Cytoplasm Of Spermatids Research Articles

Two-directional trafficking of the IFT25 protein in the developing mouse sperm flagella.

Intraflagellar transport 25 (IFT25) is a component of the IFT-B complex. In mice, even though this IFT component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of IFT in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse IFT25-GFP knock-in (KI) mouse model using the CRISPR/cas9 system, with the mouse IFT25 protein fused with a GFP tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-IFT25 and anti-GFP antibodies showed that the IFT25-GFP fusion protein was highly abundant only in the testis, which is consistent with the endogenous IFT25 protein. Examination of localization of the IFT25-GFP in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous KI mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of IFT25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching (FRAP). Kymograph and FRAP analyses demonstrate the transport of IFT25-GFP within the developing tail demonstrate no apparent preference for trafficking towards and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse IFT25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.

Eco-friendly postharvest irradiation strategy with 131I isotope for environmental management of populations of migratory locust, Locusta migratoria

Purpose Irradiation of food is promising for control of pests to minimize postharvest losses of yields and thus improvement of food safety, shelf life of produce. It is a method of choice that induces a series of lethal biochemical and molecular changes culminating into the engagement of a downstream cascade to cause abnormalities in irradiated pests. In this study, the effects of iodine-131 (131I) isotope radiation on the male gonad development of the migratory locust, Locusta migratoria, were evaluated. Materials and methods Newly emerged adult male locusts, less than one-day-old, were divided into two groups, control and irradiated. Locusts in the control group (n = 20 insects) didn’t drink irradiated water and were reared under normal environmental conditions for one week. Locusts in the irradiated group (n = 20 insects) were exposed to irradiated water at a dose of 30 mCi and they were subsequently observed until they drank the whole quantity. Results At the end of the experiment, scanning and electron microscopic examination of testes obtained from irradiated locusts revealed several major abnormalities, including malformed nuclei of spermatozoa, irregular plasma membranes, shrinkage of testicular follicles, vacuolated cytoplasm, disintegrated nebenkern and agglutinations of spermatids. Flow cytometry analysis revealed that 131I radiation induced both early and late apoptosis, but not necrosis, in testicular tissues. Testes of irradiated insects also exhibited a burst in reactive oxygen species (ROS), as indicated by significant elevation in amounts of malondialdehyde (MDA), a marker for peroxidation of lipids. In contrast, irradiation coincided with significant reductions in activities of enzymatic antioxidant biomarkers. Relative to controls, a three-fold upregulation of expression of mRNA of heat shock protein, Hsp90, was observed in testicular tissue of irradiated locusts. 131I-irradiated insects exhibited genotoxicity, as indicated by significant increases in various indicators of DNA damage by the comet assay, including tail length (7.80 ± 0.80 µm; p < .01), olive tail moment (40.37 ± 8.08; p < .01) and tail DNA intensity % (5.1 ± 0.51; p < .01), in testicular cells compared to the controls. Conclusion This is the first report on elucidation of I131-irradiation-mediated histopathological, biochemical and molecular mechanisms in gonads of male L. migratoria. Herein, the findings underscore the utility of 131I radiation as an eco-friendly postharvest strategy for management of insect pests and in particular for control of populations of L. migratoria.

TSKS localizes to nuage in spermatids and regulates cytoplasmic elimination during spermiation

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

WDR87 interacts with CFAP47 protein in the middle piece of spermatozoa flagella to participate in sperm tail assembly.

Spermatogenesis is a complex process that includes spermatogonia self-renewal, spermatocyte meiosis and spermatozoa assembly. Recent studies have revealed that WD40-repeat domain-containing (WDR) proteins play important roles in spermatocyte division, spermatozoa flagella assembly and head shaping. In this study, we investigated the expression pattern of WDR87 and found that it was highly expressed in the testis of both humans and mice. Immunofluorescence staining revealed that mouse WDR87 was distributed in the perinuclear cytoplasm of primary spermatocytes, secondary spermatocytes and round spermatids. In the spermiogenesis stage, with extension of the nucleus, WDR87 migrated to the manchette and finally localized to the middle piece of the spermatozoa tail. Furthermore, we identified a cilia- and flagella-associated protein, CFAP47, which interacted with WDR87 in the flagellar midpiece of the spermatozoa, suggesting that WDR87 may be associated with multiple morphological abnormalities of the flagella (MMAF). Subsequently, we screened gene mutations in seven MMAF individuals and found two novel mutations in CFAP47 (c.706G>A, Val236Met; c.1337C>T, Thr446Met) in one case. Immunoblotting and immunofluorescence revealed that CFAP47 was dramatically reduced in spermatozoa from the CFAP47-mutated man. Meanwhile, the expression of WDR87 was also significantly decreased, and weak signals were detected adjacent to the spermatozoa nuclei, indicating that CFAP47 was necessary for WDR87 transportation during spermatozoa flagella biogenesis. These data indicate that WDR87 is located in the middle piece of the sperm tail and interacts with CFAP47 to form a complex which is involved in spermatozoa tail assembly.

