Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

Conventional IVF in horses remains challenging. In particular, stallion sperm fails to penetrate the zona pellucida, possibly due to incomplete invitro sperm capacitation. Therefore, there is a need to elucidate, in horses, molecules with a proven role during capacitation in other mammals. Our laboratory has described the relevance of the endocannabinoid system in capacitation of bovine and murine sperm. We reported that anandamide (AEA), an endocannabinoid present in follicular and oviducal fluids, induced capacitation-associated events. The aims of this work were to characterise the localization of cannabinoid receptors in equine sperm and to evaluate the effects of AEA on levels of tyrosine-phosphorylated proteins (pY) and substrates phosphorylated by protein kinase A (pPKA). Both cannabinoid receptors (CB1, CB2, TRPV1) and pPKA and pY were localised in sperm by indirect immunofluorescence. Sperm (15×106mL−1) were incubated, at 38.5°C in air, in modified Tyrode’s-albumin-lactate-pyruvate (TALP) with 25mM NaHCO3, 5mM dextrose and 1mgmL−1 polyvinyl alcohol (PVA; TALP-Bic-PVA) or TALP-Bic-PVA supplemented with AEA (0.1, 1, 10, 100nM, and 1µM) for 4h. After incubation, Western blot was used to determine levels of pY and pPKA in 4.5×106 sperm. Cryopreserved sperm samples from three stallions were evaluated. The normality of data distributions and homoscedasticity were verified with the Shapiro-Wilk and Levene tests, respectively. Data were analysed by one-way ANOVA and Bonferroni post hoc test, with P<0.05 considered significant. Based on immunofluorescence, CB1 was mainly localised in the post-acrosomal region and flagellum (93.4%±5.5, mean±s.d.), CB2 in the post-acrosomal region and middle piece (89.9%±28.3), and TRPV1 in the post-acrosomal region and flagellum (89.3%±9). Sperm positive for pPKA had fluorescence in the middle piece and principal piece of the flagellum. Incubation with 1nM AEA for 4h induced a 61% increase in pPKA levels compared with TALP-Bic-PVA medium alone, with no induction of pY levels in any treatment. In conclusion, cannabinoid receptors were present in equine sperm, and incubation with AEA induced an increase in PKA activity, an essential event associated with sperm capacitation. To our knowledge, this was the first report describing the presence of receptors of the endocannabinoid system in equine sperm and the potential role of AEA in the acquisition of sperm fertilizing ability.

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Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽

10.1530/reprod/120.2.325 ◽

2000 ◽

pp. 325-335 ◽

Cited By ~ 29

Author(s):

A Calvo ◽

LM Pastor ◽

S Bonet ◽

E Pinart ◽

M Ventura

Keyword(s):

Ricinus Communis ◽

Lectin Histochemistry ◽

Apical Part ◽

Seminiferous Tubules ◽

In Situ Characterization ◽

Intense Staining ◽

Terminal Galactose ◽

Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

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Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised

Scientific Reports ◽

10.1038/s41598-021-83929-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rasoul Kowsar ◽

Shahrzad Ronasi ◽

Nima Sadeghi ◽

Khaled Sadeghi ◽

Akio Miyamoto

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Dna Fragmentation ◽

Acrosome Reaction ◽

Developmental Competence ◽

Ros Production ◽

Sperm Capacitation ◽

Bovine Sperm ◽

Epidermal Growth ◽

High Level

AbstractUpon insemination, sperm cells are exposed to components of the female reproductive tract (FRT) fluids, such as urea and epidermal growth factor (EGF). It has been shown that both urea and EGF use EGF receptor signaling and produce reactive oxygen species (ROS) that are required at certain levels for sperm capacitation and acrosome reaction. We therefore hypothesized that during bovine sperm capacitation, a high level of urea and EGF could interfere with sperm function through overproduction of ROS. High-level urea (40 mg/dl urea is equal to 18.8 mg/dl of blood urea nitrogen) significantly increased ROS production and TUNEL-positive sperm (sperm DNA fragmentation, sDF) percentage, but decreased HOS test score, progressive motility, acrosome reaction and capacitation. The EGF reversed the negative effects of urea on all sperm parameters, with the exception of ROS production and DNA fragmentation, which were higher in urea-EGF-incubated sperm than in control-sperm. The developmental competence of oocytes inseminated with urea-EGF-incubated sperm was significantly reduced compared to the control. A close association of ROS production or sDF with 0-pronuclear and sperm non-capacitation rates was found in the network analysis. In conclusion, EGF enhanced urea-reduced sperm motility; however, it failed to reduce urea-increased sperm ROS or sDF levels and to enhance subsequent oocyte competence. The data suggests that any study to improve sperm quality should be followed by a follow-up assessment of the fertilization outcome.

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Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Zygote ◽

10.1017/s0967199402002137 ◽

2002 ◽

Vol 10 (2) ◽

pp. 95-104 ◽

Cited By ~ 16

Author(s):

Mike Katayama ◽

Takashi Miyano ◽

Masashi Miyake ◽

Seishiro Kato

Keyword(s):

Plasma Membrane ◽

Intracytoplasmic Sperm Injection ◽

Acrosome Reaction ◽

Sperm Chromatin ◽

Oocyte Activation ◽

Freezing And Thawing ◽

Chromatin Decondensation ◽

Progesterone Treatment ◽

Pronuclear Formation ◽

Boar Spermatozoa

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.

