The Sperm Protein Spaca6 is Essential for Fertilization in Zebrafish

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine sperm lacking SPACA6, which was reported to be able to bind but unable to fuse with oocytes. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.

Sperm Selection for ICSI: Do We Have a Winner?

In assisted reproductive technology (ART), the aim of sperm cells’ preparation is to select competent spermatozoa with the highest fertilization potential and in this context, the intracytoplasmic sperm injection (ICSI) represents the most applied technique for fertilization. This makes the process of identifying the perfect spermatozoa extremely important. A number of methods have now been developed to mimic some of the natural selection processes that exist in the female reproductive tract. Although many studies have been conducted to identify the election technique, many doubts and disagreements still remain. In this review, we will discuss all the sperm cell selection techniques currently available for ICSI, starting from the most basic methodologies and continuing with those techniques suitable for sperm cells with reduced motility. Furthermore, different techniques that exploit some sperm membrane characteristics and the most advanced strategy for sperm selection based on microfluidics, will be examined. Finally, a new sperm selection method based on a micro swim-up directly on the ICSI dish will be analyzed. Eventually, advantages and disadvantages of each technique will be debated, trying to draw reasonable conclusions on their efficacy in order to establish the gold standard method.

Integrity of Sperm Cell Membrane in the Semen of Crossbred and Purebred Boars during Storage at 17 °C: Heterosis Effects

The aim of the study was to assess changes in the integrity of sperm cell membranes during the storage of semen collected from Duroc × Pietrain crossbred boars and purebred boars of the component breeds. To compare the cell membrane integrity of sperm heads in crossbred and purebred boars, heterosis effects were estimated. The study was conducted on 48 ejaculates collected from Duroc × Pietrain crossbred boars and from purebred Duroc and Pietrain boars used for artificial insemination. Microscope slides were prepared from each ejaculate for the evaluation of the cell membrane integrity of the sperm, at 1, 24, 48, 72, and 96 h after collection of the ejaculate. Diluted ejaculates were stored at 17 °C. Sperm membrane integrity was analysed by two methods: SYBR-14/PI and eosin–nigrosin. Our results showed that the cell membrane integrity of sperm heads changed with storage time, but the extent of the changes varied depending on the genetic group of boars. The semen of Duroc × Pietrain crossbreds was clearly seen to be less sensitive to storage conditions than that of boars of the parent breeds, which was confirmed by the calculated heterosis effects. The percentage of sperm with an intact cell membrane was higher in crossbred boars than in purebred boars (p ≤ 0.05). In addition, significantly fewer moribund sperm spermatozoa and spermatozoa with a damaged cell membrane were observed in crossbred boars (p ≤ 0.05). In the semen of purebred Duroc and Pietrain boars, the cell membrane integrity of the sperm should be assessed more often during storage than in the semen of Duroc × Pietrain crossbred boars. This study provides valuable information for the development and implementation of semen quality monitoring in crossbred boars and boars of the parent breeds during storage at 17 °C with respect to the cell membrane structure of sperm heads. The evaluation methods used effectively identify damage to the cell membranes of the sperm during semen storage.

The sperm protein Spaca6 is essential for fertilization in zebrafish

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine SPACA6, which was reported not to be required for sperm-egg membrane binding but necessary for fusion. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.

Fractionated Seminal Plasma of Boar Ejaculates Analyzed by LC–MS/MS: Its Effects on Post-Thaw Semen Quality

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC–MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC–MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

Conserved Mechanism of Bicarbonate-Induced Sensitization of CatSper Channels in Human and Mouse Sperm

To fertilize an egg, mammalian sperm must undergo capacitation in the female genital tract. A key contributor to capacitation is the calcium (Ca2+) channel CatSper, which is activated by membrane depolarization and intracellular alkalinization. In mouse epididymal sperm, membrane depolarization by exposure to high KCl triggers Ca2+ entry through CatSper only in alkaline conditions (pH 8.6) or after in vitro incubation with bicarbonate (HCO3–) and bovine serum albumin (capacitating conditions). However, in ejaculated human sperm, membrane depolarization triggers Ca2+ entry through CatSper in non-capacitating conditions and at lower pH (&lt; pH 7.4) than is required in mouse sperm. Here, we aimed to determine the mechanism(s) by which CatSper is activated in mouse and human sperm. We exposed ejaculated mouse and human sperm to high KCl to depolarize the membrane and found that intracellular Ca2+ concentration increased at pH 7.4 in sperm from both species. Conversely, intracellular Ca2+ concentration did not increase under these conditions in mouse epididymal or human epididymal sperm. Furthermore, pre-incubation with HCO3– triggered an intracellular Ca2+ concentration increase in response to KCl in human epididymal sperm. Treatment with protein kinase A (PKA) inhibitors during exposure to HCO3– inhibited Ca2+ concentration increases in mouse epididymal sperm and in both mouse and human ejaculated sperm. Finally, we show that soluble adenylyl cyclase and increased intracellular pH are required for the intracellular Ca2+ concentration increase in both human and mouse sperm. In summary, our results suggest that a conserved mechanism of activation of CatSper channels is present in both human and mouse sperm. In this mechanism, HCO3– in semen activates the soluble adenylyl cyclase/protein kinase A pathway, which leads to increased intracellular pH and sensitizes CatSper channels to respond to membrane depolarization to allow Ca2+ influx. This indirect mechanism of CatSper sensitization might be an early event capacitation that occurs as soon as the sperm contact the semen.