Zona Pellucida sperm-binding protein 3 receptor distribution during Gopc-/- globozoospermic spermatogenesis.

Globozoospermia is a type of teratozoospermia characterized by round morphology of the sperm head. Gopc-/- infertile globozoospermic murine model has failures during spermiogenesis, such as the incorrect biogenesis of the acrosome, disorganized acroplaxome and manchette, round nuclei and spiral flagella. In this study, Western blot, RT-PCR, immunohistochemistry and immunogold were done for the localization of the acrosome protein Zona Pellucida sperm-binding protein 3 receptor (ZP3R), also called sp56, in wild type and Gopc-/- mice testis. The ZP3R protein was located in the acrosome and pseudo-acrosome vesicles of wild type and Gopc-/- mice, respectively. Also, it is distributed through the cytoplasm of the haploid spermatids only. The incorrect spermiogenesis of Gopc-/- mice causes a deregulation in the expression of ZP3R in the globozoospermic spermatids. Our results suggest that although the lack of GOPC causes a failure during the transport of the pre-acrosomal vesicles, the acrosome protein ZP3R is localized in the acrosome and is distributed through the cytoplasm only during spermiogenesis. Furthermore, the failure in spermiogenesis does not impair the synthesis of ZP3R and its localization in the pre-acrosomal vesicles.

PIWIs maintain testis apoptosis to remove abnormal germ cells in Eriocheir sinensis.

PIWI proteins play important roles in germline development in the mammals. However, the functions of PIWIs in crustaceans remain unknown. In the present study, we identified three Piwis from the testis of Eriocheir sinensis (E. sinensis). Three Piwi genes encoded proteins with typical features of PIWI subfamilies and were highly expressed in the testis. Three PIWIs could be detected in the cytoplasm of spermatocytes and spermatids, while in spermatozoa, we could only detect PIWI1 and PIWI3 in the nucleus. The knockdown of PIWIs by dsRNA significantly affected the formation of the nuclei in spermatozoa, which resulted in deviant and irregular nuclei. PIWI defects significantly inhibited the apoptosis of abnormal germ cells through the caspase-dependent apoptosis pathway and p53 pathway. Knockdown of PIWIs inhibited the expression of caspase (Casp) 3, 7, 8, and p53 without affecting Bcl2 (B-cell lymphoma gene 2), Bax (B-cell lymphoma-2-associated X), and BaxI (B-cell lymphoma-2-associated X inhibitor), which further significantly increased abnormal spermatozoa in the knockdown-group crabs. These results show a new role of PIWI proteins in crustaceans that is different from that in mammals. In summary, PIWIs play roles in the formation of the germline nucleus and can maintain apoptosis in abnormal germ cells to remove abnormal germ cells in E. sinensis.

Ultrastructure evidence for vesicles and double-membrane structures involved in cytoplasmic elimination during spermiogenesis in large yellow croaker, Larimichthys crocea (Teleostei, Perciformes, Scienidae)

Spermatids eliminate excess cytoplasm to form streamlined sperm during spermiogenesis, which mechanism is insufficiently elucidated in fish. In this study, we investigated the cytoplasmic elimination procedure in spermatid during spermiogenesis in the large yellow croaker (Larimichthys crocea) using transmission electron microscopy. The early spermatid is subrotund with a centrally located nucleus. With further development, nucleus polarizes into one side of the cell while the cytoplasm with numerous vesicles near the membrane migrates to the caudal region. Furthermore, exocytosis-like structures were detected in middle spermatid. In late spermatid, the vesicles are reduced and rarely observed. These findings indicate that vesicles may be involved in cytoplasmic elimination possibly via exocytosis. In the later spermatid, a double-membrane, autophagosome-like structure envelopes the cytoplasm, which may develop into a single-membrane structure, and gets discarded from the cell as a residual body from the caudal region. This suggests its potential functions in the formation of residual body and cytoplasmic elimination. Overall, our results revealed that polarized development of spermatid causes polarized distribution of cytoplasm necessary for cytoplasmic elimination. Moreover, they provide ultrastructure evidence for vesicles and double-membrane structures involved in discarding spermatid cytoplasm in large yellow croaker, thus offering novel insights into cytoplasmic elimination during spermiogenesis in fish.

