Effect of anisosmotic stress on the structural and functional integrity of the plasma and acrosomal membrane of ram sperm

The integrity of the plasma membrane (MP) and the acrosome (MA) have been two of the most studied seminal evaluation parameters due to their role as a cell boundary and because they are responsible for interactions between cells effective. To assessing more objectively the effects of osmotic stress on the integrity of the PM and MA, as well as the rate of change that occurred during seminal cryopreservation, five freshly collected ejaculates were evaluated, refrigerated at 5 ºC and thawed per ram/session during 5 consecutive weeks. Using eosin-nigrosin (EN) staining, vitality (VIT), morpho abnormalities and cellular response were evaluated after performing osmotic resistance (ORT) and endosmosis (HOST) tests. The direct effect of anysosmosis and cryopreservation on the dependent variables were analyzed using the GLM procedure (SAS®) and when differences were observed, the effects were quantified using the LSMEANS. All the sperm quality values studied were significantly affected (P <0.001) by cryopreservation (VIT, ORT, HOST). The ORT demonstrated how the acrosome was one of the structures most affected by cryopreservation (P <0.001). In conclusion, the present study confirms that anysosmotic stress affects the sperm cell in an important way, compromising the reference values that quantify semen quality, especially MA and MP.

αV Integrin Expression and Localization in Male Germ Cells

Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

Collapsed mitochondrial cristae in goat spermatozoa due to mercury result in lethality and compromised motility along with altered kinematic patterns

Earlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following in vitro exposure to different concentrations (0.031–1.25 µg/ml) of mercuric chloride for 15 min and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability and motility of sperms was significantly reduced along with altered kinematic patterns, significant increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group, there was significant increase in expression of Bcl-2 in mercury-treated groups. Transmission electron microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to mitochondria seems to be responsible for alterations in functional and kinematic attributes of spermatozoa.

Effect of Addition Palmitic Acid and Vitamin E to the Tris-Egg Yolk Diluter in Canine Semen Cryopreservation

Background: Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal’s species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing.Materials, Methods & Results: Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog’s semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn´t exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2.Discussion: The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100 µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.Keywords: dog, semen, antioxidants, cryopreservation.Descritores: cão, sêmen, antioxidantes, criopreservação.

Seminal plasma interference in the kinetics and viability of cryopreserved canine sperm

Most researchers have frozen dog semen using methodologies described for other species. Consequently, these studies have shown that the thawed semen of dogs is of low quality, with conception rates lower than that of other species. Therefore, in this study, we evaluated different freezing protocols for canine semen, using three adult Bulldog Campeiro males, aged 2 to 5 years, with proven fertility. Five semen samples were collected from each animal using the penile bulb digital manipulation method. The collected samples were divided into: group 1, the samples were diluted directly in Botudog® commercial freezing medium (Botupharma Biotecnologia Animal) and group 2, the samples were centrifuged at 600 g for 10 min and the pellet was resuspended in Botudog®, totaling a concentration end of 200 x 106 sperm per ml. The samples were packaged in 0.5 mL straws at a concentration of 100 x 106 viable sperm. The samples remained for 1 h in stabilization at 4 °C and transferred to nitrogen vapor for 20 min, immersed in nitrogen, stored in a cryogenic cylinder, and thawed at 46 °C for 20 s. It was found that the total motility (MT, %), path speed (VAP, μm/s), progressive motility (PM, %), progressive linear speed (VSL; µm/s), curvilinear speed (VCL; µm/s), linearity (LIN, %), percentage of fast sperm (RAP, %), and plasma and acrosomal membrane integrity were higher in group 1, with samples not being centrifuged. These data demonstrate that the canine semen freezing protocol, using the Botudog® diluent, does not recommend the centrifugation of the ejaculate, prior to freezing.

