Spermatogenesis is regulated by hormones, local regulatory factors in the testes and specific gene expression of spermatogenic cells in humans. In this study, we have detected the expression of the deleted in azoospermia (DAZ), the DAZ-like autosome (DAZL1), and the protamine-2 genes in spermatogenic cells. Spermatogenesis in 38 male infertility patients was evaluated by the semen analysis and histological examination. Patients were diagnosed as Sertoli cell-only syndrome (n = 20), maturation arrest (n = 6), hypospermatogenesis (n = 6), and obstructive azoospermic patients with normal spermatogenesis (n = 6). After microscopic observation of the wet preparation of the testis tissues, seminiferous tubule contents were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis of DAZ, DAZL1 and protamine-2. In cases with Sertoli-cell only syndrome, we found spermatogenic cells in 30% of patients (6/20) by the wet preparation method. There was no difference between the histology and the wet preparation results in maturation arrest and obstructive azoospermia; however, in one case of hypospermatogenesis, spermatozoa were not detectable by the wet preparation method. Using in-situ hybridization with DAZ and protamine-2 ribonuclear probes, we confirmed spermatogenic cell-specific expression of DAZ (spermatogonia/early spermatocyte) and protamine-2 (spermatid/spermatozoon). DAZ and protamine-2 expression can therefore be considered spermatogenic cell markers and could be useful in molecular diagnosis of spermatogenesis. In 13 patients with spermatozoa under the wet preparation, the expression of DAZ, DAZL1 and protamine-2 was detected in all the preparations. In one wet preparation showing only spermatogonia/spermatocyte, only DAZ and DAZL1 RNA were detected. In 14 wet preparations showing no spermatogenic cells, DAZ, DAZL1 and protamine-2 were not detected except in one preparation where DAZL1 expression was detected. In 10 wet preparations representing spermatogonia/spermatocyte to spermatids, but showing no spermaozoa, DAZ and DAZL1 were detected in eight and nine preparations respectively, and protamine-2 was detected in six preparations. These results of gene expression were similar to the wet preparation results. RT-PCR for DAZ, DAZL1 and protamine-2 was informative for the existence of germ cells, germ cell physiology and differentiation. From these results, we suggest that the analysis of DAZ, DAZL1 and protamine-2 expression by RT-PCR and wet preparation might offer a better method for finding the spermatogenic cells compared to the histological method.

T grew optimally at 25 C and pH 7.5-8.5 in the presence of 1.0-3.0 % (w/v) sea salts. The major cellular fatty acids (>10 %) were C 16 : 1 !6c and/or C 16 : 1 !7c, C 16 : 0 , and C 18 : 1 !6c and/or C 18 : 1 !7c. The genomic DNA G+C content was 39.7 mol%. On the basis of the phylogenetic, genomic, chemotaxonomic and phenotypic data presented, we propose the name Pseudoalteromonas neustonica sp. nov. with the type strain PAMC 28425 T (=KCCM 43187 T =JCM 31286 T ).The genus Pseudoalteromonas was established by recombining 11 species of the genus Alteromonas and 1 species of the genus Pseudomonas into 12 species of the genus Pseudoalteromonas based on a wealth of phylogenetic analyses of nearly complete 16S rRNA gene sequences (Gauthier et al., 1995). The genus Pseudoalteromonas is classified in the family Alteromonadaceae, and members of this genus are Gramstain negative, aerobic, motile and have C 16 : 0 , C 16 : 1 !7c, C 17 : 1 !8c and C 18 : 1 !7c as major fatty acids and genomic G +C contents of 38-48 mol% (Bowman & McMeekin, 2005). At the time of writing, the genus Pseudoalteromonas comprises 41 species and 2 subspecies with validly published names (List of Prokaryotic Names with Standing in Nomenclature, http://www.bacterio.net/pseudoalteromonas.html; Park et al., 2016;Ying et al., 2016). Most species of the genus Pseudoalteromonas have been isolated from various marine habitats including seawater, sediment, marine invertebrates, algae and sea ice (Bowman & McMeekin, 2005). With cultivation-independent molecular techniques, pseudoalteromonads are also known as a ubiquitous marine group extending from the sea surface microlayer (SSM) to deep-sea sediment, and from equatorial to polar regions (Cui et al., 2008;Cunliffe & Murrell, 2009;Wietz et al., 2010).Here, we isolated a bacterial strain, which is the first representative of the genus Pseudoalteromonas isolated from Antarctic SSM to our knowledge, and performed a polyphasic taxonomic analysis to determine the taxonomic position of the strain.A SSM sample was taken using a customized SSM sampler employing a type of rotating drum (Harvey 1966) covered with polycarbonate from the Terra Nova Bay (75.29 S Abbreviations: ANI, average nucleotide identity; GGDC, genome-togenome distance calculation; SSM, sea surface microlayer.The GenBank/EMBL/DBBJ accession number for the 16S rRNA gene sequence and the draft genome sequence of strain PAMC 28425 T are KU716039 and BDDS01000000, respectively.Two supplementary tables and one supplementary figure are available with the online Supplementary Material.

