Spermatozoa from male mice with infertility due to Odf4 deficiency can fertilize oocytes by in vitro fertilization.

Purpose: The purpose of this study is to confirm whether in vitro fertilization (IVF) with spermatozoa from Odf4-deficient infertile males (Odf4 -/- spermatozoa) can lead to the development of zygotes, which was reported in a previous in vivo study. Methods: In vitro capacitation and IVF were performed using Odf4 -/- spermatozoa in a small drop of TYH medium with pyruvate and glucose, for 60 min or up to 4 days. A capacitation test was performed by immunoblotting using an anti-p-Tyr antibody. A sperm movement test was performed using a computer-assisted sperm motility analysis system (SMAS). An IVF fertilization test was also performed to evaluate zygote production. Videos were taken by a DMi8 stereomicroscope equipped with a high-speed camera. Results: In in vitro condition, Odf4 -/- spermatozoa with hairpin flagella harboring large cytoplasmic droplets (CDs) underwent capacitation, about 30% of large CDs were removed from spermatozoa, and the flagella became straight (capacitation test). The Odf4 -/- spermatozoa with straight flagella swam forward (movement test) and fertilized Odf4 +/+ oocytes, which eventually developed into zygotes (fertilization test). Conclusions: By conventional IVF, spermatozoa from Odf4-deficient male mice can fertilize oocytes that then develop into zygotes. These findings can be translated to human males with infertility caused by ODF4 deficiency.

The association of ODF4 with AK1 and AK2 in mice is essential for fertility through its contribution to flagellar shape.

Normal sperm flagellar shape and movement are essential for fertilization. The integral protein outer dense fiber 4 (ODF4) localizes to ODFs, but its function remains unclear. Adenylate kinase (AK) is a phosphotransferase that catalyzes the interconversion and controls the concentration equilibrium of adenine nucleotides. AK shuttles ATP to energy-consuming sites. Here, we report on the relationship of flagellar shape and movement with ODF4, AK1 and AK2 by using Odf4-deletion (Odf4-/-) mice. Soluble ODF4 is coimmunoprecipitated with AK1 and AK2 in Odf4+/+ spermatozoa. ODF4, AK1 and AK2 localize to whole flagella (plasmalemma, mitochondria, ODFs, and residual cytoplasmic droplets (CDs)), principal pieces, and midpieces, respectively. Odf4-/- sperm flagella lose ODF4 and reduce AK1 and AK2 but produce ATP. The flagellum is bent (hairpin flagellum) with a large CD in the midpiece. There is no motility in the midpiece, but the principal piece is motile. Odf4-/- spermatozoa progress backward and fail to ascend in the uterus. Thus, Odf4-/- males are infertile owing to abnormal flagellar shape and movement caused mainly by the loss of ODF4 with AK1 and AK2. This study is supported by the rescue experiment; the abnormalities and male infertility caused by Odf4 deletion were reversed by Odf4 restoration.

Decrease of lysyl hydroxylase 2 activity causes abnormal collagen molecular phenotypes, defective mineralization and compromised mechanical properties of bone.

Lysyl hydroxylase 2 (LH2) is an enzyme that catalyzes the hydroxylation of lysine (Lys) residues in fibrillar collagen telopeptides, a critical post-translational modification for the stability of intermolecular cross-links. Though abnormal LH2 activities have been implicated in various diseases including Bruck syndrome, the molecular basis of the pathologies is still not well understood. Since LH2 null mice die at early embryonic stage, we generated LH2 heterozygous (LH2+/-) mice in which LH2 level is significantly diminished, and characterized collagen and bone phenotypes using femurs. Compared to the wild-type (WT), LH2+/- collagen showed a significant decrease in the ratio of hydroxylysine (Hyl)- to the Lys-aldehyde-derived collagen cross-links without affecting the total number of aldehydes involved in cross-links. Mass spectrometric analysis revealed that, in LH2+/- type I collagen, the extent of hydroxylation of all telopeptidyl Lys residues was significantly decreased. In the helical domain, Lys hydroxylation at the cross-linking sites was either unaffected or slightly lower, but other sites were significantly diminished compared to WT. In LH2+/- femurs, mineral densities of cortical and cancellous bones were significantly decreased and the mechanical properties of cortical bones evaluated by nanoindentation analysis were compromised. When cultured, LH2+/- osteoblasts poorly produced mineralized nodules compared to WT osteoblasts. These data provide insight into the functionality of LH2 in collagen molecular phenotype and its critical role in bone matrix mineralization and mechanical properties.