Subcellular localization of the mouse PRAMEL1 and PRAMEX1 reveals multifaceted roles in the nucleus and cytoplasm of germ cells during spermatogenesis

BackgroundPreferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal gametogenic tissues and a variety of tumors. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins that provide a versatile structural framework for the formation of protein–protein interactions. As a nuclear receptor transcriptional regulator, PRAME has been extensively studied in cancer biology and is believed to play a role in cancer cell proliferation by suppressing retinoic acid (RA) signaling. The role of the PRAME gene family in germline development and spermatogenesis has been recently confirmed by a gene knockout approach. To further understand how PRAME proteins are involved in germ cell development at a subcellular level, we have conducted a systematic immunogold electron microscopy (IEM) analysis on testis sections of adult mice with gene-specific antibodies from two members of the mouse Prame gene family: Pramel1 and Pramex1. Pramel1 is autosomal, while Pramex1 is X-linked, both genes are exclusively expressed in the testis.ResultsOur IEM data revealed that both PRAMEL1 and PRAMEX1 proteins were localized in various cell organelles in different development stages of spermatogenic cells, including the nucleus, rER, Golgi, mitochondria, germ granules [intermitochondrial cement (IMC) and chromatoid body (CB)], centrioles, manchette, and flagellum. Unlike other germ cell-specific makers, such as DDX4, whose proteins are evenly distributed in the expressed-organelle(s), both PRAMEL1 and PRAMEX1 proteins tend to aggregate together to form clusters of protein complexes. These complexes were highly enriched in the nucleus and cytoplasm (especially in germ granules) of spermatocytes and spermatids. Furthermore, dynamic distribution of the PRAMEL1 protein complexes were observed in the microtubule-based organelles, such as acroplaxome, manchette, and flagellum, as well as in the nuclear envelope and nuclear pore. Dual staining with PRAMEL1 and KIF17B antibodies further revealed that the PRAMEL1 and KIF17B proteins were co-localized in germ granules.ConclusionOur IEM data suggest that the PRAMEL1 and PRAMEX1 proteins are not only involved in transcriptional regulation in the nucleus, but may also participate in nucleocytoplasmic transport, and in the formation and function of germ cell-specific organelles during spermatogenesis.

Silica nanoparticles inhibiting the differentiation of round spermatid and chromatin remodeling of haploid period via MIWI in mice

Researches have shown that silica nanoparticles (SiNPs) could reduce both the quantity and quality of sperm. However, the mechanism of toxicity induced by SiNPs in the male reproductive system is still unclear. In this study, male mice were randomly divided into a control group, and SiNPs treated group (20 mg/kg dose; n = 30 per group). Half of the mice per group were sacrificed on 35 days and the remaining on 50 days of the SiNPs exposure. SiNPs were found to decrease sperm count and mobility, increase the sperm abnormality rate, and damage the testes' structure. Furthermore, SiNPs decreased the protein levels of Protamine 1(PRM1) and elevated the histones' levels and suppressed the chromatin condensation of sperm. There was a significant reduction of the ubiquitinated H2A (ubH2A)/H2B (ubH2B) and RING finger protein 8 (RNF8) levels in the spermatid nucleus, while the RNF8 level in the spermatid cytoplasm increased evidently. The protein expression levels of PIWI-like protein 1(MIWI) in the late spermatids significantly increased on day 35 of SiNPs exposure. After 15 days of the withdrawal, the sperm parameters and protamine levels, and histones in the epididymal sperm were unrecovered; however, the changes in testis induced by SiNPs were recovered. Our results suggested that SiNPs could decrease the RNF8 level in the nucleus of spermatid either by upregulating of the expression of MIWI or by inhibiting its degradation. This resulted in the detention of RNF8 in the cytoplasm that maybe inhibited the RNF8-mediated ubiquitination of ubH2A and ubH2B. These events culminated in creating obstacles during the H2A and H2B removal and chromatin condensation, thereby suppressing the differentiation of round spermatids and chromatin remodeling, which compromised the sperm quality and quantity.

