Late Spermatid Stage Research Articles

A postmeiotically bifurcated roadmap of honeybee spermatogenesis marked by phylogenetically restricted genes.

Haploid males of hymenopteran species produce gametes through an abortive meiosis I followed by meiosis II that can either be symmetric or asymmetric in different species. Thus, one spermatocyte could give rise to two spermatids with either equal or unequal amounts of cytoplasm. It is currently unknown what molecular features accompany these postmeiotic sperm cells especially in species with asymmetric meiosis II such as bees. Here we present testis single-cell RNA sequencing datasets from the honeybee (Apis mellifera) drones of 3 and 14 days after emergence (3d and 14d). We show that, while 3d testes exhibit active, ongoing spermatogenesis, 14d testes only have late-stage spermatids. We identify a postmeiotic bifurcation in the transcriptional roadmap during spermatogenesis, with cells progressing toward the annotated spermatids (SPT) and small spermatids (sSPT), respectively. Despite an overall similarity in their transcriptomic profiles, sSPTs express the fewest genes and the least RNA content among all the sperm cell types. Intriguingly, sSPTs exhibit a relatively high expression level for Hymenoptera-restricted genes and a high mutation load, suggesting that the special meiosis II during spermatogenesis in the honeybee is accompanied by phylogenetically young gene activities.

Lhb−/−Lhr−/− Double Mutant Mice Phenocopy Lhb−/− or Lhr−/− Single Mutants and Display Defects in Leydig Cells and Steroidogenesis

In the mouse, two distinct populations of Leydig cells arise during testis development. Fetal Leydig cells arise from a stem cell population and produce T required for masculinization. It is debated whether they persist in the adult testis. A second adult Leydig stem cell population gives rise to progenitor-immature-mature adult type Leydig cells that produce T in response to LH to maintain spermatogenesis. In testis of adult null male mice lacking either only LH (Lhb-/-) or LHR (Lhr-/-), mature Leydig cells are absent but fetal Leydig cells persist. Thus, it is not clear whether other ligands signal via LHRs in Lhb null mice or LH signals via other receptors in the absence of LHR in Lhr null mice. Moreover, it is not clear whether truncated LHR isoforms generated from the same Lhr gene promoter encode functionally relevant LH receptors. To determine the in vivo roles of LH-LHR signaling pathway in the Leydig cell lineage, we generated double null mutant mice lacking both LH Ligand and all forms of LHR. Phenotypic analysis indicated testis morpho-histological characteristics are identical among double null and single mutants which all showed poorly developed interstitium with a reduction in Leydig cell number and absence of late stage spermatids. Gene expression analyses confirmed that the majority of the T biosynthesis pathway enzyme-encoding mRNAs expressed in Leydig cells were all suppressed. Expression of thrombospondin-2, a fetal Leydig cell marker gene was upregulated in single and double null mutants indicating that fetal Leydig cells originate and develop independent of LH-LHR signaling pathway in vivo. Serum and intratesticular T levels were similarly suppressed in single and double mutants. Consequently, expression of AR-regulated genes in Sertoli and germ cells were similarly affected in single and double mutants without any evidence of any additive effect in the combined absence of both LH and LHR. Our studies unequivocally provide genetic evidence that in the mouse testis, fetal Leydig cells do not require LH-LHR signaling pathway and a one-to-one LH ligand-LHR signaling pathway exists in vivo to regulate adult Leydig cell lineage and spermatogenesis.

Assessment of age-related morphological changes in the testes of post-hatch light ecotype Nigerian indigenous chicken.

