Heterotrimeric Kinesin II is required for flagellar assembly and elongation of nuclear morphology during spermiogenesis in Schmidtea mediterranea.

Development of sperm requires microtubule-based movements that drive assembly of a compact head and flagellated tails. Much is known about how flagella are built given their shared molecular core with motile cilia, but less is known about the mechanisms that shape the sperm head. The Kinesin Superfamily Protein 3A (KIF3A) pairs off with a second motor protein (KIF3B) and the Kinesin Associated Protein 3 (KAP3) to form Heterotrimeric Kinesin II. This complex drives intraflagellar transport (IFT) along microtubules during ciliary assembly. We show that KIF3A and KAP3 orthologs in Schmidtea mediterranea are required for axonemal assembly and nuclear elongation during spermiogenesis. Expression of Smed-KAP3 is enriched during planarian spermatogenesis with transcript abundance peaking in spermatocyte and spermatid cells. Disruption of Smed-kif3A or Smed-KAP3 expression by RNA-interference results in loss of spermatozoa and accumulation of unelongated spermatids. Confocal microscopy of planarian testis lobes stained with alpha-tubulin antibodies revealed that spermatids with disrupted Kinesin II function fail to assemble flagella, and visualization with 4',6-diamidino-2-phenylindole (DAPI) revealed reduced nuclear elongation. Disruption of Smed-kif3A or Smed-KAP3 expression also resulted in edema, reduced locomotion, and loss of epidermal cilia, which corroborates with somatic phenotypes previously reported for Smed-kif3B. These findings demonstrate that heterotrimeric Kinesin II drives assembly of cilia and flagella, as well as rearrangements of nuclear morphology in developing sperm. Prolonged activity of heterotrimeric Kinesin II in manchette-like structures with extended presence during spermiogenesis is hypothesized to result in the exaggerated nuclear elongation observed in sperm of turbellarians and other lophotrochozoans.

Sperm morphometry is affected by increased inbreeding in the Retinta cattle breed: A molecular approach.

The effect of inbreeding depression on sperm motility is well documented, but its influence on sperm morphometry has been scarcely examined to date. Here, we combined the use of computer-assisted sperm morphometry analysis (CASMA) with a SNP-based genomic approach to determine and characterize the effect of inbreeding on the sperm shape of a highly inbred cattle population. We determined seven morphometric parameters on frozen-thawed sperm samples of 57 Retinta bulls: length (L, µm), width (W, µm), area (A, µm2 ), perimeter (P, µm), ellipticity (ELI; L/W), elongation (L-W)/(L + W) and perimeter-to-area shape factor (p2a; P2 /4 × π × A). The comparison of highly inbred (HI) and lowly inbreed (LI) individuals based on runs of homozygosity (ROH) inbreeding values (F ROH ) showed no differences between groups. An additional two-step unsupervised sperm subpopulation analysis based on morphometric parameters showed significant differences in the abundance of different sperm subpopulations between groups (p < 0.05). This analysis revealed that HI bulls harbored a higher percentage of narrow-head sperm as opposed to the higher percentage of large- and round-headed sperm detected in LI. A further genomic characterization revealed 23 regions differentially affected by inbreeding in both groups, detecting six genes (SPAG6, ARMC3, PARK7, VAMP3, DYNLRB2, and PHF7) previously related to different spermatogenesis-associated processes.

Arylsulfatase A Remodeling during Human Sperm In Vitro Capacitation Using Field Emission Scanning Electron Microscopy (FE-SEM).

Capacitation drives sperm biophysical and biochemical changes for sperm-oocyte interactions. It is a well-known fact that the molecular complex arylsulfatase A (ARSA), hyaluronidase sperm adhesion molecule 1 (SPAM1), and heat shock protein 2 (HSPA2) plays a significant role in sperm-zona pellucida (ZP) binding. However, the time-dependent capacitation effects on the sperm surface ARSA presence and specific topographic distributions remain to be elucidated. Here, we quantified the ARSA density and specific membrane domain locations before (US) and after in vitro capacitation (one and four hours; CS1-CS4) in human sperm using high-resolution field emission scanning electron microscopy (FE-SEM) and immunogold labeling. Our results showed a significant and progressive capacitation-mediated increase of labeled spermatozoa from the US (37%) to CS4 (100%) physiological conditions. In addition, surface mapping revealed a close relationship between the ARSA residues and their acrosomal repositioning. Compared with the ARSA surface heterogeneous distribution found in US, the CS1-4 conditions exhibited clustering on the peri-acrosomal region, showing that time-dependent capacitation also induced a ARSA residue dramatic translocation on sperm surfaces. Our findings provide novel insights into the molecular remodeling events preceding sperm-oocyte interactions.

