Sperm Activation Research Articles

Runjing Decoction alleviated cyclophosphamide-induced oligoasthenospermia rats by inhibiting cell apoptosis via RXFP1/AKT/FOXO1 pathway.

Runjing Decoction (RJD) is a prescription of traditional Chinese medicine for the treatment of oligoasthenospermia. However, the molecular mechanism of RJD on oligoasthenospermia still remains unknown. A model of oligoasthenospermia was induced in 30 Sprague Dawley rats by intraperitoneal injection of cyclophosphamide at 35mg/kg per day for 5days and treated by intragastric RJD (13.5g/kg) or L-carnitine (100mg/kg) for 14days. The body weight, testis and epididymis weight, grade A spermatozoa, grade B spermatozoa, the percentage of sperm forward motility (PR%), the sperm activity rate and the sperm density of rats were evaluated before and after RJD treatment. The testis apoptosis was determined by TUNEL staining. The expressions of RXFP1, FoxO1, PI3K, Akt, Bax and Bcl-2 were determined by qRT-PCR and Western blot, respectively. After RJD treatment, the grade A spermatozoa, sperm PR%, sperm activity and sperm density were significantly increased relative to those in model rats. Cell apoptosis of testis tissue was reversed by RJD. RJD suppressed cell apoptosis, inhibited the expression of RXFP1, FOXO1, PI3K, AKT and Bax, and promoted the expression levels of Bcl-2 in testicular tissue of oligoasthenospermia rats. RJD could alleviate sperm quality and testis damage in oligoasthenospermia rats by inhibiting RXFP1/AKT/FOXO1 pathway.

Vaginal Probiotic Lactobacillus crispatus Seems to Inhibit Sperm Activity and Subsequently Reduces Pregnancies in Rat.

BackgroundThe vaginal microbiota is associated with the health of the female reproductive system and the offspring. Lactobacillus crispatus belongs to one of the most important vaginal probiotics, while its role in the agglutination and immobilization of human sperm, fertility, and offspring health is unclear.MethodsAdherence assays, sperm motility assays, and Ca2+-detecting assays were used to analyze the adherence properties and sperm motility of L. crispatus Lcr-MH175, attenuated Salmonella typhimurium VNP20009, engineered S. typhimurium VNP20009 DNase I, and Escherichia coli O157:H7 in vitro. The rat reproductive model was further developed to study the role of L. crispatus on reproduction and offspring health, using high-throughput sequencing, real-time PCR, and molecular biology techniques.ResultsOur results indicated that L. crispatus, VNP20009, VNP20009 DNase I, and E. coli O157:H7 significantly inhibited the sperm motility in vitro via adversely affecting the sperm intracellular Ca2+ concentration and showed a high adhesion to sperms. The in vivo results indicated that L. crispatus and other tested bacteria greatly reduced the pregnancy rates, but L. crispatus had a positive effect on maternal health and offspring development. Moreover, the transplantation of L. crispatus could sustain a normal bacterial composition of the vaginal microbiota in healthy rats and markedly reduced the expression of uterine inflammatory factors (toll-like receptor-4/nuclear factor kappa-B, tumor necrosis factor-α, production of interleukin-1β, etc.) and apoptosis factors (Fas Ligand, Bcl-2-associated X protein/B cell lymphoma-2, etc.) compared with the other tested strains.ConclusionOur study demonstrated that the vaginal probiotic L. crispatus greatly affected the sperm activity and could also reduce pregnancies through its adhesion property, which might account for some unexplained infertility. Therefore, more caution should be paid when using L. crispatus as a vaginal viable preparation in women of child-bearing age, especially for women whose partners have abnormal sperms.

Expression of miR-135a-5p and its target gene JAK2 in spermatozoa of patients with asthenozoospermia.

