila, María José Contreras, Fernanda Fuentes, María Elena Arias and Ricardo Felmer

Int. J. Mol. Sci. 2023, 24(15), 11947; https://doi.org/10.3390/ijms241511947 - 26 Jul 2023

Cited by 1 | Viewed by 1089

Abstract

Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes. Full article

(This article belongs to the Special Issue Molecular Mechanisms Involved in Sperm Development, Maturation, and Fertilization 2.0)

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