High-throughput sperm assay using label-free microscopy: morphometric comparison between different sperm structures of boar and stallion spermatozoa.

The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our knowledge, this is the first reported comparison when using AFM and high-sensitivity interferometric microscopy (such as SLIM) to evaluate spermatozoa. Results indicate that with the use of SLIM microscopy there is similar nanoscale sensitivity as with use of AFM while there is approximately 1,000 times greater throughput with use of SLIM. With SLIM, there is also allowace for the measurement of the dry mass (non-aqueous content) of spermatozoa, which may be a new label-free marker for sperm viability. In the second part of this study, there was analysis of two sperm populations. There were interesting correlations between the different compartments of the sperm and the dry mass in both boars and stallions. Furthermore, there was a correlation between the dry mass of the sperm head and the length and width of the acrosome in both boars and stallions. This correlation is positive in boars while it is negative in stallions.

Relationship of sperm plasma membrane and acrosomal integrities with sperm morphometry in Bos taurus.

To date, sperm morphometric studies have assessed whole sperm populations without considering sperm function. The aim of this study was to evaluate the relationship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls. To this end, sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes, including Hoechst 33343, carboxyfluorescein diacetate, and propidium iodide. This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometrics of the sperm head, nucleus, and acrosome using a specific plug-in module created on ImageJ. Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome (P < 0.001). In the case of spermatozoa with an intact acrosome, those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane (P < 0.001). No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations. There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for intact spermatozoa. These correlations were particularly strong for the morphometric parameters of the sperm acrosome. We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

Micromorphological and ultrastructural description of spermatozoa from squirrel monkeys (Saimiri collinsi Osgood, 1916).

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

The perforatorium and postacrosomal sheath of rat spermatozoa share common developmental origins and protein constituents†.

The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the subacrosomal layer (SAL) and the postacrosomal sheath (PAS). In falciform spermatozoa, a third region of the PT emerges that extends beyond the nuclear apex called the perforatorium. The formation of the SAL and PAS differs, with the former assembling early in spermiogenesis concomitant with acrosome formation, and the latter dependent on manchette descent during spermatid elongation. The perforatorium also forms during the elongation phase of spermiogenesis, suggesting that like the PAS, its assembly is facilitated by the manchette. The temporal similarity in biogenesis between the PAS and perforatorium led us to compare their molecular composition using cell fractionation and immunodetection techniques. Although the perforatorium is predominantly composed of its endemic protein FABP9/PERF15, immunolocalization indicates that it also shares proteins with the PAS. These include WBP2NL/PAWP, WBP2, GSTO2, and core histones, which have been implicated in early fertilization and zygotic events. The compositional homogeny between the PAS and perforatorium supports our observation that their development is linked. Immunocytochemistry indicates that both PAS and perforatorial biogenesis depend on the transport and deposition of cytosolic proteins by the microtubular manchette. Proteins translocated from the manchette pass ventrally along the spermatid head into the apical perforatorial space prior to PAS deposition in the wake of manchette descent. Our findings demonstrate that the perforatorium and PAS share a mechanism of developmental assembly and thereby contain common proteins that facilitate fertilization.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo.

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l -/-; Prm2 S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l -/- mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

salto/CG13164 is required for sperm head morphogenesis in Drosophila.

Producing mature spermatozoa is essential for sexual reproduction in metazoans. Spermiogenesis involves dramatic cell morphological changes going from sperm tail elongation and nuclear reshaping to cell membrane remodeling during sperm individualization and release. The sperm manchette plays a critical scaffolding function during nuclear remodeling by linking the nuclear lamina to the cytoskeleton. Here, we describe the role of an uncharacterized protein in Drosophila, salto/CG13164, involved in nuclear shaping and spermatid individualization. Salto has dynamic localization during spermatid differentiation, being progressively relocated from the sperm-nuclear dense body, which is equivalent to the mammalian sperm manchette, to the centriolar adjunct and acrosomal cap during spermiogenesis. salto-null male flies are sterile and exhibit complete spermatid individualization defects. salto-deficient spermatids show coiled spermatid nuclei at late maturation stages and stalled individualization complexes. Our work sheds light on a novel component involved in cytoskeleton-based cell-morphological changes during spermiogenesis.