Study of Sertoli cells and spermatogenic cells in rats with experimentally induced metabolic syndrome and after balneophysiotherapy

Introduction. Metabolic syndrome (MS) can cause impaired spermatogenesis and a decrease in sperm counts. However, the details of the effect of MS on developing spermatogenic cells remain unclear. Difficulties in solving this problem, the inconsistency of published clinical data, indicate the advisability of using experimental models to solve this urgent problem of andrology and reproductology.The study objective is to describe to investigate the specifics of the course of meiotic prophase I and the activity of the processes of phagocytosis and autophagy in Sertoli cells of rats with experimentally induced MS and in the course of therapeutic and prophylactic procedures during the development of experimental MS.Materials and methods. The animals were divided into three groups, each of which included four sexually mature male rats: 1st group – males fed a standard diet; 2nd group – males receiving a diet high in fat and fructose for 60 days; 3rd group – males with MS receiving sulphate mineral waters therapy, low-intensity ultrahigh frequency electromagnetic radiation therapy. Testicular cells were examined using light and transmission electron microscopy. For the first time in animals with MS, an immunocytochemical study of the peculiarities of chromosome synapsis in prophase I of meiosis was carried out on the basis of analysis of spread synaptonemal complexes of meiotic chromosomes and immunocytochemical analysis of Sertoli cells and spermatogenic cells in squashed preparations of seminiferous tubules. The parametric Student’s t-test and the nonparametric Mann–Whitney U-test were used for statistical data processing.Results. As a result of a histological study of the structure of the seminiferous tubules of animals of three groups, a statistically significant decrease in the indices of the spermatogenesis index in 2nd and 3rd groups compared to the control was revealed. Immunomorphologically, in the spread nuclei of primary spermatocytes of rats of the 2nd and 3rd groups, violations of the architectonics of nuclei, the formation of synaptonemal complexes fragments and circular synaptonemal complexes, numerous atypical inclusions were found. Signs of pachytene arrest were found in 40–50 % of spermatocyte nuclei. In the study of squashed cells preparations of the seminiferous tubules of rats of the 2nd and 3rd groups, signs of phagocytosed synaptonemal complexes were found in the cytoplasm of Sertoli cells, which were confirmed using antibodies to the SCP3 protein. Thus, evidence for the phagocytosis of degenerating primary spermatocytes by Sertoli cells has been obtained. In Sertoli cells, spermatocytes and spermatids, many autophagosomes are found, using LC3B protein marker. The presence of autophagosomes in Sertoli cells and spermatogenic cells in animals of these two groups was also confirmed by electron microscopy. In male rats of the 2nd group, significant disturbances in the structure of the pachytene nuclei were revealed. In the cytoplasm of Sertoli cells and spermatids of rats of the 2nd group, lipid droplets, numerous phagolysosomes containing cell detritus were revealed. Structural damage and phagocytosis of mitochondria were found in Sertoli cells and spermatocytes. Аutophagy in Sertoli cells were most distinctive in animals of the 3rd group.Conclusion. In male rats with experimental MS, significant disturbances in the structure of the nuclei of meiotic cells, a high content of primary spermatocytes with signs of pachytene arrest were revealed. The results obtained are in good agreement with the data of other authors, who revealed a decrease in the number of spermatozoa in the epididymis of rats and mice when modeling MS. It is assumed that the activation of autophagy is an important factor in supporting the viability of Sertoli cells and supporting the viability of germ cells in stressful situations, including MS. Apparently, autophagy is an adaptive mechanism that removes the remnants of apoptotic spermatogenic cells that are selected as a result of MS development.

Process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei: Polarized development of the spermatid and discarding of the residual body.