This study aimed to identify the molecular mechanism by which JAK2 mRNA and microRNA-135a-5p (miR-135a-5p) affect asthenozoospermia. The expression levels of JAK2 mRNA in the spermatozoa of 30 asthenozoospermia patients and 30 normal controls were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). MiR-135a-5p that targeted JAK2 mRNA was predicted by bioinformatics. A dual luciferase reporter vector containing miR-135a-5p 3'UTR was constructed. The binding of miR-135a-5p to JAK2 mRNA was verified by luciferase reporter assay. The protein expression levels of JAK2 and STAT3 in spermatozoa were examined by Western bolt. The relative expression levels of JAK2 mRNA and protein in the asthenozoospermia group were significantly lower than those of the normal group. MiR-135a-5p overexpression inhibited JAK2 mRNA and protein expression by targeting JAK2 mRNA 3'UTR. Correlation analysis showed that miR-135a-5p level was significantly negatively correlated with progressive sperm motility, while JAK2 mRNA level was significantly positively correlated with progressive sperm activity. Low expression of JAK2 mRNA and high expression of miR-135a-5p were related to asthenozoospermia and male infertility.

Competitive CatSper Activators of Progesterone from Rhynchosia volubilis.

The root Rhynchosia volubilis was widely used for contraception in folk medicine, although its molecular mechanism on antifertility has not yet been revealed. In human sperm, it was reported that the cation channel of sperm, an indispensable cation channel for the fertilization process, could be regulated by various steroid-like compounds in plants. Interestingly, these nonphysiological ligands would also disturb the activation of the cation channel of sperm induced by progesterone. Therefore, this study aimed to explore whether the compounds in R. volubilis affect the physiological regulation of the cation channel of sperm. The bioguided isolation of the whole herb of R. volubilis has resulted in the novel discovery of five new prenylated isoflavonoids, rhynchones A – E ( 1 – 5 ), a new natural product, 5′- O -methylphaseolinisoflavan ( 6 ) ( 1 H and 13 C NMR data, Supporting Information), together with twelve known compounds ( 7 – 18 ). Their structures were established by extensive spectroscopic analyses and drawing a comparison with literature data, while their absolute configurations were determined by electronic circular dichroism calculations. The experiments of intracellular Ca 2+ signals and patch clamping recordings showed that rhynchone A ( 1 ) significantly reduced cation channel of sperm activation by competing with progesterone. In conclusion, our findings indicat that rhynchone A might act as a contraceptive compound by impairing the activation of the cation channel of sperm and thus prevent fertilization.

A differential proteomic study reveals the downregulation of several plasma membrane Ca2+-binding proteins in the sperm of Atlantic salmon (Salmo salar) following cold storage

Sperm motility is a key event in fertilization, which is regulated by different factors. Among the factors that most affect sperm motility in fish are the external concentrations of Ca2+, the influence of which is highly regulated by various plasma membrane Ca2+-binding proteins. Concentrations of this cation have also been shown to be one of the most important factors affecting motility in cold-stored sperm cells. Taking into account the aforementioned aspects, we carried out a differential proteomic study on Salmo salar sperm stored at 4 °C with the aim of evaluating the protein profile on day 0 and day 14. The results of our study showed that 401 proteins were significantly downregulated (p < 0.05) on day 14, where four of them are key in the sperm motility of Salmo salar. The results of this study will allow a better understanding of the sperm activation mechanisms of Salmo salar, which will be of great importance in the design of future cold storage strategies for sperm preservation.

Cryopreservation of semen of initial forms and chimeras of birds of two breeds: Sussex light and Poltava clay