CDC42 Negatively Regulates Testis-Specific SEPT12 Polymerization.

Septin (SEPT) genes encode well-preserved polymerizing GTP-binding cytoskeletal proteins. The cellular functions of SEPTs consist of mitosis, cytoskeletal remodeling, cell polarity, and vesicle trafficking through interactions with various types of cytoskeletons. We discovered that mutated SEPTIN12 in different codons resulted in teratozoospermia or oligozoospermia. In mouse models with a defective Septin12 allele, sperm morphology was abnormal, sperm count decreased, and sperms were immotile. However, the regulators of SEPT12 are completely unknown. Some studies have indicated that CDC42 negatively regulates the polymerization of SEPT2/6/7 complexes in mammalian cell lines. In this study, we investigated whether CDC42 modulates SEPT12 polymerization and is involved in the terminal differentiation of male germ cells. First, through scanning electron microscopy analysis, we determined that the loss of Septin12 caused defective sperm heads. This indicated that Septin12 is critical for the formation of sperm heads. Second, CDC42 and SEPT12 were similarly localized in the perinuclear regions of the manchette at the head of elongating spermatids, neck region of elongated spermatids, and midpiece of mature spermatozoa. Third, wild-type CDC42 and CDC42Q61L (a constitutive-acting-mutant) substantially repressed SEPT12 polymerization, but CDC42T17N (a dominant-negative-acting mutant) did not, as evident through ectopic expression analysis. We concluded that CDC42 negatively regulates SEPT12 polymerization and is involved in terminal structure formation of sperm heads.

SPATC1L maintains the integrity of the sperm head-tail junction.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity.

Sperm head morphology is associated with sperm swimming speed: A comparative study of songbirds using electron microscopy.

Sperm exhibit extraordinary levels of morphological diversification across the animal kingdom. In songbirds, sperm have a helically shaped head incorporating a distinct acrosomal membrane or "helical keel," the form and extent of which varies across species. The functional significance of this helical shape, however, remains unknown. Using scanning electron microscopy, we quantified inter- and intraspecific variation in sperm head morphology across 36 songbird species (Passeriformes: Passerida). Using phylogenetic comparative methods, we investigated the relationship between sperm head morphology and both sperm swimming speed and the frequency of extra-pair young (EPY). We found that species whose sperm had a relatively more pronounced helical form (i.e., long acrosome, short nucleus, wide helical membrane, and a more pronounced waveform along the sperm head "core") had faster-swimming sperm. We found no evidence of a relationship between interspecific variation in sperm head morphology and EPY, although we did find that among- and within-male variation in sperm head traits were negatively correlated with EPY. Applying principles of fluid mechanics, we discuss how the helical form of the sperm head may influence swimming speed, and suggest that further studies considering aspects of sperm morphology beyond sperm length are needed to improve our understanding of sperm structure-function relationships.

An illustration of human sperm morphology and their functional ability among different group of subfertile males.

Condensed sperm chromatin is a prerequisite for natural fertilization. Some reports suggested the prevalence of chromatin condensation defects in teratozoospermia cases with head anomalies; conversely, earlier studies exemplified its occurrence in morphologically normal spermatozoa too. The aim of this study was to compare the condensation defects in correlation with head anomalies among different groups of subfertile males and its impact on the rate of fertilization in assisted reproduction procedures. Ultrastructure analysis of spermatozoa through scanning electron microscopy and atomic force microscopy could facilitate an in-depth evaluation of sperm morphology. Nuclear condensation defects (%) in spermatozoa were analyzed in 666 subjects, and its effect on the rate of fertilization was analyzed in 116 IVF and 90 intracytoplasmic sperm injection cases. There was no correlation of condensation defects with head anomalies (%). Student's t-test showed no significant changes in mean values of condensation defects in abnormal semen samples in comparison with the normal group. Condensation defects were observed in normal spermatozoa too, which was negatively associated with the rate of fertilization in IVF (p < 0.01), but intracytoplasmic sperm injection outcome remained unaffected. Ultrastructure study revealed sperm morphological features in height, amplitude, and three-dimensional views in atomic force microscopy images presenting surface topography, roughness property of head, and compact arrangement of mitochondria over axoneme with height profile at nanoscale. In pathological forms, surface roughness and nuclear thickness were marked higher than the normal spermatozoa. Thus, percentage of normal spermatozoa with condensation defects could be a predictive factor for the rate of fertilization in IVF. From diverse shapes of nucleus in AFM imaging, it could be predicted that defective nuclear shaping might be impeding the activity of some proteins/ biological motors, those regulate the proper Golgi spreading over peri-nuclear theca.