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Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Gamete Research ◽

10.1002/mrd.1120110205 ◽

1985 ◽

Vol 11 (2) ◽

pp. 143-155 ◽

Cited By ~ 5

Author(s):

Adriana C. Hinrichsen ◽

Edda Töpfer-Petersen ◽

Thomas Dietl ◽

Christian Schmoeckel ◽

Wolf-B. Schill

Keyword(s):

Acrosomal Membrane ◽

Immunological Approach ◽

Boar Spermatozoa

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Antioxidant effect of Mn2+ on capacitation and acrosome reaction of fresh and chilled cattle bull semen

Veterinary Science Development ◽

10.4081/vsd.2011.3479 ◽

2012 ◽

Vol 1 (1) ◽

pp. 18

Author(s):

Amrit Kaur Bansal ◽

Ranjna Sundhey Cheema ◽

Vinod Kumar Gandotra

Keyword(s):

Reactive Oxygen Species ◽

Lipid Peroxidation ◽

Acrosome Reaction ◽

Reactive Oxygen ◽

Antioxidant Effect ◽

Oxygen Species ◽

Sperm Capacitation ◽

Bull Semen

The aim of this paper was to investigate the antioxidant effect of Mn2+ (200 mM) on the sperm capacitation and acrosome reaction of fresh and chilled cattle bull semen. It has been found that Mn2+ supplementation improves (P≤0.05) the motility at 0, 2, 4 and 6 h of incubation. MDA (malondialdehyde), end product of lipid peroxidation, decreases significantly (P≤0.05) with the supplementation of manganese at 0- and 6-hr of incubation both in fresh and chilled semen. Manganese also increases acrosome reaction significantly (P≤0.05) both in fresh and chilled semen at 0, 4 and 6 h of incubation. Therefore, our findings suggest the role of Mn2+supplementation in improving the quality of cattle bull semen by its scavenging property<em> i.e.</em> reduction in the production of reactive oxygen species during its storage at 4°C or incubation at 37°C for capacitation.

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Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

Asian Journal of Andrology ◽

10.1038/aja.2012.81 ◽

2012 ◽

Vol 14 (6) ◽

pp. 816-821 ◽

Cited By ~ 110

Author(s):

Debby Ickowicz ◽

Maya Finkelstein ◽

Haim Breitbart

Keyword(s):

Protein Kinases ◽

Acrosome Reaction ◽

Sperm Capacitation

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Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Reproduction ◽

10.1530/rep-20-0123 ◽

2020 ◽

Vol 160 (5) ◽

pp. 725-735

Author(s):

Julieta Gabriela Hamze ◽

María Jiménez-Movilla ◽

Raquel Romar

Keyword(s):

Acrosome Reaction ◽

Cumulus Cell ◽

Cumulus Cells ◽

In Vitro Fertilisation ◽

Sperm Binding ◽

Fertilisation Rate ◽

Gamete Recognition ◽

Boar Spermatozoa

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

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Sperm Capacitation, the Acrosome Reaction, and Fertilization

Reproductive Endocrinology and Infertility ◽

10.1007/978-1-4419-1436-1_25 ◽

2010 ◽

pp. 389-421 ◽

Cited By ~ 1

Author(s):

Peter Sutovsky

Keyword(s):

Acrosome Reaction ◽

Sperm Capacitation

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Perfluorooctane Sulfonate (PFOS) and Perfluorohexane Sulfonate (PFHxS) Alters Protein Phosphorylation, Increase ROS Levels and DNA Fragmentation during In Vitro Capacitation of Boar Spermatozoa

Animals ◽

10.3390/ani10101934 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1934

Author(s):

Iván Oseguera-López ◽

Serafín Pérez-Cerezales ◽

Paola Berenice Ortiz-Sánchez ◽

Oscar Mondragon-Payne ◽

Raúl Sánchez-Sánchez ◽

...

Keyword(s):

Dna Fragmentation ◽

Acrosome Reaction ◽

Perfluorooctane Sulfonate ◽

Toxic Effects ◽

Toxic Compounds ◽

Worldwide Distribution ◽

Lack Of Information ◽

Boar Spermatozoa ◽

Vital Parameters

Perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) are toxic and bioaccumulative, included in the Stockholm Convention’s list as persistent organic pollutants. Due to their toxicity, worldwide distribution, and lack of information in spermatozoa physiology during pre-fertilization processes, the present study seeks to analyze the toxic effects and possible alterations caused by the presence of these compounds in boar sperm during the in vitro capacitation. The spermatozoa capacitation was performed in supplemented TALP-Hepes media and mean lethal concentration values of 460.55 μM for PFOS, and 1930.60 μM for PFHxS were obtained. Results by chlortetracycline staining showed that intracellular Ca2+ patterns bound to membrane proteins were scarcely affected by PFOS. The spontaneous acrosome reaction determined by FITC-PNA was significantly reduced by PFOS and slightly increased by PFHxS. Both toxic compounds significantly alter the normal capacitation process from 30 min of exposure. An increase in ROS production was observed by flow cytometry and considerable DNA fragmentation by the comet assay. The immunocytochemistry showed a decrease of tyrosine phosphorylation in proteins of the equatorial and acrosomal zone of the spermatozoa head. In conclusion, PFOS and PFHxS have toxic effects on the sperm, causing mortality and altering vital parameters for proper sperm capacitation.

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