The elimination of the spermatid cytoplasm during spermiogenesis enables the sperm to acquire a streamlined architecture, which allows for unhindered swimming. While this process has been well described in vertebrates, it has rarely been reported in invertebrates. In this study, we observed the process of cytoplasm elimination during spermiogenesis in Octopus tankahkeei (Mollusca, Cephalopoda) using light microscopy, transmission electron microscopy, and immunofluorescence. In the early spermatid, the cell is circular, and the nucleus is centrally located. With spermatid development, the cell becomes polarized. The nucleus gradually elongates and moves toward the end of the cell where the tail is forming. As a result, the cytoplasm moves past the nucleus at the anterior region of the future sperm head (the foreside of the acrosome). Following this, during the late stage of spermiogenesis, the cytoplasm condenses and collects on the foreside of the acrosome until finally the residual body is discarded from the top of the sperm head. This represents a distinct directionality for the development of cytoplasmic polarity and discarding of residual body compared with that reported for vertebrates (in which the cytoplasm of the elongating spermatids is polarized toward the caudal region). The fact that the cytoplasm also becomes concentrated suggests that water pumps may be involved in the elimination of water from the cytoplasm before the residual body is discarded. Furthermore, we found that microtubules, forming a manchette-like structure, are involved not only in reshaping of the nucleus but also in the transport of mitochondria and vesicles to the foreside of the acrosome, subsequently allowing them to be discarded with the residual body. This study broadens our understanding of the development of polarization and elimination of cytoplasm from spermatids in animals.

Expression and localization of alpha-tubulin N-acetyltransferase 1 in the reproductive system of male mice.

The structure of microtubules is essential for the fertilizing ability of spermatozoa. Acetylation of α-tubulin plays an important role in flagellar elongation and spermatozoa motility. Previous reports have suggested that alpha-tubulin N-acetyltransferase 1 (ATAT1) is the main acetyltransferase involved in the acetylation of α-tubulin. Although ATAT1 is reported to express in the testis, no information is available regarding its expression in elongated spermatids, epididymis, and mature spermatozoa. Hence, it remains unclear whether ATAT1 is involved in spermatozoa maturation and capacitation. Therefore, we evaluated the expression of ATAT1 in the mouse male reproductive system using immunostaining and western blotting. Our results showed that ATAT1 was expressed in spermatids during spermiogenesis in mouse testes, but its expression varied according to the seminiferous tubule stage. We observed ATAT1 in the cytoplasm of round spermatids, the flagella of elongated spermatids, and in the cytoplasm of step 16 spermatids, just before its release into the lumen. In addition, ATAT1 was expressed in epithelial cells of the epididymis. In spermatozoa of the cauda epididymis, ATAT1 expression was primarily observed in the midpiece of the spermatozoa. The localization of ATAT1 protein in the male germline was observed during spermiogenesis as well as during spermatozoa maturation. Our results suggest that ATAT1 may be involved in the formation of flagella and in the acetylation process, which has attracted attention in recent years regarding male infertility.

The effect of major salivary glands hypertrophy on rat’s spermatogenic epithelium ultrastructure

Purpose. The aim of this study was to ascertain the characteristics of major salivary glands endocrine effect on spermatogenesis. Materials and methods. Mature white outbred male rats (2 months, 153 ± 18 g) consisted of the following groups (each containing 30 rats): intact, control, and group of rats subjected to multiple amputation of incisors. To achieve hypertrophy of major salivary glands multiple amputation of incisors was performed: incisors were cut to a level of 1-2 mm above the gingival margin under ether anesthesia once every 3 days within 2 weeks. Animals of the control group were anesthetized with ether at the same time. Rats were sacrificed by CO 2 asphyxia after 2, 3, 4, 6, 8 and 10 weeks after the first amputation of incisors. Fragments of the rat testes were examined on a JEM-1400 “JEOL” (Japan) transmission electron microscope. On electron microscopy images the specific vacuolization of the cytoplasm of Sertoli cells, spermatogonia, spermatocytes and spermatids (standard units) was analyzed by the point counting method. In spermatogenic cells the proportion of mitochondria (%) with morphological signs of swelling was assessed. Results. Transient ultrastructural changes of Sertoli and spermatogenic cells develop in the rats convoluted seminiferous tubules as a result of multiple amputation of the incisors, such as phagosomes and pronounced vacuolization in the Sertoli cells cytoplasm, cytoplasm vacuolization and mitochondrial swelling in spermatogenic cells. Sporadic spermatogenic cells with signs of nuclear (chromatin fragmentation, its condensation on the periphery of the nucleus) and cytoplasm (destruction of membrane organelles) destruction appeared as a result of multiple incisors’ amputation. Ultrastructural changes of Sertoli and spermatogenic cells are most pronounced at 2-3 weeks, decrease at 4 week and are completely leveled by the 6th week of the experiment. Conclusion. Hypertrophy of major salivary glands, caused by multiple amputations of incisors, has similar to sialoadenectomy effect on the spermatogenic epithelium. Multiple incisors’ amputation cause transient depression of granular convoluted cells function. Probably submandibular gland granular convoluted tubules cells endocrine factors make the greatest contribution to the regulation of spermatogenesis in rats.