Progesterone stimulates the long-distance migration of capacitated ram spermatozoa through viscous media under geotactic condition.

Forward progressive motility of spermatozoa is an essential prerequisite for reproductive success, and sperm navigation is assisted by guidance mechanisms that may depend on micro-environmental factors. In the present study, we performed an integrated analysis of long-distance ram sperm migration in vitro that combined two environmental factors (10 µM progesterone and a geotactic effect) and the physiological status of the cells (capacitation treatment). A penetration assay was used in which spermatozoa had to travel 20 mm in a viscous medium (two media of differing viscosity: acrylamide and hyaluronic acid) through a tube device. The number of migrating spermatozoa, the physiology of the cells (motility analyzed using a CASA system; acrosomal status, viability and active mitochondria evaluated by flow cytometry; DNA fragmentation index calculated by quantitative PCR) and the morphometry of sperm heads (performed using an image analysis system) were evaluated after long-distance sperm migration. Ram sperm capacitation significantly stimulates cell migration through viscous media under geotactic conditions, and this effect is enhanced by progesterone induction. The rheological characteristics of viscous media have a marked impact on ram sperm migration, and acrylamide more favorably facilitates navigation over a large distance. The migrating spermatozoa are morphologically better adapted (high ellipticity) for displacement in viscous media and exhibit remarkably depleted mitochondrial membrane potential.

Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Microscopic sperm head damage and abnormalities as heat stress indicators in Australian Merino rams (Ovis aries) in Northern Patagonia, Argentina / Danos e anormalidades microscópicas das cabeças espermáticas como indicadores de estresse por calor em carneiros Merino Australiano (Ovis aries) da Patagônia Norte, Argentina

In Northern Patagonia, the mating season starts on March 15th, when rams are submitted to summer temperatures. Exposure of rams to heat stress increases the prevalence of microscopic damage to spermatozoa, morphological abnormalities, and reductions in fertility. This study assesses the adaptive capabilities of six unshorn and six shorn Australian Merino rams, half of which were treated in a heat chamber for eight hours for five days, gradually reaching a temperature of up to 40 °C. Microscopic damage, abnormalities and ultramicroscopic alterations of the plasma membrane and the acrosome of sperm head were analysed. There were significant differences in the percentage of tailless spermatozoa and proximal cytoplasmic droplets between post-treatment periods. Temperature primarily affected the shorn rams and the sperm heads during spermiogenesis. Submicroscopic alterations were observed when the plasma membrane was present in the anterior segment. These alterations can be intact, waved, or dilated. When the plasma membrane was absent, the acrosome might be intact, dilated, and waved. In addition, the outer acrosomal membrane may completely lose its contents or have a nude nucleus. The plasma membrane assumes a waved shape as a result of the effect of temperature on the epididymis. According to this study, the tailless head, proximal cytoplasmic droplets, and the ultramicroscopic categories studied were robust indicators of semen heat stress. After ten weeks, the sperm head recovered its normal shape. Unshorn rams are better adapted to summer heat stress than shorn ones. Microscopy and transmission electron microscopy alterations have been shown to be excellent indicators of thermal stress in Australian Merino rams and may be useful tools to help sheep farmers choose when to begin the mating season, which will vary depending on the environmental conditions of the summer.(AU)