Beneficial Role of Ginger Powder (Zingiber officinale) against Acephate-induced Reprotoxicity in Adult Male Rats

Aims: The present study was aimed to investigate the protective role of ginger against acephate-induced testicular toxicity in adult rats. Methodology: Rats were allocated into four groups where animals in group I served as controls, while animals in group II, III and group IV were treated as experimental rats. Rats in groups II, III and IV were treated with acephate (50mg/kg body weight), ginger (100mg/kg body weight) and combination of both acephate and ginger, respectively over a period of 60 days. After completion of experimental period sperm count, sperm viability, sperm motility, sperm membrane integrity, testicular steroidogenic marker enzymes (3β-HSD and 17β-HSD, serum testosterone and testicular architecture was performed in both control and experimental rats. Results: Relative weights of reproductive organs, sperm count, sperm viability, sperm motility and sperm membrane integrity were significantly decreased in acephate treated rats over controls. Acephate administration also reduced the circulatory levels of testosterone associated with a significant reduction in the testicular steroidogenic marker enzymes (3β-HSD and 17β-HSD) in rats. The testicular architecture was disrupted in acephate intoxicated rats. In contrast, ginger administration significantly recovered the acephate-induced suppressed selected reproductive parameters with increased circulatory levels of testosterone and restoration of sperm endpoints in as compared to acephate alone treated rats. No significant changes were observed in any of the selected reproductive endpoints in ginger treated rats as compared to controls. Conclusion: The results can be concluded that supplementation of ginger mitigates the negative effects of acephate on male reproductive health via amelioration of testicular setroidogenesis and spermatogenesis and epididymal sperm maturation events in rats.

Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility

Pregnancy rates in ewes are markedly low after cervical insemination with frozen-thawed sperm. Sensitivity of ram sperm to freeze-thawing is related to the lipid composition of the membrane, particularly to its low sterol content. Recently, we proved that sterol content of ram sperm can be increased by treatment with methyl-β-cyclodextrin-sterol complexes and we provided mechanistic based evidence on the differential behavior of cholesterol and desmosterol in the ram sperm membrane. In the present study, we evaluated the role of increasing cholesterol and desmosterol content of ram sperm before cryopreservation, on the extent and distribution of sterols, cryocapacitation status, acrosome integrity, DNA damage associated with apoptosis and fertility competence in vitro and in vivo of post-thawed sperm. After freeze-thawing, similar levels of sterol content were evidenced in control sperm cells and in those pre-incubated with either cholesterol or desmosterol. Still, moderately higher levels of sterols were registered in treated sperm compared to the control, indicating no physiological excess of sterols after thawing or sterol losses that exceed the control. Live cell imaging of fluorescent cholesterol evidenced the presence of sperm sub-populations differentially affected by freeze-thawing. Similar unimodal frequency profiles were observed between sterol-enriched groups, while the control exhibited a sub-population of sperm compatible with low sterol content. Tyrosine phosphorylation was significantly lower when ram sperm incorporated cholesterol compared to the control. No difference in this capacitation parameter was found between the latter and desmosterol-enriched sperm. The percentage of sperm with damaged acrosomes post-thawing, assessed by a fluorescent lectin, was reduced in sperm that incorporated sterols before freezing, irrespective of the sterol class. These results suggest that sterols exert a stabilizing effect on the acrosome. No differences were found in levels of apoptotic DNA fragmentation among experimental groups. As to fertility trials, desmosterol-enriched sperm gave rise to higher rates of in vitro activated oocytes by heterologous fertilization and to significantly lower pregnancy loss in vivo. Our research provides new insights on sterol incorporation into ram sperm prior to cryopreservation, in particular on the additional benefit of incorporating desmosterol as a strategy to improve fertility outcome.

Assessment of quality in specific fractions of Large White Yorkshire boar semen

Boar semen is voluminous and ejaculated as jets or fractions of pre-sperm, sperm rich (SRF) and post-sperm rich fractions. Recent studies have reported more resilient characteristics of sperm in initial portions of SRF towards cold shock and cryopreservation. The present study was conducted to assess the quality of specific fractions of SRF, namely, first 10mL of SRF (F1) and rest of SRF (F2) in Large white Yorkshire (LWY) boar semen. Ejaculates were collected using gloved-hand technique and were subjected to quality assessments of volume, pH, sperm progressive motility, concentration, plasma membrane integrity, abnormality, acrosome integrity and sperm membrane cholesterol. Upon statistical analysis, significant differences were noticed in volume, pH, sperm concentration and sperm membrane cholesterol between fractions of the ejaculate.