The age-related morphological changes of the testes in light ecotype Nigerian indigenous chicken were evaluated in this study using gross anatomical, histological and histomorphometric techniques. The results showed that the testes of 3- to 9-month-old birds were light pink while testes of sexually mature chicken were creamy white in colour. The left and right testicular weight, length, diameter, circumference and the organosomatic indices increased significantly (p<.05) with increasing age across the groups. Although the mean tubular diameter and epithelial height of the left and right seminiferous tubules increased significantly (p<.05) with age, the tubular diameter, epithelial height and luminal diameter did not vary significantly (p>.05) between the left and right testes of all the groups. The one-cell layer thick germinal epithelium of the left testes at 3 to 6months old showed islands of cell proliferation that contained spermatogonia and spermatocytes. At 6 to 9months, the left testes exhibited numerous early spermatids with occasional occurrence of late stage spermatids while the right testes showed scanty early stage spermatids. At 12 to 18months, the germinal epithelia of both left and right testes were characterized by the presence of Sertoli cells, spermatogonia, primary spermatocytes, numerous early and late stage spermatids as well as spermatozoa. In conclusion, the morphological features highlighted in the present study show that at pre-pubertal periods, the left testes may develop faster than the right testes. However, both left and right testes may participate actively in the production of spermatozoa during the post-pubertal life.

In Vivo Knockdown of the IP3R1 Calcium Channel Disrupts ER and Actin Morphology, as well as TBC Bulb Maturation in the Apical Processes of Sertoli Cells in Rat Testes

Spermatogenesis is the process by which sperm cells are produced, but at one phase of this process, late stage spermatids are temporarily attached to the apex of Sertoli cells in the seminiferous epithelium. Late spermatids are anchored by large adhesion like junction complexes termed ‘ectoplasmic specializations’ (ESs) that are proposed to be disassembled by novel endocytic structures only found in mammalian Sertoli cells, called ‘tubulobulbar complexes’ (TBCs). Both ESs and TBCs are heavily actin related, with parallel actin arrays and dendritic actin networks respectively being hallmarks of their identities. The endoplasmic reticulum (ER) in the Sertoli cell apical processes surrounding the late spermatids forms an extensive and continuous network. Cisternae of this network form part of the structure of the ESs, participate in a membrane contact site with the bulbs of TBCs, and have a parallel alignment with the surrounding Sertoli cell plasma membrane. Previously, we've localized the IP3R calcium channel to the TBC bulb‐ER contacts as well as other calcium exchange machinery to various parts of the ER in the apical process. Calcium release and exchange have been implicated in processes related to both actin dynamics as well as in fusion and acidification of endocytic components such as endosomes. We've proposed that calcium exchange may be a crucial mechanism in regulating both the actin networks of ESs and TBCs, as well as in facilitating the maturation and fusion of TBC bulbs as they develop into putative endosomes. If calcium exchange from the ER is responsible for regulating the local actin networks and maturation of endocytic components in the apical process, then knocking down the primary calcium release channel of the ER should alter local actin and TBC bulb morphology. To test this, we injected the testes of Sprague Dawley rats with siRNAs against IP3R1 (ITPR1) and collected tissues at 1 day, 2 day, and 3 days post injection and processed the tissues for either western blot, immunofluorescence, or electron microscopy. ITPR1 siRNA treated tissues showed significant morphological alterations to the ER, actin, and TBC bulbs in the apical processes of late spermatids compared to controls. This data supports the hypothesis that ER calcium exchange is an integral component to actin dynamics and TBC bulb maturation in the apical processes of Sertoli cells.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant awarded to AWV; Alexander Graham Bell Scholarship (NSERC) awarded to AAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis

A systematic interrogation of male germ cells is key to complete understanding of molecular mechanisms governing spermatogenesis and the development of new strategies for infertility therapies and male contraception. Here we develop an approach to purify all types of homogeneous spermatogenic cells by combining transgenic labeling and synchronization of the cycle of the seminiferous epithelium, and subsequent single-cell RNA-sequencing. We reveal extensive and previously uncharacterized dynamic processes and molecular signatures in gene expression, as well as specific patterns of alternative splicing, and novel regulators for specific stages of male germ cell development. Our transcriptomics analyses led us to discover discriminative markers for isolating round spermatids at specific stages, and different embryo developmental potentials between early and late stage spermatids, providing evidence that maturation of round spermatids impacts on embryo development. This work provides valuable insights into mammalian spermatogenesis, and a comprehensive resource for future studies towards the complete elucidation of gametogenesis.