Mutator Foci Are Regulated by Developmental Stage, RNA, and the Germline Cell Cycle in Caenorhabditis elegans.

RNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

Spermatogenesis is normal in Tex33 knockout mice.

Testis expressed gene 33 (Tex33) is a recently reported testis-specific gene and it is evolutionarily conserved in vertebrates. The Tex33 expression is found in cytoplasm of round spermatids in Mus musculus. However, the in vivo function of Tex33 remains unknown. In this study, we made a 62bp in frame deletion on Exon2 of Tex33 gene by CRISPR/Cas9 in C57B/L6 mouse, which cause frame shift mutation of Tex33 gene. Tex33-/-adult male were fertile, and there is no significant change on litter size compared with male wildtype (Tex33+/+) adult. Besides, no overt differences were found in testis/body weight ratios, testicular/epididymal tissue morphology, sperm counts, sperm morphology and spermatozoa motility in adult Tex33-/-male mice (N = 3), in comparison with Tex33+/+ adult (N = 3). TUNEL assay also indicates the germ cells apoptosis ratio was not significantly changed in adult Tex33-/- adult male mouse testis (N = 3), compared with adult Tex33+/+ male (N = 3). Importantly, the first wave of elongating spermatids formation happens in 5w old mice. We find that the first wave of spermiogenesis is not disrupted in both 5-week-old Tex33+/+ and Tex33-/-male mouse testes and three hallmarks of spermatogenesis, PLZF,γ-H2AX and TNP1, are all detectable in seminiferous tubule. All results indicate that Tex33 is a redundant gene to spermatogenesis. This study can help other researchers avoid repetitive works on redundant genes.

Gene expression patterns and protein cellular localization suggest a novel role for DAZL in developing Tibetan sheep testes

Deleted in azoospermia-like (DAZL) is essential for mammalian spermatogenesis as it regulates proliferation, development, maturation and functional maintenance of male germ cells. Its expression and regulation vary with different species or in the same animal at different developmental stages, and despite its importance, very little is known about its roles in sheep, especially Tibetan sheep. To investigate the expression patterns and regulatory roles of DZAL in Tibetan sheep testis, testicular tissue was isolated from sheep at three crucial development stages: 3 months old, 1 year old and 3 years old. Using quantitative real-time PCR and Western blot, we found that DAZL mRNA first decreased and then increased with advancing age, while DAZL protein exhibited an opposite expression pattern, with first increased and subsequently decreased levels. Immunohistochemistry and immunofluorescence revealed that DAZL protein was located predominantly in the cytoplasm of Leydig cells and in both the cytoplasm and nucleus of spermatids. ELISA indicated that testosterone content within developing testes was first enhanced and then declined. Our results, taken together, demonstrate, for the first time, that DAZL gene is involved in Tibetan sheep spermatogenesis by regulating the development of spermatids in post-pubertal rams, along with a novel role in functional maintenance of Leydig cells in postnatal rams.

Close association between vasa-positive germ plasm granules and mitochondria correlates with cytoplasmic localization of 12S and 16S mtrRNAs during zebrafish spermatogenesis

The phenomenon of the cytoplasmic localisation of mitochondrial ribosomal subunits (12 S mitochondrial rRNA and 16 S mitochondrial rRNA) has been discovered by scientific teams working with spermatogenic cells of mice. Previous reports showed that the release of mitochondrial substance occurs during interaction of mitochondria with the germ plasm granules (GG). To determine if the interplay between the vasa-positive GG and the mitochondria is associated with cytoplasmic localisation of mtrRNAs, we studied the spermatogenic cells of zebrafish, Danio rerio. It was revealed that in type A undifferentiated spermatogonia the GG did not contact mitochondria, and the extra-mitochondrial localisation of the mtrRNAs was not found. In type A differentiated spermatogonia, the amount of GG in contact with mitochondria increased, but the extra-mitochondrial localisation of the mtrRNAs was not found either. In type B late spermatogonia, which are pre-meiotic cells, the GG/mitochondrion complexes were typically found in contact with the nucleus. This stage was associated with the intra-mitochondrial localisation of GG-originated vasa and extra-mitochondrial localisation of 12 S mtrRNA and 16 S mtrRNA. Until the onset of meiosis, which was determined by the observation of synaptonemal complexes in zygotene-pachytene spermatocytes I, the GG/mitochondrion complexes disappeared, but both types of mtrRNAs persisted in the cytoplasm of spermatids and spermatozoa.