Dlec1 is required for spermatogenesis and male fertility in mice.

Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1-/- mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet-Biedl Syndrome (BBS) protein complex subunits, as well as α- and ß-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.

Cleavage of SPACA1 regulates assembly of sperm-egg membrane fusion machinery in mature spermatozoa†.

The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

Odf2 haploinsufficiency causes a new type of decapitated and decaudated spermatozoa, Odf2-DDS, in mice.

Outer dense fibre 2 (Odf2 or ODF2) is a cytoskeletal protein required for flagella (tail)-beating and stability to transport sperm cells from testes to the eggs. There are infertile males, including human patients, who have a high percentage of decapitated and decaudated spermatozoa (DDS), whose semen contains abnormal spermatozoa with tailless heads and headless tails due to head-neck separation. DDS is untreatable in reproductive medicine. We report for the first time a new type of Odf2-DDS in heterozygous mutant Odf2+/- mice. Odf2+/- males were infertile due to haploinsufficiency caused by heterozygous deletion of the Odf2 gene, encoding the Odf2 proteins. Odf2 haploinsufficiency induced sperm neck-midpiece separation, a new type of head-tail separation, leading to the generation of headneck sperm cells or headnecks composed of heads with necks and neckless tails composed of only the main parts of tails. The headnecks were immotile but alive and capable of producing offspring by intracytoplasmic headneck sperm injection (ICSI). The neckless tails were motile and could induce capacitation but had no significant forward motility. Further studies are necessary to show that ICSI in humans, using headneck sperm cells, is viable and could be an alternative for infertile patients suffering from Odf2-DDS.

Usp26 mutation in mice leads to defective spermatogenesis depending on genetic background.

Spermatogenesis is a reproductive system process that produces sperm. Ubiquitin specific peptidase 26 (USP26) is an X chromosome-linked deubiquitinase that is specifically expressed in the testes. It has long been controversial whether USP26 variants are associated with human male infertility. Thus, in the present study, we introduced a mutation into the Usp26 gene in mice and found that Usp26 mutant males backcrossed to a DBA/2 background, but not a C57BL/6 background, were sterile or subfertile and had atrophic testes. These findings indicate that the effects of the Usp26 mutation on male reproductive capacity were influenced by genetic background. Sperm in the cauda epididymis of Usp26 mutant mice backcrossed to a DBA/2 background were decreased in number and showed a malformed head morphology compared to those of wild-type mice. Additionally, histological examinations of the testes revealed that the number of round and elongated spermatids were dramatically reduced in Usp26 mutant mice. The mutant mice exhibited unsynapsed chromosomes in pachynema and defective chiasma formation in diplonema, which presumably resulted in apoptosis of metaphase spermatocytes and subsequent decrease of spermatids. Taken together, these results indicate that the deficiencies in fertility and spermatogenesis caused by mutation of Usp26 were dependent on genetic background.

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.

Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality.

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2-/-) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2-/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.

Deletion of Eqtn in mice reduces male fertility and sperm-egg adhesion.

A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn - / - ) mouse model and found that the EQTN-KO males have reduced fertility and sperm-egg adhesion, while the EQTN-KO females are fertile. Eqtn - / - sperm were normal in morphology and motility. Eqtn - / - -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn - / - with Eqtn +/+ -Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn - / - males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn - / - sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn - / - sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 - / - ) was more severe compared with that of Eqtn - / - males. Eqtn - / - -Tg (Eqtn) males, which were generated to rescue Eqtn - / - males, restored the reduced fertility.

Blocking Liver Autophagy Accelerates Apoptosis and Mitochondrial Injury in Hepatocytes and Reduces Time to Mortality in a Murine Sepsis Model.

