Sperm Principal Piece Research Articles

A variant in sperm-specific glycolytic enzyme enolase 4 (ENO4) causes human male infertility.

Although defects in sperm morphology and physiology lead to male infertility, in many instances, the exact disruption of molecular pathways in a given patient is often unknown. The glycolytic pathway is an essential process to supply energy in sperm cell motility. Enolase 4 (ENO4) is crucial for the glycolytic process, which provides the energy for sperm cells in motility. ENO4 is located in the sperm principal piece and is essential for the motility and organization of the sperm flagellum. In the present study, we characterized a family with asthenozoospermia and abnormal sperm morphology as a result of a variant in the enolase 4 (ENO4) gene. Computer-assisted semen analysis, papanicolaou smear staining and scanning electron microscopy were used to examine sperm motility and morphology for semen analysis in patients. For genetic analysis, whole-exome sequencing followed by Sanger sequencing was performed. Two brothers in a consanguineous family were being clinically investigated for sperm motility and morphology issues. Genetic analysis by whole-exome sequencing revealed a homozygous variant [c.293A>G, p.(Lys98Arg)] in the ENO4 gene that segregated with infertility in the family, shared by affected but not controls. In view of the association of asthenozoospermia and abnormal sperm morphology in Eno4 knockout mice, we consider this to be the first report describing the involvement of ENO4 gene in human male infertility. We also explore the possible involvement of another variant in explaining other phenotypic features in this family.

Phosphoproteomics for the identification of new mechanisms of cryodamage: the role of SPATA18 in the control of stallion sperm function†.

Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protein 4 (AKAP4), A-kinase anchoring protein 3 (AKAP3) and the Family with sequence similarity 71 member B (FAM71B). These proteins belong to the gene ontology (GO) terms sperm fibrous sheath (GO: 0035686), and sperm principal piece (GO: 0097228). The regulatory interactions between kinases and phosphorylation sites on the proteins that were affected most were also investigated, and the potential kinases (based on human orthologs) involved in the regulation of these phosphoproteins identified were: PKCß for SPATA18 and GSK3ß for CABYR. Kinase inhibition assays were also conducted showing that kinases phosphorylating the above-mentioned proteins play an important role in their activity and thus, phosphorylation controls the activity of these proteins and their role in the regulation of the functionality and viability of stallion spermatozoa. In conclusion, the data reported here contribute to the understanding of the fact that the dephosphorylation of certain proteins is a molecular lesion induced by cryopreservation in the stallion spermatozoa.

Isoform-specific requirement for GSK3α in sperm for male fertility.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3β, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3β is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3β, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3β are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3β does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.

Comparison of the external physical damages between laser-assisted and mechanical immobilized human sperm using scanning electronic microscopy

We aim to visualize the external physical damages and distinct external phenotypic effects between mechanical and laser-assisted immobilized human spermatozoa using scanning electronic microscopy (SEM). Human spermatozoa were immobilized mechanically or with laser assistance for SEM examination and the membrane integrities were checked on both types of immobilized spermatozoa. We found evidence of external damages at SEM level on mechanically kinked sperm, but not on laser-assisted immobilized sperm. Although no external damage was found on laser-assist immobilized sperm, there were two distinct types of morphological changes when spermatozoa were stricken by infra-red laser. Coiled tails were immediately formed when Laser pulse was applied to the sperm end piece area, whereas laser applied to the sperm principal piece area resulted in a sharp bend of sperm tails. Sperm immobilized by laser did not exhibit any morphological change if the laser did not hit within the on-screen central target zone or if the laser hit the sperm mid piece or head. Our modified membrane integrity assay revealed that the external membrane of more than half of the laser-assisted immobilized sperm remained intact. In conclusion, mechanical immobilization produced membrane damages whilst laser-assisted immobilization did not result in any external membrane damages besides morphological changes at SEM level.

Sperm structure and sperm motility of the African and Rockhopper penguins with special reference to multiple axonemes of the flagellum

This study evaluated the semen of two penguin species from separate genera with reference to unique features in sperm structure using light microscopy and transmission electron microscopy. Ejaculates from African penguin (n = 51) and Rockhopper penguin (n = 9) contained on average more than 60% motile spermatozoa and a sperm concentration of 3274 × 106/ml and 1423 × 106/ml, respectively. The percentage progressive motility was similar for the two species as well as all the kinematics parameters. The sperm morphology of these two penguin species is almost identical and largely resembles that of non-passerine birds in terms of the filiform head, small acrosome and mid-piece containing 13 spherical mitochondria, arranged around the proximal and distal centrioles in a single helix. Apart from a shorter mid-piece, penguin sperm morphometrics were similar to other non-passerine birds. The ultrastructure of the sperm principal piece revealed the typical 9 + 2 microtubular arrangement without any outer dense fibres. An unusual feature in both African and Rockhopper penguin spermatozoa was the occurrence of multiple axonemes contained in one plasmalemma in 4% of spermatozoa. These double, triple and quadruple axonemal arrangements have not been described previously albeit multiple tails were reported in other bird species. It is unclear whether such a unique structural feature will be of any advantage for sperm motility and might rather be a result of the absence of sperm competition. Multiple axonemes found in penguin flagella could be an apomorphism that distinguish them from other bird spermatozoa.

