The First Identification of Homomorphic XY Sex Chromosomes by Integrating Cytogenetic and Transcriptomic Approaches in Plestiodon elegans (Scincidae).

The sex chromosomes of skinks are usually poorly differentiated and hardly distinguished by cytogenetic methods. Therefore, identifying sex chromosomes in species lacking easily recognizable heteromorphic sex chromosomes is necessary to fully understand sex chromosome diversity. In this paper, we employed cytogenetics, sex quantification of genes, and transcriptomic approaches to characterize the sex chromosomes in Plestiodon elegans. Cytogenetic examination of metaphases revealed a diploid number of 2n = 26, consisting of 12 macrochromosomes and 14 microchromosomes, with no significant heteromorphic chromosome pairs, speculating that the sex chromosomes may be homomorphic or poorly differentiated. The results of the sex quantification of genes showed that Calumenin (calu), COPI coat complex subunit γ 2 (copg2), and Smoothened (smo) were at half the dose in males as in females, suggesting that they are on the X chromosome. Transcriptomic data analysis from the gonads yielded the excess expression male-specific genes (n = 16), in which five PCR molecular markers were developed. Restricting the observed heterozygosity to males suggests the presence of homomorphic sex chromosomes in P. elegans, XX/XY. This is the first breakthrough in the study of the sex chromosomes of Plestiodon.

Oviductal sperm storage in the Chinese pond turtle, Mauremys reevesii, depends on androgen-based promotion of the BCL 2 anti-apoptotic pathway.

CONTEXT: Sperm storage is a complex and highly coordinated process that is regulated by a variety of factors. The BCL 2 protein family plays a key role in regulating apoptosis, and determines sperm survival. AIMS: The objective of this study was to explore the correlation between sperm storage and the BCL 2 protein family in the oviduct of Mauremys reevesii . METHODS: Hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques were used to investigate three parts of the reproductive tract (isthmus, uterus and vagina) of mated and unmated female M. reevesii . KEY RESULTS: Hematoxylin-eosin staining revealed many sperm stored in the oviduct. IHC showed positive immunostaining for the BCL 2 and BAX proteins in epithelial ciliated and glandular cells. RT-qPCR indicated that the mRNA expressions of anti-apoptotic genes (BCL 2 , MCL 1 , BCL- W , BCL-XL ) and the androgen receptor (AR) were significantly higher in mated turtles than unmated turtles. However, the expression of pro-apoptotic genes (BAX , BAD , BID and CASPASE 3 ) showed the opposite relationship. CONCLUSIONS: These results suggest that sperm entering the oviduct can promote the synthesis of anti-apoptotic genes to protect themselves from various degradation factors. IMPLICATIONS: These findings will help researchers understand the mechanisms of sperm storage.

Sperm storage in the oviduct of the Chinese pond turtle Mauremys reevesii depends on oestrogen-based suppression of the TLR2/4 immune pathway.

The long-term storage of spermatozoa in the female reproductive tract is limited by the innate immune system. Oestrogen plays a role in regulating the innate immune system. Thus, exploring the expression of genes in the Toll-like receptor (TLR) 2/4 pathway and oestrogen receptors in the oviduct of Mauremys reevesii could contribute to our understanding of the mechanism of sperm storage. In this study, three parts of the oviduct (isthmus, uterus and vagina) in three mated and unmated female turtles were used to perform immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). Immunohistochemistry revealed that the TLR2/4 protein was mainly distributed in epithelial tissues and glandular cell membranes, and that TLR2/4 levels in the oviduct were significantly decreased in mated compared with unmated turtles. Real-time qPCR indicated that TLR2/4, myeloid differentiation factor 88 (MyD88), interleukin 1 receptor associated kinase 4 (IRAK4), TNF receptor associated factor 6 (TRAF6), interferon regulatory factor 3 (IRF3) and interleukin 6 (IL6) mRNA expression was significantly higher in the oviduct of unmated than mated turtles, whereas the opposite was true for the expression of oestrogen receptor 1 (ESR1) and progesterone receptor (PGR). These results indicate that when spermatozoa are stored in the oviduct, an increase in oestrogen suppresses the immune response induced by the TLR2/4 pathway so that spermatozoa are not removed as a foreign substance, but stored until fertilisation. The findings of this study are relevant to our understanding of the relationship between sperm storage and the innate immune system in the oviduct of reptiles.

The complete mitochondrial genome of the Odorrana schmackeri (Anura, Ranidae).

The complete mitochondrial genome of O. schmackeri has been sequenced and characterized in this study. The mitogenome is a circular molecule of 18 610 bp in length, containing 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 21 transfer RNA (tRNA) genes and a non-coding D-loop region (control region). Its gene arrangements are identical to the typical neobatrachian-type except for the loss of tRNAHis gene. Our data provide a useful resource for the phylogenetic studies of genus Odorrana.

[Molecular cloning, recombinant expression and characterization of lysozyme from Chinese shrimp Fenneropenaeus chinensis].

Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme (FcLyz in short). The full length of the gene was of 709 bp, and the open reading frame (477 bp) encoded 158 amino acids. The predicted protein had a signal peptide (-1--18 residue) and molecular weight of the mature protein (residue 1-140) was of 16.2 kD. A Lyz 1 domain (residue 1-130) in the lysozyme was found by SMART analysis. The results of semiquantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-beta-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria, and MIC reached 3.43 micromol/L, and relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important molecules against pathogens in innate immunity of the shrimp.