Autophagy plays an important role in cell survival, sequestering, and degrading a wide variety of substrates. Although an increase of autophagosomes in liver has been reported in sepsis patients as well as in septic mice, the influence of autophagy on liver injury, the interaction between autophagy, and other types of cell death in sepsis remain unclear. The aim of this study was to elucidate the contribution of liver autophagy to the pathophysiology of sepsis. We performed a cecal ligation and puncture on liver-specific autophagy-deficient (Alb-Cre/Atg5) mice (6-8-week-old male). When compared with controls (C57BL/6), we found a significant accumulation of p62 in the liver and demonstrated a greater number of cleaved caspase-3 immunoreactive hepatocytes in these knockout (KO) mice. Additionally, we confirmed a significant increase in autophagic vacuoles in the control mice relative to KO mice; in contrast, cell shrinkage and nuclear fragmentation (morphological characteristics of apoptosis) were preferentially seen in the KO mice by transmission electron microscopy. Severe mitochondrial damage was also prominent in KO mice, relative to controls, associated with an increase of reactive oxygen species in hepatocytes. Serum aspartate transaminase levels (P = 0.005) and serum interleukin-6 levels (P = 0.020) were significantly increased in the KO mice compared with controls. Deficiency of autophagy in liver significantly decreased survival in the murine sepsis model (P = 0.025). In conclusion, blocking liver autophagy accelerates time to mortality in the murine sepsis model, suggesting that liver autophagy plays a protective role for organ failure through degradation of damaged mitochondria, as well as prevention of apoptosis.

Subcellular localization of MS4A13 isoform 2 in mouse spermatozoa.

To identify upregulated genes during the development of spermatozoa, we performed PCR-selected subtraction analysis of testes RNA samples from 10-day-old and 12-week-old shrews. A transcript, highly homologous to two mouse transcripts, Ms4a13-1 and Ms4a13-2, was differentially regulated. Ms4a13-2, but not Ms4a13-1, was shown to be primarily expressed in mouse testes in an age-dependent manner. Ms4a13-2 cDNA contains an open-reading frame of 522 nucleotides, encoding a protein of 174 amino acids, with predicted molecular mass, 19,345 Da. MS4A13-2 protein was expressed along the periphery of nuclei of round and elongated spermatids (steps 3-16) in adult mouse testes, and in the equatorial region of the heads of fresh mature mouse spermatozoa. In addition, MS4A13-2 was found to localize to the outer acrosomal membrane in the equatorial region of heads in fresh spermatozoa. In acrosome-reacted spermatozoa, the MS4A13-2 expression extended to the entire sperm head including the postacrosomal region and acrosomal cap. MS4A family proteins are known to facilitate intracellular protein-protein interactions as ion channel/adaptor proteins by oligomerization, and have important regulatory roles in cellular growth, survival and activation. We report that the MS4A family member, MS4A13-2, may form oligomers in sperm membranes, which may be involved in an interaction with the zona pellucida or cumulus during fertilization.

EPC1/TIP60-Mediated Histone Acetylation Facilitates Spermiogenesis in Mice.

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.

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.

Suppression of T Cell Autophagy Results in Decreased Viability and Function of T Cells Through Accelerated Apoptosis in a Murine Sepsis Model.

OBJECTIVE: While type 1 programmed cell death (apoptosis) of T cells leads to immunosuppression in sepsis, a crosstalk between apoptosis and autophagy (type 2 programmed cell death) has not been shown. The aim of this study is to elucidate the details of the interaction between autophagy and immunosuppression. DESIGN: Laboratory investigation in the murine sepsis model. SETTING: University laboratory. SUBJECTS: Six- to 8-week-old male mice. INTERVENTIONS: We investigated the kinetics of autophagy in T cells from spleen in a cecal ligation and puncture model with green fluorescent protein-microtubule-associated protein light chain 3 transgenic mice. We analyzed apoptosis, mitochondrial homeostasis and cytokine production in T cells, and survival rate after cecal ligation and puncture using T cell-specific autophagy-deficient mice. MEASUREMENTS AND MAIN RESULTS: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4 T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4 T cells from the cecal ligation and puncture-operated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model. CONCLUSIONS: We demonstrated that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4 T cells in the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis.

A critical role of solute carrier 22a14 in sperm motility and male fertility in mice.

We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14-/- cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14-/- spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14-/- sperm cells: the annulus, a ring-like structure at the mid-piece-principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa.