Purpose: evaluation of the quality of seed chimer and source forms before and after cryopreservation.Materials and methods. For research, the breed of Sussex chickens and Poltava clay. Using these rocks, chimeras were obtained by the method of transplantation of blastodermal cells. An assessment of the quality of sperm of the roosters was carried out under a microscope at a temperature of 42 degrees. At various times, the activity of native sperm and its concentration was determined. Cryoconservation was carried out in small granules. The defrosting of the granules was produced on a heated metal plate at a temperature of 60 ° C.Results. A comparative analysis of the volume of the ejaculate showed that Himer on average it was somewhat larger than that of the initial forms. The activity of sperms in native genital cells was sufficiently high in all studied samples, and was 80% or more. The concentration of sperm retained the same trend and practically did not differ from each other in all studied samples. The activity of frozen-fatty seed of the initial breeds and their chimer was significantly different. The maximum activity was observed in the Sussex breed and reliably below (p≤ 0.01) it has the remaining experimental groups. The activity of frozen-fatty seed turned out to be equal in chimeric organisms and Poltava clay, although Himer recipients were sussexes. It should be noted that the swing of oscillations according to the degree of activity of frozen-fatty seed in chimeric birds was significant (from 10 to 40 points), and the number of observations is small.Conclusion. The study assess the quality of seed chimer and the source forms before and after cryopreservation. It was demonstrated that after defrosting the activity of frozen-fatty seed of the initial rocks and their chimer was different.

Sperm DNA fragmentation and total antiradical activity of sperm in men with exaggerated anamnesis

Various intracellular and extracellular stimuli can promote excessive accumulation of reactive oxygen species and lead to detrimental effects on sperm function, in particular, damage to sperm DNA, thus often resulting into infertility. The aim of the study was to assess the degree of sperm DNA fragmentation by flow cytometry and to study total antiradical activity of sperm in men with infertility and chronic prostatitis using spectrophotometric technique. A total of 75 men were examined. The comparison group consisted of 45 men with chronic prostatitis beyond exacerbation. The observation group included 30 men with chronic prostatitis in remission state. We have found that in the subjects during the period of acute inflammation, pathological fragmentation of sperm DNA was revealed, being statistically significant (p = 0.004). It was registered 1.7 times more often than in the examined men during the remission of chronic prostatitis. Increased fragmentation of the sperm DNA was noted in 50% of men from comparison group. In the patients from observation group, the degree of sperm DNA fragmentation over reference ranges was observed in 80% of the subjects. In men diagnosed with chronic prostatitis at the exacerbation stage, there is a statistically significant (p 0.05) relationship between sperm DNA fragmentation and development of acute inflammatory process. It was shown that in the subjects from observation group, the mean value of antiradical sperm activity did not show statistically significant differences (p = 0.378) compared with results found in men from the comparison group. The presented results suggest a higher degree of sperm DNA fragmentation in the examined patients with chronic prostatitis at the stage of decompensation, which may point to excessively increased production of free radicals during the acute inflammatory process. Obviously, exacerbation of chronic prostatitis contributes to imbalance in the redox homeostatic system in men, thus creating prerequisites for excess of reactive oxygen species and significant fragmentation of sperm DNA. The identified changes, i.e., an increased sperm DNA fragmentation levels and a deficiency of total antiradical activity of sperm in men with chronic urogenital diseases, may play a pathogenetic role in occurrence and further development of infertility. The degree of sperm DNA fragmentation and markers of redox processes may serve as informative activity indices of the inflammatory process.

Breeding of Cav2.3 deficient mice reveals Mendelian inheritance in contrast to complex inheritance in Cav3.2 null mutant breeding

High voltage-activated Cav2.3 R-type Ca2+ channels and low voltage-activated Cav3.2 T-type Ca2+ channels were reported to be involved in numerous physiological and pathophysiological processes. Many of these findings are based on studies in Cav2.3 and Cav3.2 deficient mice. Recently, it has been proposed that inbreeding of Cav2.3 and Cav3.2 deficient mice exhibits significant deviation from Mendelian inheritance and might be an indication for potential prenatal lethality in these lines. In our study, we analyzed 926 offspring from Cav3.2 breedings and 1142 offspring from Cav2.3 breedings. Our results demonstrate that breeding of Cav2.3 deficient mice shows typical Mendelian inheritance and that there is no indication of prenatal lethality. In contrast, Cav3.2 breeding exhibits a complex inheritance pattern. It might be speculated that the differences in inheritance, particularly for Cav2.3 breeding, are related to other factors, such as genetic specificities of the mutant lines, compensatory mechanisms and altered sperm activity.

