Subfamily hypostominae: similarities and differences in testicular structure of amazonian fish

Background Hypostominae is a subfamily of the family Loricariidae that has a great diversity of species. Accordingly, testicular studies in fish can contribute to the phylogeny and taxonomy of species and to the comparison of reproductive aspects between species. Thus, this work aimed to characterize the testicular morphology and spermatogenesis of the Hypostominae species Baryancistrus xanthellus, Peckoltia oligospila and Hypancistrus zebra. Results B. xanthellus, P. oligospila and H. zebra had an anastomosed tubular type of testis. The germinal epithelium was continuous with unrestricted spermatogonia, and the proliferative, meiotic and spermiogenic phases were defined in all species. In the spermiogenic phase, spermatids were classified as initial, intermediate and final. Only in B. xanthellus in the final phase was there nuclear rotation. The sperm for the three species had a head with an oval shape and a single flagellum composed of the short midpiece, principal piece and end piece. B. xanthellus and P. oligospila showed a cylindrical flagellum and H. zebra showed projections that produced a flattened appearance. Conclusions On the basis testicular structure and ultrastructural characteristics of the germ cells, there was a greater relationship between B. xanthelus and P. oligospila, while H. zebra had particular characteristics. These aspects show that despite belonging to the same subfamily, the species have distinct biological characteristics.

Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits

Bottlenose dolphin (Tursiops truncatus) males follow many reproductive strategies to ensure their paternity. However, little is known about the sperm traits, including morphometric features, that contribute to their reproductive success. Our aim was to study dolphin sperm morphometry (a total of 13 parameters) in two adult males to evaluate (i) presumptive sperm subpopulations, (ii) the correlation of sperm morphometry with testosterone levels and (iii) the effect of refrigerated storage on the sperm morphometry. Sperm populations were classified into four principal components (PCs) based on morphometry (>94% of cumulative variance). The PCs clustered into two different sperm subpopulations, which differed between males. Furthermore, the levels of serum testosterone were positively correlated with the length of the midpiece but negatively correlated with head width and the principal piece, flagellum and total sperm lengths. Most of the sperm morphometric parameters changed during the storage period (day 1 vs. day 7), but only the principal piece length was affected by the storage temperature (5 °C vs. 15 °C). This is the first study to identify dolphin sperm subpopulations based on morphometry and the influence of serum testosterone and refrigeration on sperm morphometry.

Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece–principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1−/− mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

103 Characterization of the receptors of the endocannabinoid system in equine sperm: Possible role of anandamide in sperm function

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.

Cdc42 activity is essential for the interplay between cAMP/PKA pathway and CatSper function

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+-dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by sAC, providing a new regulatory mechanism for the stimulation of CatSper by the cAMP/PKA-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

Ultrastructure of Spermatozoa of Elaphe schrenckii (Reptilia, Squamata)

Elaphe schrenckii (Serpentes, Colubridae), a kind of large nonvenomous snakes and great significance to maintain the stability of ecosystem in China. We provide detailed descriptions of the sperm microstructure and ultrastructure of E. schrenckii, experimented by light microscope and transmission electron microscope. The spermatozoon of E. schrenckii is filiform and consists of head and tail regions. The cross-section of acrosomal vesicle is always rounded and divided into medulla inside and cortex outside. The ultrastructure of acrosome complex observed the unilateral ridge, the single perforatorium, the perforatorium base plate, the epinuclear lucent zone, the subacrosomal space and the nuclear fossa at the end of nucleus connect the neck region. The neck region is short with the stratified laminar structure and observed the distal centriole and the proximal centriole are perpendicular and both consisted of nine triplets. Midpiece is long and observed the extracellular microtubules, the multilaminar membranec, the mitochondria with the dense bodies discontinuity distribting, the fibrous sheath, and the axoneme. The principal piece is after the annulus with no mitochondrias and the end piece with no mitochondrias neither the fibrous sheath. Our study contrasted the spermatozoa ultrastructure of 8 species belong to 5 families and 6 genera and added the sperm measurement compare, summarized that three Colubridae snakes are more similar than others momentarily but some specific characteristics in E. schrenckii and proved that the ultrastructure of sperm related to phylogeny in some ways.

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg’s salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg’s salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-h incubation and localized to the principal piece in some sperm within 24 h. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.

Localisation and function of glucose transporter GLUT1 in chicken (Gallus gallus domesticus) spermatozoa: relationship between ATP production pathways and flagellar motility

Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter 1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1 was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during incubation for 0–80min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.

Elucidating the Role of K+ Channels during In Vitro Capacitation of Boar Spermatozoa: Do SLO1 Channels Play a Crucial Role?

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K+-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.