Calcium and lipid exchange machinery in Sertoli cells

Sperm production (spermatogenesis) takes place in the seminiferous tubules of the testis. Specialized junction complexes termed ‘ectoplasmic specializations' (ESs) in the seminiferous epithelium have several key functions. ESs between Sertoli cells disassemble during the movement of early spermatogenic cells towards the apex of the epithelium and those ESs between a Sertoli cell and late stage spermatids disassemble during sperm release. There is evidence that structures called ‘Tubulobulbar Complexes' (TBCs) are endocytic structures responsible for the internalization and eventual degradation of junctions associated with ESs, allowing for early spermatocyte translocation from the basal epithelium and release of late spermatids from the apical epithelium. One key feature of both ESs and TBCs is their extensive association with peripheral ER. ESs are tripartite in structure consisting of Sertoli cell plasma membrane (PM), a layer of actin bundles, and an overlying leaflet of ER. TBCs are double membrane invaginations that appear as ESs are beginning to break down. They are long, endocytic in‐growths, with a swelled bulb region that contains an extensive ER‐PM contact site. TBC bulbs eventually ‘bud’, fuse, and become endosomes. Little is understood about the function of the ER at both ESs and TBCs, though it is well established that ER contact sites in other cells function in both calcium signalling and lipid exchange. If ER in ESs and ER‐TBC contacts function in calcium and lipid exchange, then known calcium signalling machinery and lipid exchange proteins should be localized to these structures. Using confocal imaging, we localized the CACNA1H voltage gated calcium channel to the PM of apical ESs, and the VAPA integral ER protein, which is associated with lipid synthesis and exchange machinery, to the apical ER as well as the ER in basal regions known to contain ESs. This data adds further weight to the hypothesis that calcium exchange is integral to regulation of ES and TBC structure, as well as begins our exploration into the possible role of lipid exchange at these sites.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (155397‐2013 to A. Wayne Vogl)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oocyte-activating capacity of fresh and frozen-thawed spermatids in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation.

Evidence for protandry in Polydactylus quadrifilis in the Kwanza Estuary, Angola, and its implications for local fisheries

A total of 141 Polydactylus quadrifilis were sampled from the Kwanza Estuary in Angola ranging in size from 436 to 1360 mm fork-length (FL). Of these, 124 were male, six intersex and 11 female. Female fish were significantly longer (mm, FL) and heavier (kg) than males and had significantly higher gonadosomatic indices (GSI’s) than those of males and intersex fish. Transitional (intersex) gonads were delimited, with testicular and ovarian regions separated by connective tissue. The first signs of ovarian tissue appeared on the outer ventral surface of the gonad. A second layer of ovarian tissue was first noticeable at either end of the initial ovarian region and developed back towards the centre of the ventral wall to form a luminal space. Early-stage oocytes were commonly found in the outer area of male regions and residual late-stage spermatids and spermatozoa were found in the luminal space of ovarian regions, suggesting a process of sex change from the outside inwards. A loss of male function was noted with increased ovarian prevalence. Based on this evidence it is suggested that P. quadrifilis in the Kwanza Estuary are protandrous. Owing to the reliance of P. quadrifilis on large highly fecund females for egg production, it is likely that they will be sensitive to fishing practices that target larger individuals within the population.

Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin

Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.

Dynamic transcription and expression patterns of KIF3A and KIF3B genes during spermiogenesis in the shrimp, Palaemon carincauda

Spermiogenesis is a highly ordered and complex process in the male germ cell differentiation. The microtubule-based motor proteins KIF3A and KIF3B are required for the progression of the stages of spermiogenesis. In this study, the main goal was to determine whether KIF3A and KIF3B have a key role in spermiogenesis in Palaemon carincauda. The complete cDNA of KIF3A/3B from the testis of P. carincauda was cloned by using PCR and rapid amplification of cDNA ends (RACE). The predicted secondary and tertiary structures of KIF3A/3B contained three domains which were the: a) head region, b) stalk region, and c) tail region. Real-time quantitative PCR (qPCR) results revealed that KIF3A and KIF3B mRNAs were obtained for all the tissues examined, with the greatest gene expression in the testis. In situ hybridization indicated the KIF3A and KIF3B mRNAs were distributed in the periphery of the nuclear in the early spermatid of spermiogenesis. In the middle and late spermatid stages, KIF3A and KIF3B mRNAs were gradually upregulated and assembled to one side where acrosome biogenesis begins. In the mature sperm, KIF3A and KIF3B mRNAs were distributed in the acrosome cap and spike. Immunofluorescence studies indicated that KIF3A, tubulin, mitochondria, and Golgi were co-localized in different stages during spermiogenesis in P. carincauda. The temporal and spatial gene expression dynamics of KIF3A/3B indicate that KIF3A and KIF3B proteins may be involved in acrosome formation and nucleus shaping. Moreover, these proteins can transport the mitochondria and Golgi that facilitate acrosome formation in P. carincauda.