Прооксидантно-антиоксидантний гомеостаз та відтворювальна здатність кнурів-плідників за впливу цитрату міді

Peroxide oxidation processes play a leading role in ensuring the motility, survival and fertilizing ability of sperm. A special role is given to limiting antioxidants (vitamins, amino acids, microelements). Therefore, the development of standardized feeding programs to provide antioxidant nutrition is one of the effective methods of reproductive biotechnology. The aim of the study was to determine the effect of copper citrate on the quality of sperm production and the formation of prooxidant-antioxidant homeostasis in sperm of boars. The experiment used adult boars of a large white breed, analogs in age, live weight and quality of sperm products. Experimental groups were fed copper citrate above the norm by 10% and 20%. It has been determined that feeding combined feed to boars with the addition of this compound in an amount of 10% above norm probably increases the weight of ejaculate by 12.5% (P&lt;0.05), the sperm motility and survival by 6.5% (P&lt;0.01) and 13.5% (P&lt;0.001), respectively. Such changes in sperm occur against the background of an increase in SOD activity by 80.6% (P&lt;0.05), a decrease in catalase by 43.5% (P&lt;0.05), a slowing down of peroxidation processes — a decrease in diene conjugates and TBA-active compounds. The additional introduction to the diet of copper citrate by 20% more than normal increases the concentration of spermatozoa by 13.2% (P&lt;0.01), the number of live spermatozoa by 20.7% (P&lt;0,01), with a simultaneous decrease in their survival, due to the acceleration of peroxidation processes — an increase in the content of diene conjugates, TBA-active compounds and DAA and a decrease in reduced glutathione. It has been found out that the fertilizing ability of sperm significantly depended on the amount of fed microelement. Sows inseminated with sperm of boars receiving copper supplement in the diet by 10%, had higher fertility rates by 7.1%, multifertility by 3.6%, and a litter weight at weaning by 8.8%. The additional administration of copper citrate reduced the fertility of sperm by 20%, as the fertility rate of sows of III group was the lowest and was 7.7% and 14.3% lower compared to I and II groups. A similar trend occurred in terms of high fertility, a litter weight at birth and weaning. Therefore, the additional feeding of a small amount of copper has a positive effect on the functional activity of sperm and the processes of normal fertilization, growth and development of embryos and newborn piglets by optimizing the formation of prooxidant-antioxidant homeostasis.

Electromagnetic Radiations on the Functional Potential of Spermatozoa

The growing exposure to digital communication system and tools leads to radiation toxicity to the users. Unaware of the safety measures even a kid at the age of one starts operating digital gadgets emitting Radio frequencyelectromagnetic radiations. Radiations from mobile phones laptops, note pad, Wi-Fi or other devices are reported to be harmful beyond the permissible limit. So there is a growing concern for the overall health, reproductive, and hormonal functions. Experimental studies were conducted by using a Wi-Fi network active laptop and live spermatozoa. The exposure of sperms to the source of EMF showed that the activity of the live sperms got reduced and mortality was observed depending on the exposure duration and the distance from EMF sources. After 5h of exposure to RF-EMF source the semen quality changed when compared with control. The vitality of sperm in the control was 95±1.0 after 2 hr and it was reduced to 60±1.5 percent at a distance of 1cm and 55±2.2 percent at a distance of 10 cm. After 5 hour exposure, the vitality was reduced to 63±1.86 at 1 cm distance and 70±1.42 at 10 cm distance. The reduction in vitality of sperm after exposure to RF-EMF source for 2 hour was 35% at 1cm and 40% at 10cm distance.

Identification and Synthesis of DDI-6, a Quinolinol Analog Capable of Activating Both Caenorhabditis elegans and Mouse Spermatozoa.