Acrosome markers of human sperm.

Molecular biomarkers that can assess sperm acrosome status are very useful for evaluating sperm quality in the field of assisted reproductive technology. In this review, we introduce and discuss the localization and function of acrosomal proteins that have been well studied. Journal databases were searched using keywords, including "human acrosome", "localization", "fertilization-related protein", "acrosomal membrane", "acrosomal matrix", "acrosome reaction", "knockout mouse", and "acrosome marker".

Interaction between basigin and monocarboxylate transporter 2 in the mouse testes and spermatozoa.

Basigin is a member of the immunoglobulin superfamily and plays various important roles in biological events including spermatogenesis. To examine the basigin molecular variants during spermatogenesis and sperm maturation in the mouse, immunoprecipitated basigin samples from testis and epididymal spermatozoa were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results demonstrated that basigin molecules from the testis and spermatozoa were separable into two major bands and that the differences in the molecular sizes were possibly because of an endoproteolytic cleavage. Since basigin is known to be a chaperone for the monocarboxylate transporter 1 (MCT1), the localization of basigin, MCT1 and MCT2 was examined during postnatal testicular development. Immunohistochemical studies showed different expression patterns of MCT1 and MCT2. MCT1 was localized on the surface of spermatogonia, spermatocytes, and spermatids. In contrast, MCT2 appeared on the principal piece of spermatozoa in the testis, where basigin was also observed. In mature epididymal spermatozoa, MCT2 was located on the midpiece, where basigin co-localized with MCT2 but not with MCT1. Furthermore, MCT2 was immunoprecipitated with basigin in mouse testes and sperm. These results suggest that basigin has a functional role as a binding partner with MCT2 in testicular and epididymal spermatozoa.

Immunohistochemical localization of GLUT3, MCT1, and MCT2 in the testes of mice and rats: the use of different energy sources in spermatogenesis.

Lactate represents a preferential energy substrate of germ cells rather than glucose. Testicular Sertoli cells are believed to produce lactate and pyruvate and to supply these to germ cells, particularly spermatocytes and spermatids. Monocarboxylate transporter (MCT), responsible for the transport of lactate and other monocarboxylates via the cell membrane, is abundant in the testes and sperm (MCT1, MCT2, and MCT4). For the uptake of glucose, germ cells within the seminiferous tubules and sperm have been known to intensely express GLUT3. The present study investigated expression profiles of MCTs and GLUTs and revealed their cellular and subcellular localization in the mouse and rat testis. An in situ hybridization analysis showed significant expressions of MCT1, MCT2, and GLUT3 mRNA in the testis. Immunohistochemically, spermatogonia, spermatocytes, and spermatids expressed MCT1 on their cell surfaces in a stage-dependent manner: in some seminiferous tubules, an intense expression of MCT1 was unique to the spermatogonia. MCT2 was restricted to the tails of elongated spermatids and sperm. An intense immunoreactivity for GLUT3 was shared by spermatocytes, spermatids, and sperm. Sertoli cells were devoid of any immunoreactivities for MCT1, MCT2, and GLUT3. The predominant energy source of germ cells may be lactate and other monocarboxylates--especially for spermatogonia, but glucose and other hexoses may be responsible for an energy supply to spermatocytes and spermatids.

Analysis of the complexity of the sperm acrosomal membrane by super-resolution stimulated emission depletion microscopy compared with transmission electron microscopy.

The acrosome is a Golgi-derived sperm cell organelle enclosed by a continuous acrosomal membrane. The acrosomal membrane complexes with surrounding matrices containing molecules necessary for fertilization; however, the complex of acrosomal membrane and associating matrices (CAMAM) has not been visualized in detail under living conditions. Here, we analyzed the CAMAM at the nanometer level using super-resolution stimulated emission depletion (STED) fluorescence microscopy and equatorin-enhanced green fluorescent protein transgenic mice. The STED images were compared with the corresponding images taken by immunoelectron microscopy. Consequently, the substructure of CAMAM could be differentiated at nanometer-scale resolution by STED microscopy without the need for sectioning. The information obtained in this study will be beneficial not only for understanding the molecular mechanism of fertilization but also for cell imaging under living conditions.