KIFC1 is essential for acrosome formation and nuclear shaping during spermiogenesis in the lobster Procambarus clarkii.

In order to study the function of kinesin-14 motor protein KIFC1 during spermatogenesis of Procambarus clarkii, the full length of kifc1 was cloned from testes cDNA using Rapid-Amplification of cDNA Ends (RACE). The deduced KIFC1 protein sequence showed the highest similarity between Procambarus clarkii and Eriocheir senensis (similarity rate as 64%). According to the results of in situ hybridization (ISH), the kifc1 mRNA was gathered in the acrosome location above nucleus in the mid- and late-stage spermatids. Immunofluorescence results were partly consistent with the ISH in middle spermatids, while in the late spermatids the KIFC1 was distributed around the nucleus which had large deformation and formed four to six nuclear arms. In the mature sperm, KIFC1 and microtubules were distributed around the sperm, playing a role in maintaining the sperm morphology and normal function. Overexpression of P. clarkii kifc1 in GC1 cells for 24 hours resulted in disorganization of microtubules which changed the cell morphology from circular and spherical into fusiform. In addition, the overexpression also resulted in triple centrosomes during mitosis which eventually led to cell apoptosis. RNAi experiments showed that decreased KIFC1 protein levels resulted in total inhibition of spermatogenesis, with only mature sperm found in the RNAi-testis, implying an indispensable role of KIFC1 during P. clarkii spermiogenesis.

Breast cancer resistance protein (Bcrp) and the testis—an unexpected turn of events

Breast cancer resistance protein (Bcrp) is an ATP-dependent efflux drug transporter. It has a diverse spectrum of hydrophilic and hydrophobic substrates ranging from anticancer, antiviral and antihypertensive drugs, to organic anions, antibiotics, phytoestrogens (e.g., genistein, daidzein, coumestrol), xenoestrogens and steroids (e.g., dehydroepiandrosterone sulfate). Bcrp is an integral membrane protein in cancer and normal cells within multiple organs (e.g., brain, placenta, intestine and testis) that maintains cellular homeostasis by extruding drugs and harmful substances from the inside of cells. In the brain, Bcrp is a major component of the blood-brain barrier located on endothelial cells near tight junctions (TJs). However, Bcrp is absent at the Sertoli cell blood-testis barrier (BTB); instead, it is localized almost exclusively to the endothelial TJ in microvessels in the interstitium and the peritubular myoid cells in the tunica propria. Recent studies have shown that Bcrp is also expressed stage specifically and spatiotemporally by Sertoli and germ cells in the seminiferous epithelium of rat testes, limited only to a testis-specific cell adhesion ultrastructure known as the apical ectoplasmic specialisation (ES) in stage VI-early VIII tubules. These findings suggest that Bcrp is equipped by late spermatids and Sertoli cells to protect late-stage spermatids completing spermiogenesis. Furthermore, Bcrp was found to be associated with F (filamentous)-actin and several actin regulatory proteins at the apical ES and might be involved in the organisation of actin filaments at the apical ES in stage VII-VIII tubules. These findings will be carefully evaluated in this brief review.