Sperm activation is an essential process by which the male gametes become capable of fertilization. Because the process in Caenorhabditis elegans is readily reproducible in vitro, this organism serves as an excellent model to investigate it. C. elegans sperm activation in vivo occurs during spermiogenesis. Membranous organelles (MOs) contained within spermatids fuse with the plasma membrane, resulting in extracellular release of their contents and relocation of some proteins indispensable for fertilization from the MO membrane onto the sperm surface. Intriguingly, these cytological alternations are exhibited similarly in mouse spermatozoa during the acrosome reaction, which also represents a form of sperm activation, prompting us to hypothesize that C. elegans and mice share a common mechanism for sperm activation. To explore this, we first screened a chemical library to identify compounds that activate C. elegans spermatozoa. Because a quinolinol analog named DDI-6 seemed to be a candidate sperm activator, we synthesized it to use for further analyses. This involved direct dechlorination and hydrogenolysis of commercially available 5-chloro-8-quinolinol, both of which are key steps to yield 1,2,3,4-tetrahydro-8-quinolinol, and we subsequently introduced the sulfonamide group to the compound. When C. elegans spermatids were stimulated with solvent alone or the newly synthesized DDI-6, approx. 3% and approx. 28% of spermatids became MO-fused spermatozoa, respectively. Moreover, DDI-6 triggered the acrosome reaction in approx. 20% of mouse spermatozoa, while approx. 12% became acrosome-reacted after mock stimulation. Thus, DDI-6 serves as a moderately effective activator for both C. elegans and mouse spermatozoa.

Motility activation and short-term storage of silver carp, Hypopthalmichthys molitrix, sperm

Sperm activation and short-term storage have important implications in breeding programs. Although silver carp, Hypophthalmicthys molitrix, is an important aquaculture species in South Asia, information on sperm biology and storage is limited. The study aims to test the efficacy of NaCl solutions for activation and short-term storage of silver carp sperm. We found that the motility of silver carp sperm lasted for 45 sec after activation in distilled water. Sperm motility was higher within the first 10 sec of sperm activation, but sperm movement decreased with increasing activation time. When sperm was suspended a graded dilution of NaCl solutions (17 mM to 188 mM), sperm was immobilized at 154mM NaCl (287 mOsmol kg–1). The efficacy of sperm immobilizing solutions was validated during short-term storage at 4°C. A total of 7.1 ± 3.1% sperm was motile in 154 mM NaCl after 5 days of storage. Sperm motility lasted up to 2 days when sperm diluted in 103, 120, 171 and 188 mM NaCl solutions. The osmotic balance between seminal plasma and NaCl solution prepared at 154 mM prevented sperm activation and provided a suitable media for short-term storage. A 154 mM NaCl solution can be used for sperm collection and short-term storage of silver carp sperm.

Glutathione Content and Glutathione Peroxidase Activity of Sperm in Males with Unexplained Infertility

Summary The study aimed to investigate glutathione peroxidase (GPx) activity and glutathione (GSH) levels in the sperm of patients with unexplained infertility. The sperm samples were collected from subjects with normal semen parameters divided into fertile and infertile groups. Sperm analysis was performed according to the 2010 WHO criteria. Measurement of the GPx activity and GSH were performed by enzymatic assay kits. The higher enzymatic activity recorded in spermatozoa and seminal plasma in the infertile group was close to the significant one – p=0.054 for seminal plasma andp= 0.086 for the spermatozoa.GSH levels were higher in the fertile group in the seminal plasma (p=0.045). Defining the causes of unexplained infertility requires the addition of oxidative stress. In patients with unexplained infertility, the level of glutathione is reduced, and the activity of one of the significant enzyme antioxidants GPx is not changed significantly and even shows a tendency to rise.

Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)).

Simple SummaryFor scientific studies on the zebrafish model, simple and routine reproductive procedures should be used to ensure stable and repeatable results. When the milt is collected, spermatozoa are spontaneously activated by urine or excrement (low osmolarity) which routinely contaminates the samples, because of the minuscule size of the fish body. Therefore, whenever milt is collected from a zebrafish for short-term milt preservation and artificial fertilization, milt must be collected into an immobilizing solution, which because of its high osmolarity stops the movement of spermatozoa and keeps the sperm immobile until fertilization. Usually, the spermatozoa showed forward movement during the 35 s period following dilution in water. The sperm concentration ranged from 0.08 to 3.52 × 109/mL with a volume from 0.1 to 2.0 μL per male. The most suitable extender proved to be E400, which allowed storage of sperm for fertilization for 6 to 12 h at a temperature of 0–2 °C. To achieve a good level of fertilization and hatchability, a test tube with a precisely defined amount of sperm with extender, eggs and activating solution proved to be the most effective.The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0–2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40–60%, respectively, with milt stored for 1.5 h in the E400 extender at 0–2 °C.