Na Patagônia Norte, os ovinos têm sua estação de acasalamento iniciada em 15 de março, portanto, ficam sujeitos às temperaturas do verão. A exposição de carneiros a estresse térmico aumenta a prevalência de danos microscópicos e anomalias morfológicas nos espermatozoides, que implica uma redução na fertilidade. Este trabalho avaliou a capacidade adaptativa de carneiros Merino Australiano com lã (N = 6) e tosquiados (N = 6): metade ficou ao ar livre e outra metade foi mantida em uma câmara climática por oito horas, durante cinco dias, chegando gradualmente a uma temperatura máxima de 40 °C. Foram analisados danos microscópicos, anormalidades e alterações ultramicroscópicas da membrana plasmática e do acrossoma da cabeça dos espermatozoides. Os resultados microscópicos confirmaram a existência de diferença significativa na porcentagem de espermatozoides sem cauda e com gota citoplasmática proximal, entre os ejaculados pós-tratamento. A temperatura afetou os carneiros tosquiados, principalmente a cabeça de seus espermatozoides, durante a espermatogênese. Alterações submicroscópicas foram observados na membrana plasmática quando ela estava presente no segmento anterior: quando não intacta, ficava ondulada ou dilatada. Quando a membrana plasmática estava ausente, o acrossoma podia se apresentar ondulado ou dilatado. Além disso, sob efeito do calor, a membrana acrossomal externa pode perder completamente seu conteúdo ou apresentar núcleo desnudo. A membrana plasmática assume uma forma ondulada pelo efeito da temperatura no epidídimo. Depois de dez semanas, a cabeça dos espermatozoides recuperou sua forma normal. Como demonstrado neste estudo, a cabeça sem cauda, as gotas citoplasmáticas proximais e as categorias ultramicroscópicas estudadas são indicadores do efeito do estresse térmico no sêmen, e os carneiros com maior cobertura de lã se adaptam melhor ao estresse por calor. Alterações de microscopia e de microscopia eletrônica de transmissão têm se mostrado excelentes indicadores de estresse por calor em carneiros Merino Australiano e podem ser ferramentas úteis para ajudar criadores de ovelhas a escolher quando começar a época de acasalamento, o que irá variar de acordo com as condições ambientais do verão.(AU)

La capacitación y la reacción acrosómica se asocian con cambios en la localización del ácido siálico y en la morfometría de la región cefálica del espermatozoide humano / Capacitation and acrosome reaction are associated with changes in sialic acid location and head morphometry in human sperm

Objetivo. Evaluar los cambios de distribución del ácido siálico durante el proceso de capacitación y reacción acrosómica in vitro y estudiar las modificaciones morfométricas en estas condiciones fisiológicas en la región cefálica del espermatozoide humano. Material y método. En este estudio prospectivo, evaluamos 6 muestras espermáticas normozoospérmicas. La distribución de ácido siálico se evaluó mediante la lectina Wheat germ agglutinin en diferentes condiciones fisiológicas: antes, después de la capacitación y tras la reacción acrosómica. La forma y el tamaño cefálico de cada estadio se estudiaron mediante métodos de morfometría geométrica. Resultados. Tras la capacitación, un 73,07±21,43% de espermatozoides presentaron ácido siálico en la región acrosomal en relación directa con una expansión del acrosoma y una contracción del segmento ecuatorial. Por otra parte, después de la reacción acrosómica se registra un mayor efecto alométrico entre los estadios debido a que los espermatozoides experimentaron una marcada expansión del segmento ecuatorial. En relación con la localización de Wheat germ agglutinin, encontramos una disminución significativa en el porcentaje de espermatozoides con fluorescencia en la región acrosomal, así como un incremento del marcaje en la banda ecuatorial. Conclusiones. Nuestros resultados demuestran que la distribución de la lectina Wheat germ agglutinin covaría con importantes cambios en la morfometría de la cabeza del espermatozoide y evidencia importantes implicaciones en los procesos de capacitación y reacción acrosómica (AU)

Objective. Assess changes in sialic acid distribution during capacitation and acrosome reaction processes, and evaluate head sperm morphometrics modifications in these physiological conditions in human sperm. Material and method. In this prospective study, we included 6 normozoospermics sperm samples. Sialic acid distribution was evaluated by Wheat germ agglutinin lectin in different physiological conditions: before, after capacitation and after acrosome reaction. Head shape and size of each stage were analyzed by means of geometric morphometric methods. Results. After capacitation, 73.07±21.43% of sperm showed sialic acid in acrosomal region, linked with an acrosome expansion and equatorial segment contraction. Otherwise, after acrosome reaction higher allometric effect between stages was recorded since sperm undergo further expansion of equatorial segment. Regarding Wheat germ agglutinin location, we found that sperm percentage significant decline in acrosomal fluorescence and an increase of equatorial band labeling. Conclusions. Our findings demonstrate that modifications in Wheat germ agglutinin expression covariate with dramatic changes in sperm head morphometry, suggesting important implications in capacitation and acrosome reaction processes (AU)

Sperm morphology of predatory pirate bugs Amphiareus constrictus and Blaptostethus pallescens (Heteroptera: Anthocoridae) with phylogenetic inferences.