Chromatin remodelling initiation in spermatids: differences among human males

During the last phase of spermatogenesis, called spermiogenesis, the nucleosome-based chromatin structure is replaced by a protamine-based DNA packaging. Not much is known about the chromatin remodelling involved in humans and animals. Here, we have investigated initiation of chromatin remodelling over seven probands of which five were diagnosed with non-obstructive azoospermia (NOA) and two with obstructive azoospermia (OA) (failed vaso-vasostomy patients with proven fertility prior to vasectomy, Johnsen scores ≥9). Chromatin remodelling was studied evaluating the presence of nucleosomes, histone H3, pre-protamine 2 and protamine 1. This approach was feasible since the local initiation of nucleosome eviction in the sub acrosomal domain, which was visible in alkaline nuclear spread preparations. The patterns of nucleosome and H3 loss were largely congruent. Nucleus wide incorporation of protamine 1 could already be observed at the late round spermatid stage. Both for nucleosome loss and for protamine 1 incorporation, there was distinct variation within and between probands. This did not relate to the efficiency of sperm production per meiocyte. Pre-protamine 2 was always confined to the subacrosomal domain, confirming the role of this area in chromatin remodelling.

Breast cancer resistance protein regulates apical ectoplasmic specialization dynamics stage specifically in the rat testis

Drug transporters determine the bioavailability of drugs in the testis behind the blood-testis barrier (BTB). Thus, they are crucial for male contraceptive development if these drugs (e.g., adjudin) exert their effects behind the BTB. Herein breast cancer resistance protein (Bcrp), an efflux drug transporter, was found to be expressed by both Sertoli and germ cells. Interestingly, Bcrp was not a component of the Sertoli cell BTB. Instead, it was highly expressed by peritubular myoid cells at the tunica propria and also endothelial cells of the microvessels in the interstitium at all stages of the epithelial cycle. Unexpectedly, Bcrp was found to be expressed at the Sertoli-step 18-19 spermatid interface but limited to stage VI-early VIII tubules, and an integrated component of the apical ectoplasmic specialization (apical ES). Apparently, Bcrp is being used by late-stage spermatids to safeguard their completion of spermiogenesis by preventing harmful drugs to enter these cells while they transform to spermatozoa. Also, the association of Bcrp with actin, Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein), and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to induce branched actin polymerization) at the apical ES suggest that Bcrp may be involved in regulating the organization of actin filament bundles at the site. Indeed, a knockdown of Bcrp by RNAi in the testis perturbed the apical ES function, disrupting spermatid polarity and adhesion. In summary, Bcrp is a regulator of the F-actin-rich apical ES in the testis.

Age-dependent alterations in spermatogenesis initchymice

Spermatogenesis is an intricate process in which spermatogonial stem cells divide and differentiate to produce mature sperm. This process strongly depends on protein turnover both in the developing germ cells and the supportive Sertoli cells, and recent evidence has demonstrated the role of the ubiquitin-proteasome system in this protein turnover in the testis. Itch, an E3 ligase important in the immune system, has been implicated in regulating the blood testis barrier. Although the specific role of Itch during spermatogenesis is not yet well understood, its ubiquitous expression and wide array of functional targets suggest multiple and tissue-specific roles. Here the testes of mice that lack Itch protein are evaluated at two developmental time points: peri-pubertal postnatal day (PND) 28 and adult PND 56. Itchy mice demonstrate an increased germ cell apoptotic index compared with wild type C57BL/6J mice at both PND 28 and PND 56. A corresponding 27% reduction in the total number of spermatid heads produced in PND 56 itchy mice was also evident. A histological evaluation of itchy mice revealed a delay in spermatogenesis at PND 28 and disorganization of late stage spermatids at PND 56. An analysis of several apoptotic markers revealed an age-dependent change in cleaved caspase 9, an intrinsic apoptosis mediator. The breeding success of the itchy mice was also significantly decreased, possibly due to a developmental defect. Taken together, these findings indicate that Itch is required for functional spermatogenesis, and that it may play differing cellular roles during development.

ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.

Myosin Va Participates in Acrosomal Formation and Nuclear Morphogenesis during Spermatogenesis of Chinese Mitten Crab Eriocheir sinensis

BackgroundThe Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis.Methodology/Principal FindingsWestern blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT) and nucleus.Conclusions/SignificanceOur study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis.