Role of Spata34 in cell proliferation and its expression pattern in postnatal development of rat testis.

Spata34 is a testis-specific-expressed gene which exerts diverse functions in testis development. This study intends to examine the expression profiles of Spata34 in postnatal rat testis, and explore its potential roles in cell proliferation in vitro. We found that the mRNA and protein expression levels of Spata34 were developmentally upregulated in rat testes during the early 1-7 postnatal weeks using real-time polymerase chain reaction and western blotting. Immunohistochemical results indicated that Spata34 protein was mainly detected in the nuclear and cytoplasm of spermatocytes and round spermatids. The possible function of Spata34 in cellular proliferation was analyzed using cell counting kit, colony formation and flow cytometry assays. Our results showed that overexpression of Spata34 in multipotent adult germline stem cell lines (maGSC129SV) cells markedly facilitated cell proliferation with a large increase in cell numbers in S phase of cell cycle. While knockdown of Spata34 expression by specific siRNA suppressed the growth of maGSC129SV cells and triggered cell-cycle arrest at G1/S phase transition, which was related to the elevation of p21 and p27 and decrease of Cyclin D1 and Cyclin D-dependent kinase 4. Altogether, our results indicated that the Spata34 gene evokes unique expression patterns during postnatal development of the rat testis, and for the first time, unravels the function of Spata34 on regulating cell-cycle progress through p21 and p27 pathway.

Deciphering the Dark Proteome: Use of the Testis and Characterization of Two Dark Proteins

For the C-HPP consortium, dark proteins include not only uPE1, but also missing proteins (MPs, PE2-4), smORFs, proteins from lncRNAs, and products from uncharacterized transcripts. Here, we investigated the expression of dark proteins in the human testis by combining public mRNA and protein expression data for several tissues and performing LC-MS/MS analysis of testis protein extracts. Most uncharacterized proteins are highly expressed in the testis. Thirty could be identified in our data set, of which two were selected for further analyses: (1) A0AOU1RQG5, a putative cancer/testis antigen specifically expressed in the testis, where it accumulates in the cytoplasm of elongated spermatids; and (2) PNMA6E, which is enriched in the testis, where it is found in the germ cell nuclei during most stages of spermatogenesis. Both proteins are coded on Chromosome X. Finally, we studied the expression of other dark proteins, uPE1 and MPs, in a series of human tissues. Most were highly expressed in the testis at both the mRNA and protein levels. The testis appears to be a relevant organ to study the dark proteome, which may have a function related to spermatogenesis and germ cell differentiation. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD009598.

LRRC8/VRAC anion channels are required for late stages of spermatid development in mice

Spermatogenesis is a highly complex developmental process that occurs primarily in seminiferous tubules of the testes and requires additional maturation steps in the epididymis and beyond. Mutations in many different genes can lead to defective spermatozoa and hence to male infertility. Some of these genes encode for ion channels and transporters that play roles in various processes such as cellular ion homeostasis, signal transduction, sperm motility, and the acrosome reaction. Here we show that germ cell–specific, but not Sertoli cell–specific, disruption of Lrrc8a leads to abnormal sperm and male infertility in mice. LRRC8A (leucine-rich repeat containing 8A) is the only obligatory subunit of heteromeric volume-regulated anion channels (VRACs). Its ablation severely compromises cell volume regulation by completely abolishing the transport of anions and osmolytes through VRACs. Consistent with impaired volume regulation, the cytoplasm of late spermatids appeared swollen. These cells failed to properly reduce their cytoplasm during further development into spermatozoa and later displayed severely disorganized mitochondrial sheaths in the midpiece region, as well as angulated or coiled flagella. These changes, which progressed in severity on the way to the epididymis, resulted in dramatically reduced sperm motility. Our work shows that VRAC, probably through its role in cell volume regulation, is required in a cell-autonomous manner for proper sperm development and explains the male infertility of Lrrc8a−/− mice and the spontaneous mouse mutant ébouriffé.