A Gram-staining-negative, motile, aerobic and rod-shaped bacterial strain, PAMC 27157 T , was isolated from a melt pond on sea ice in the Chukchi Sea. Phylogenetic analysis of the 16S rRNA gene sequence of strain PAMC 27157 T revealed an affiliation to the genus Aureimonas with the closest sequence similarity (96.2 %) to that of Aureimonas phyllosphaerae. Strain PAMC 27157 T grew optimally at 30 8C and pH 7.0 in the presence of 3.5 % (w/v) NaCl. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, sulfoquinovosyldiacylglycerol and an unidentified aminolipid. The major cellular fatty acid was summed feature 8 (C 18 : 1 v7c and/or C 18 : 1 v6c, 83.1 %) and the major respiratory quinone was Q-10. The genomic DNA G+C content was 69

A Gram-stain-negative, non-motile, facultatively anaerobic and rod-shaped bacterial strain, designated PAMC 28131T, was isolated from a sea surface microlayer sample in the open water of the Pacific Ocean. Phylogenetic analysis of the 16S rRNA gene sequence of strain PAMC 28131T revealed an affiliation to the genus Sandaracinobacter with the closest species Sandaracinobacter sibiricus RB16-17T (sequence similarity of 98.2 %). Strain PAMC 28131T was able to grow optimally with 0.5–1.0 % NaCl and at pH 6.5–7.0 and 30 °C. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, an unidentified aminolipid, an unidentified glycolipid and an unidentified lipid. The major cellular fatty acids (>10 %) were C18 : 1  ω6c and/or C18 : 1  ω7c, (42.6 %), C17 : 1  ω6c (19.3 %) and C16 : 1  ω6c and/or C16 : 1  ω7c (15.8 %), and the respiratory quinone was Q-10. The genomic DNA G+C content was 65.3 mol%. The phylogenetic, phenotypic and chemotaxonomic data showed that strain PAMC 28131T could be clearly distinguished from S. sibiricus RB16-17T. Thus, strain PAMC 28131T should be classified as representing a novel species in the genus Sandaracinobacter , for which the name Sandaracinobacter neustonicus sp. nov. is proposed. The type strain is PAMC 28131T (=KCCM 43127T=JCM 30734T).

Gram-stain-negative, aerobic and rod-shaped bacterial strains, designated SSM26T and SSM44, were isolated from a sea surface microlayer sample from the Ross Sea, Antarctica. Analysis of the 16S rRNA gene sequences of strains SSM26T and SSM44 revealed a clear affiliation with the genus Pseudomonas . Based on the results of phylogenetic analysis, strains SSM26T and SSM44 showed the closest phylogenetic relationship with the species Pseudomonas sabulinigri KCTC 22137T with the 16S rRNA gene sequence similarity level of 98.5 %. Strains SSM26T and SSM44 grew optimally at 30 °C, pH 7.0–7.5 and 0.5–10.0 % NaCl (w/v). The major cellular fatty acids were C18 : 1  ω7c (31.3–34.9 %), C16 : 0 (15.5–20.2 %), summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c; 19.5–25.4 %) and C12 : 0 (6.0–9.3 %). The genomic DNA G+C content of each strain was 56.2 mol%. Genomic relatedness analyses based on the average nucleotide identity and the genome-to-genome distance showed that strains SSM26T and SSM44 constituted a single species that was clearly distinguishable from its phylogenetically close relatives. The combined phenotypic, chemotaxonomic, genomic and phylogenetic data also showed that strains SSM26T and SSM44 could be distinguished from validly published members of the genus Pseudomonas . Thus, these strains should be classified as representing a novel species in the genus Pseudomonas , for which the name Pseudomonas neustonica sp. nov. is proposed with the type strain SSM26T (=KCCM 43193T=JCM 31284T=PAMC 28426T) and a sister strain SSM44 (=KCCM 43194=JCM 31285=PAMC 28427).