The sperm morphology of two predatory bugs Amphiareus constrictus and Blaptostethus pallescens, representing the tribes Dufouriellini and Blasptostethini, respectively, was described using light and transmission electron microscopy. The spermatozoa of Amphiareus constrictus and Blaptostethus pallescens are fine and long, each measuring 216.6µm and 181.0µm in length, of which 37.0µm and 11.6µm, respectively, comprise the nuclei. When stained with DAPI (for DNA), the posterior half of the nucleus in B. pallescens exhibited low fluorescence, while in A. constrictus this feature was observed only in the last 6µm. In both species, as in Heteroptera in general, the spermatozoa have, in the head region, an acrosome and nucleus, and in the flagellar region, an axoneme with 9 accessory tubules, 9 peripheral doublets and 2 central microtubules (9+9+2 microtubules), 2 mitochondrial derivatives (MDs), and a centriolar adjunct in the nucleus-flagellum transition. However, unlike most Heteroptera, in these species, the MDs are asymmetric, and the centriolar adjunct is quite long and encompasses completely the posterior nuclear end and the anterior tips of the MDs. These features are considered as derived, thus supporting the condition derived of Anthocoridae within Cimicomorpha. In addition, several traits of the spermatozoa of these two species easily distinguish one species (and probably a tribe) from the other; for example, the difference of formats in the MDs, and the long anterior projection of the centriolar adjunct parallel to the nucleus in B. pallescens.

Disruption of Ssp411 causes impaired sperm head formation and male sterility in mice.

BACKGROUND: We previously cloned the Ssp411 gene. We found that the Ssp411 protein is predominantly expressed in elongated spermatids in the rat testis in a stage-dependent manner. Although our findings strongly suggested that Ssp411 might play an important role in mammalian spermatogenesis, this hypothesis has not been studied. METHODS: We first used real-time PCR, Western blotting and immunohistochemistry to confirm that the expression pattern of Ssp411 in several murine tissues is similar to its expression pattern in corresponding rat tissues. To better understand the roles of Ssp411 in male reproduction in vivo, we identified and characterized an Ssp411 expression-disrupted murine strain (Ssp411PB/PB) that was generated by piggyBac (PB) transposon insertion. We studied Ssp411-interacting proteins using proteome microarray, co-IP and GST pull-down assay. RESULTS: Both Ssp411 mRNA and protein were detected exclusively in spermatids after step 9 during spermiogenesis in testis. Phenotypic analysis suggested that only Ssp411PB/PB males are sterile. These males have smaller testes, reduced sperm counts, decreased sperm motility and deformed spermatozoa. Microscopy analysis indicated that the manchette, a structurally reshaped sperm head, is aberrant in Ssp411PB/PB spermatids. The results of proteome microarray analysis and GST pull-down assays suggested that Ssp411 participates the ubiquitin-proteasome system by interacting with PSMC3. This has been reported to be manchette-associated and important for the head shaping of spermatids. CONCLUSIONS: Our study suggested that Ssp411 is required for spermiogenesis. It seems to play a role in sperm head shaping. The lack of Ssp411 causes sperm deformation and results in male infertility. GENERAL SIGNIFICANCE: Ssp411PB/PB mouse strain is an animal model of idiopathic oligoasthenoteratozoospermia (iOAT), and the gene may represent a therapeutic target for iOAT patients.

Lysophosphatidic acid acyltransferase 3 tunes the membrane status of germ cells by incorporating docosahexaenoic acid during spermatogenesis.

Docosahexaenoic acid (DHA) is one of the essential ω-3 polyunsaturated fatty acids with a wide range of physiological roles important for human health. For example, DHA renders cell membranes more flexible and is therefore important for cellular function, but information on the mechanisms that control DHA levels in membranes is limited. Specifically, it is unclear which factors determine DHA incorporation into cell membranes and how DHA exerts biological effects. We found that lysophosphatidic acid acyltransferase 3 (LPAAT3) is required for producing DHA-containing phospholipids in various tissues, such as the testes and retina. In this study, we report that LPAAT3-KO mice display severe male infertility with abnormal sperm morphology. During germ cell differentiation, the expression of LPAAT3 was induced, and germ cells obtained more DHA-containing phospholipids. Loss of LPAAT3 caused drastic reduction of DHA-containing phospholipids in spermatids that led to excess cytoplasm around its head, which is normally removed by surrounding Sertoli cells via endocytosis at the final stage of spermatogenesis. In vitro liposome filtration assay raised the possibility that DHA in phospholipids promotes membrane deformation that is required for the rapid endocytosis. These data suggest that decreased membrane flexibility in LPAAT3-KO sperm impaired the efficient removal of sperm content through endocytosis. We conclude that LPAAT3-mediated enrichment of cell membranes with DHA-containing phospholipids endows these membranes with physicochemical properties needed for normal cellular processes, as exemplified by spermatogenesis.