Sperm flagellum volume determines freezability in red deer spermatozoa.

The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = −0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.

A Novel, Single, Transmembrane Protein CATSPERG Is Associated with CATSPER1 Channel Protein1

All four CATSPER channel pore-forming subunits (CATSPER1-4) are localized in the sperm principal piece. They form an alkalization-activated Ca2+-permeable channel and are required for sperm-hyperactivated motility, egg coat penetration, and male fertility. Unlike many other ion channels, the composition of the CATSPER protein complex is poorly defined. Herein, we describe the novel protein CATSPERG associated with the CATSPER complex. CATSPERG is predicted to be a single transmembrane-spanning protein with a large extracellular domain and a short intracellular tail. Like all the CATSPERs and the previously identified CATSPER-associated protein CATSPERB, CATSPERG is only expressed in testis and is localized in the sperm principal piece. In CATSPER1-deficient sperm, the CATSPERG protein (but not the K+ channel protein KCNU1) is also lost. Together with previous findings, our data suggest that the CATSPER protein complex contains pore-forming proteins and two additional proteins (CATSPERB and CATSPERG) and that the trafficking and/or assembly of these proteins depends on CATSPER1.

Calmodulin and CaMKII in the Sperm Principal Piece: Evidence for a Motility‐Related Calcium/Calmodulin Pathway

Both cyclic AMP (cAMP)/protein kinase A (PKA) and calcium (Ca(2+)) signaling pathways are known to be involved in the regulation of motility in mammalian sperm. Calmodulin (CaM) is a ubiquitous Ca(2+) sensor that has been implicated in the acrosome reaction. In this report, we identify an insoluble pool of CaM in sperm and show that the protein, in addition to its presence in the acrosome, is found in the principal piece of the flagellum. These findings are consistent with, though not proof of, the presence of a pool of CaM in the fibrous sheath. The Ca(2+)/CaM-dependent protein kinase IIbeta (CaMKIIbeta), which is a downstream target of Ca(2+)/CaM, similarly localizes to the principal piece. In addition, we confirm earlier reports that a CaM inhibitor decreases sperm motility. However, we find that this inhibition can be largely reversed by stimulation of PKA if substrates for oxidative respiration are present in the medium. Our results suggest that the Ca(2+)/CaM/CaMKII signaling pathway in the sperm principal piece is involved in regulating sperm motility, and that this pathway functions either in parallel with or upstream of the cAMP/PKA pathway.

Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines?

Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines?

Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization.

Sperm incorporation and the conversion of the sperm-derived components into zygotic structures during in vitro fertilization of bovine oocytes was explored by combining ultrastructural studies with observations of the fertilizing sperm tagged with a mitochondrion-specific vital dye MitoTracker green FM. The zygotes fertilized by the MitoTracker-labeled sperm were fixed at various times after fertilization and then processed for immunocytochemistry to examine the distribution of DNA, microtubules, and sperm tail components, including the fibrous sheath and axonemal microtubules. We show here that the complete incorporation of the sperm, but not sperm-oocyte binding and oocyte activation, depends upon the integrity of oocyte microfilaments and is inhibited by the microfilament disrupter cytochalasin B. After sperm incorporation, the mitochondria are displaced from the sperm's connecting piece, and the sperm centriole is exposed to the egg cytoplasm. This event is followed by the formation of the microtubule-based sperm aster, which is responsible for the union of male and female pronuclei. Concomitantly, the major structure of the sperm principal piece, the fibrous sheath, disappears. After the first mitosis, the compact mitochondrial sheath can be seen in one of the blastomeres. An aggregate of the sperm mitochondria is observed at the entry of the second mitosis, although they remain in the vicinity of the nucleus and can later be seen at one pole of the metaphase spindle. The mitochondrial cluster is occasionally found in one of the blastomeres in the early-stage four-cell embryos, but it is no longer detected by the beginning of the third mitotic cycle. These data suggest that the disassembly of the sperm tail during bovine fertilization occurs as a series of precisely orchestrated events involving the destruction (fibrous sheath and mitochondrial sheath) and transformation (DNA, sperm centriole) of particular sperm structures into zygotic and embryonic components.

Dimensions and allometry of testes, epididymides and spermatozoa in the domestic dog (Canis familiaris).

Dimensions of the testes, epididymides and spermatozoa were measured from a sample of 104, mainly cross-bred, domestic dogs ranging in body mass from 5 to 48 kg. Allometric analyses in relation to body mass indicated isometric changes in testicular mass but epididymal mass and the mass of the cauda epididymidis and sperm numbers in the cauda showed significant positive allometry. Total sperm length, seminiferous tubule diameter and the relative proportion of tissues in the testis showed no significant correlation with body mass, but sperm principal piece length showed a low correlation with body mass. We suggest that the positive allometry of epididymal mass and sperm numbers is related to promiscuous mating and sperm competition in stray dogs.