Retrospective health study on 278 babies conceived following cryopreserved spermatid injection into an electrically stimulated oocyte

OBJECTIVE: In order to overcome the low fertilization rate following conventional intracytoplasmic spermatid injection and the need for repeated testicular biopsies, we carried out spermatid cryopreservation and electrical stimulated oocyte activation. In this report we detail the clinical health findings of 278 babies (female: male =156:122) conceived following the use of this technique at our facility. DESIGN: Restrospective evaluation of health of babies following spermatid injection. MATERIALS AND METHODS: 1061 non-obstructive azoospermic men whose spermatogenesis had been arrested at the early or late stage spermatid level had participated in a total of 2389 treatment cycles. Each spermatogenic cell suspension was transferred into a sampling tube containing cryoprotectant (1.6mol/l ethylene glycol with 0.2mol/l sucrose). Freezing was carried out from -7°C to -30°C at a rate of -0.3°C/min. Each straw was exposed to air and then immediately inserted into a 30°C water bath. The cryoprotectant was removed in a stepwise fashion. Oocytes were transferred to a 295μM mannitol solution containing Mg++ and Ca++, and then stimulated with an alternating current of 2V/cm at 1MHz for 8s followed by a single 1.2kV/cm pulse of direct current for 99μs. Individual spermatids were drawn into an injection pipette and the spermatid nucleus along with a small amount of cytoplasm was injected into an ooplasm. RESULTS: (1) The pregnancy and abortion rates for early and late stage spermatids were 14.9% (196/1318), 35.2% (69/196), and 20.1% (215/1071), 29.8% (64/215) respectively. (2) One case each of anal atresia, mental retardation, congenital heart disease and cleft palate with cleft lip was reported. (3) In 102 cases of amniocentesis one paternally-inherited abnormal karyotype (translocation) was found. CONCLUSION: This study showed that the babies conceived after using the above mentioned technique had similar risk levels of being unhealthy as those babies conceived following unassisted natural conception.

Histopathologic patterns of testicular biopsies in infertile azoospermic men with varicocele

In azoospermic infertile men with varicocele, testicular biopsy revealed histopathologic patterns that varied from disorganized spermatogenesis with low or moderate sperm scores to early (primary spermatocytes stage) or late (spermatid stage) arrested spermatogenesis or germ cell aplasia and Sertoli cells only. Diagnostic testicular biopsy can be helpful for accurate management of azoospermic infertile men with varicoceles before surgical repair.

The risk of spermatid injection into an electrically stimulated oocytes. Reproductive health study on 251 new born babies

OBJECTIVE: To overcome the low fertilization rate following conventional intracytoplasmic spermatid injection and the need for repeated testicular biopsies, we carried out spermatid cryopreservation and electrical stimulated oocyte activation. In this report we detail the clinical health findings of 251 babies (female: male =141:110) conceived following the use of this technique at our facility. DESIGN: Restrospective evaluation of health of babies following spermatid injection. MATERIALS AND METHODS: 763 non-obstructive azoospermic men whose spermatogenesis had been arrested at the early or late stage spermatid level had participated in a total of 1889 treatment cycles. Each spermatogenic cell suspension was transferred into a sampling tube containing cryoprotectant (1.6mol/l ethylene glycol with 0.2mol/l sucrose) . Freezing was carried out from -7 oC to -30oC at a rate of -0.3oC/min. Each straw was exposed to air and then immediately inserted into a 30 oC water bath. The cryoprotectant was removed in a stepwise fashion. Oocytes were transferred to a 295μM mannitol solution containing 0.1μM of CaCl2 and 0.05μM MgCl2, and then stimulated with an alternating current of 2V/cm at 1MHz for 8s followed by a single 1.2kV/cm pulse of direct current for 99μs. Individual spermatids were drawn into an injection pipette and the spermatid nucleus along with a small amount of cytoplasm was injected into an ooplasm. RESULTS: 1. The pregnancy and abortion rates for early and late stage spermatids were 16.0% (163/1021), 30.1% (49/163), and 20.4% (176/878), 27.2% (48/176) respectively. 2. One case of anal atresia and one case of mental retardation (early spermatid origin) were found among the 251 babies. 3. In 83 cases, cytogenetic chromosomal analysis was performed on fetal cells obtained through amniocentesis, which resulted in one paternally-inherited abnormal karyotype. CONCLUSIONS: we found that this technique did not increase the risk of developing unhealthy babies more than that following unassisted natural conception.