Membrane contact site-dependent cholesterol transport regulates Na+/K+-ATPase polarization and spermiogenesis in Caenorhabditis elegans.

Spermiogenesis in nematodes is a process whereby round and quiescent spermatids differentiate into asymmetric and crawling spermatozoa. The molecular mechanism underlying this symmetry breaking remains uncharacterized. In this study, we revealed that sperm-specific Na+/K+-ATPase (NKA) is evenly distributed on the plasma membrane (PM) of Caenorhabditis elegans spermatids but is translocated to and subsequently enters the invaginated membrane of the spermatozoa cell body during sperm activation. The polarization of NKA depends on the transport of cholesterol from the PM to membranous organelles (MOs) via membrane contact sites (MCSs). The inositol 5-phosphatase CIL-1 and the MO-localized PI4P phosphatase SAC-1 may mediate PI4P metabolism to drive cholesterol countertransport via sterol/lipid transport proteins through MCSs. Furthermore, the NKA function is required for C. elegans sperm motility and reproductive success. Our data imply that the lipid dynamics mediated by MCSs might play crucial roles in the establishment of cell polarity. eGraphical abstract.

Lycopene Attenuates Hypoxia-Induced Testicular Injury by Inhibiting PROK2 Expression and Activating PI3K/AKT/mTOR Pathway in a Varicocele Adult Rat.

Purpose The aim of this study was to evaluate the effect of lycopene on hypoxia-induced testicular injury in rat model and explore the underlying mechanism. Methods Six-week-old male Wistar rats (n = 36) were randomly divided into three groups (n = 12/group): a normal group (NG, sham control), a varicocele group (VG), and a varicocele treated by lycopene group (VLG). Bilateral renal veins constriction was performed on rats in VG and VLG. Simultaneously, rats in VLG were treated to lycopene by intragastric administration. Four weeks later, sperm was collected for sperm analysis. Testes and epididymides were harvested for morphological change analysis, histologic analysis, ELISA, qRT-PCR, and western blot. Results Our observations were that lycopene improved the hypoxia-induced testicular injury in vivo. Prokineticin 2(PROK2) and prokineticin receptor 2 (PROKR2) were overexpressed in VG (P < 0.01), and lycopene inhibited the PROK2 expression (P < 0.01). Proliferating cell nuclear antigen (PCNA) and sex hormones were increased by lycopene in VLG (P < 0.05). Lycopene restored the quality and activity of sperm by blocking PROK2 expression (P < 0.05). The expression of VEGF was increased, as HIF-1/NF-κB pathway was upregulated in VLG (P < 0.05). Meanwhile, expression of pAKT/AKT in VLG was higher than that in VG (P < 0.05). In addition, lycopene reduced levels of interleukin-1β (IL-1β) and interleukin-2 (IL-2) in VLG (P < 0.05), compared to NG. Conclusions Lycopene improved the hypoxia-induced testicular injury by inhibiting the expression of PROK2 and decreasing levels of IL-1β and IL-2, which might show us a novel and promising treatment for varicocele testicular injury.

Sperm adaptation in relation to salinity in three goby species.

In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h.

Saturated fatty acids accelerate linear motility through mitochondrial ATP production in bull sperm.