Effect of Seminal Plasma Protein Fractions on Stallion Sperm Cryopreservation

Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to heparin into heparin-binding (Hep+) and heparin-non-binding (Hep−) fractions. The addition of three concentrations—125, 250, and 500 µg/mL—of each protein fraction was tested. After thawing, the following parameters were assessed: sperm motility (by CASA), plasma membrane integrity (PI staining), and acrosomal membrane integrity (PNA staining) using flow cytometry, and capacitation status (anti-phosphotyrosine antibody) using imaging-based flow cytometry. Our results showed that SP protein fractions had a significant effect on the kinematic parameters of spermatozoa and on a proportion of their subpopulations. The 125 µg/mL of Hep+ protein fraction resulted in increased linearity (LIN) and straightness (STR), moreover, with the highest values of sperm velocities (VAP, VSL), also this group contained the highest proportion of the fast sperm subpopulation. In contrast, the highest percentage of slow subpopulation was in the groups with 500 µg/mL of Hep+ fraction and 250 µg/mL of Hep− fraction. Interestingly, acrosomal membrane integrity was also highest in the groups with Hep+ fraction in concentrations of 125 µg/mL. Our results showed that the addition of protein fractions did not significantly affect the plasma membrane integrity and capacitation status of stallion spermatozoa. Moreover, our results confirmed that the effect of SP proteins on the sperm functionality is concentration-dependent, as has been reported for other species. Our study significantly contributes to the lack of studies dealing with possible use of specific stallion SP fractions in the complex puzzle of the improvement of cryopreservation protocols. It is clear that improvement in this field still needs more outputs from future studies, which should be focused on the effect of individual SP proteins on other sperm functional parameters with further implication on the success of artificial insemination in in vivo conditions.

Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood, however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%. The aimed of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in the kinetics and viability of frozen and thawed ram spermatozoa. Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups: control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated at 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics, plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated. Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP, μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLC showed decreased levels of superoxide anion formation (O2-), a free radical detrimental to spermatozoa (P < 0.05). The use of 2 mg of CLC reduce VAP (P < 0.05) and did not have any beneficial effect on the evaluated parameters. Discussion: Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclodextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H2O2. They obtained superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group. The superoxide anion (O2-) is a free radical formed from molecular oxygen by the addition of an electron. It is generated spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases, and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group. Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

Addressing the Compartmentalization of Specific Integrin Heterodimers in Mouse Sperm

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

179 Decondensation of bovine spermatozoa after dithiothreitol treatment

Dithiothreitol (DTT) has been shown to reduce protamine disulfide bonds present in the outer acrosomal membrane of sperm. These disulfide bonds provide bull sperm with greater membrane stability and an increased resistance to decondensation compared with other species. (Hutchinson et al. 2017 Biophysical J. 113, 1925-1933). This can result in asynchronous fertilization due to the sperm nucleus failing to properly decondense and form male pronuclei after assisted reproductive techniques, such as IVF or intracytoplasmic sperm injection (ICSI) (Águila et al. 2017 Reproduction 154, 307-318). By reducing these bonds and disrupting the acrosomal membrane, DTT can minimize this complication, allowing the sperm nucleus to decondense more readily. This study aimed to assess bovine sperm, treated or not with DTT, for changes in morphology and to track the progress of decondensing sperm nuclei after treatment. Frozen-thawed bovine semen, utilising only egg yolk extender, was prepared by the swim-up method in the presence or absence of DTT at 5mM. After a 1-h incubation time, permitting sperm to swim up, sperm in the top 0.5mL in each culture tube were washed by centrifugation and observed hourly. Changes in morphology of DTT-treated and nontreated sperm were observed through a combination of phase-contrast and Hoffman modulation contrast microscopy. To assess DNA dispersal, DTT-treated sperm were stained with Hoechst and viewed under fluorescence microscopy. The DTT-treated sperm exhibited time-dependent changes in morphology, including altered movement, head folding at the equatorial segment, bending and swelling of the mid-piece, and expansion of the sperm head accompanied by a cratered appearance, signalling a partially decondensed nucleus, which progresses to full decondensation, and last, complete dispersal of DNA. These changes were seen over a 7-h incubation period in HEPES-TALP. No such changes were observed in nontreated sperm. The proportion of sperm in each stage of decondensation was estimated by a single observer at hourly time points in at least 8 repetitions. Mean estimated proportions of each stage of decondensation over time was analysed through factorial ANOVA and found significant (P&lt;0.001). An estimated 90% of treated sperm displayed early changes in morphology after the second hour of incubation. Sperm displaying partial decondensation of the nucleus was estimated as 42% by the third hour and increased to 62% by the fifth hour of incubation. Fully decondensed sperm or sperm with completely dispersed DNA increased from 20% at 5h to 50% at 7h. A remaining 10% of sperm remained unaffected and showed no changes to morphology. These results were found to be in agreement with other studies and indicate that 5mM DTT treatment induces decondensation of bovine sperm nuclei, which may assist in male pronuclei formation for other assisted reproductive technologies.