PurposeThe present study was undertaken to clarify whether bovine sperm could take up fatty acids (FAs) and produce ATP to maintain linear motility.MethodsFrozen bovine semen was thawed in media containing either lipid mixture (LM) or FAs, and sperm motility was analyzed. The kinetic changes in FA levels in sperm were detected using gas chromatography‐mass spectrometry. The mitochondrial activity of sperm thawed in media containing LM or FAs was analyzed based on the fluorescence intensity of JC‐1 staining and the oxygen consumption rate. FA transporters were observed using whole‐mounted immunofluorescence.ResultsSperm linear motility was significantly (P < .05) increased after thawing in media with LM and FA. Moreover, saturated fatty acids were predominant in sperm thawed in media with LM. Notably, our study revealed that frozen bovine sperm possessed FA transporters in the midpiece where the fluorescence signals were detected after treatment with fluorescence‐tagged FA. Treatment with FA activated electron transport in mitochondria through β‐oxidation.ConclusionsSperm linear motility is facilitated by FAs in the thawing media used for frozen bovine sperm. This might provide a new approach for upgrading the artificial insemination technique used in both livestock animals and human infertility care.

Rhamdia quelen's semen pre-cryopreservation processing and sperm viability time after thawing / Processo de pré-criopreservação de sêmen de Rhamdia quelen e tempo de viabilidade do esperma após o descongelamento

The study’s aim was to evaluate the sperm movement in the semen of Rhamdia quelen, cryopreserved at different times of exposure to nitrogen vapor, besides different equilibration times and with different concentrations of methanol, and in thawed semen kept at 25ºC for different periods of time. Three distinct experiments were carried out. In the first experiment, fresh semen aliquots were diluted in a solution containing 5% D-Fructose, 5% milk powder (Nestle®, Ninho Fortificado®) and 10% methanol, filled into 0.25 mL straws and immediately kept in nitrogen vapor by 0,5; 1.0; 4.0; 8.0; 12.0 and 18.0h. After these times, the straws were transferred to the liquid nitrogen (-196°C). After 72 h, the semen was thawed by immersing the straws in water at (25 ° C) for 10 seconds. The sperm activation was evaluated by diluting the thawed semen in distilled water in the proportion of 1:250 (semen:activating solution; v/v). Motility and spermatic velocity in thawed semen were measured using the CASA system. In the second experiment, the fresh semen was diluted in solutions with glucose and powdered milk (Nestle®, Ninho Fortificado®) and 5.0; 7.5; 10.0; 12.5% methanol. Immediately after, it was filled into 0.25mL straws and kept at 25ºC by equilibrium times corresponding to 0, 20, 40 and 60 minutes. Subsequently, part of the straws was used for spermatic evaluation before cryopreservation and others were submitted to cryopreservation in nitrogen vapor for 30 minutes and after transferred to liquid nitrogen (-196°C). The processes of thawing and sperm evaluation were performed as described above. In the third experiment, the cryopreserved semen was thawed and kept at 25°C for 0.0; 7,5; 15,0; 22.5 and 30.0 minutes. After each time, the spermatic movement was evaluated as described above. The exposure time of straws to nitrogen vapor and the kept time of the thawed semen at 25ºC did not affect (p &gt; 0.05) the sperm movement. On the other hand, methanol concentrations and equilibrium time (p &lt; 0.05) influenced the spermatic movement interactively both, before and after cryopreservation. The best results were achieved when the semen was cryopreserved immediately after dilution. The cryopreservation of Rhamdia quelen semen can be successfully performed when the semen is diluted in a solution containing 5% D-Fructose, 5% milk powder and 11.66% of methanol and immediately filled into 0.25 mL straws and kept in nitrogen vapor by 30 min before being transferred to liquid nitrogen. In addition, after thawing, the semen can be kept at 25ºC for up to 30 minutes without damage to spermatic movement.

V-Set and Immunoglobulin Domain-Containing 1 (VSIG1), Predominantly Expressed in Testicular Germ Cells, Is Dispensable for Spermatogenesis and Male Fertility in Mice.

Simple SummaryV-set and immunoglobulin domain-containing 1 (VSIG1) is a newly discovered member of the junctional adhesion molecule (JAM) family encoded by a gene located on the X chromosome in humans and mice. Expression of VSIG1 in normal mammalian tissues was originally reported to be highly tissue-specific and was detected in organs such as the testis. However, as little is known about its physiological function, we aimed to investigate the function of VSIG1 in mammalian spermatogenesis and fertilization.To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.