A Male Reproduction-Related Kazal-Type Peptidase Inhibitor Gene in the Prawn, Macrobrachium rosenbergii: Molecular Characterization and Expression Patterns

Abstract

Peptidase inhibitors in the male reproductive tract are well known in mammals, in which they play roles in protecting the tract epithelium against proteolytic damage or in regulating the fertilization process. By screening the subtracted cDNA clones enriched for male reproductive tract-specific transcripts, one clone encoding a putative protein that showed significant similarity to Kazal-type peptidase inhibitor (KPI) was obtained. This is the first report of an invertebrate in which a male reproductive tract-specific KPI gene has been identified and characterized. The gene contains a 405-bp open reading frame (ORF), a 72 bp 5' untranslated region (UTR), and a 259 bp 3' UTR. The conceptually translated protein consisted of a 21-amino-acid signal peptide and a 113-amino-acid mature polypeptide with two Kazal-type domains (named after the discoverer). Significant levels of the mRNA were observed only in the male reproductive tract, while mRNA expression was not detected in any other tissues tested. The transcription of the gene remained constant during maturation, although not in the postlarval stage. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.

Temporal Silencing of an Androgenic Gland-Specific Insulin-Like Gene Affecting Phenotypical Gender Differences and Spermatogenesis

“…M. rosenbergii sperm duct specifically expressed gene Mar-SRR, accession no. DQ066890 (41), was used as the internal control using specific forward (5Ј-TCTCTGAAGCTGCAAGT-GAT TTAC-3Ј) and reverse (5Ј-AATCT GGGTCATTCTCCTGAT TGG-3Ј) primers. RT-PCR products were electrophoresed in 1.2% agarose, and visualized by exposure to UV light and ethidium bromide.…”

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: Comparative analysis of structure and activities

“…A primary focus in our laboratory has been investigating the mechanisms of reproduction in crustaceans. We previously reported that the gene encoding MRPINK from the prawn M. rosenbergii was involved in the fertilization process through its inhibitory activity against the prawn sperm [16,30]. In the present study, we successfully isolated two KPI genes from the male reproductive tract of the oriental river prawn M. nipponense and Chinese mitten crab E. sinensis.…”

“…In our previous study, the KPI MRPINK from the prawn M. rosenbergii [30] was identified and characterized as having an inhibitory effect on both the gelatinolytic activity and proteolytic activity of prawn sperm. In addition, MRPINK specifically blocked the activity of M. rosenbergii sperm gelatinase (MSG) [16].…”

“…These KPIs are all expressed outside the male reproductive system and are believed to be involved in the innate immune response [25]. However, although numerous malespecific KPIs have been identified in vertebrates, including human [26], mouse [27], monkey [28], and turkey [29], only one crustacean male reproduction-related Kazal-type peptidase inhibitor (MRPINK) has been isolated from the prawn Macrobrachium rosenbergii [30].…”

MSG is involved in sperm gelatinolytic activity in the prawn, Macrobrachium rosenbergii

“…In our previous report, a male reproduction-related Kazal-type peptidase inhibitor (MRPINK) was isolated from the giant freshwater prawn, Macrobrachium rosenbergii [16], and was shown to have an inhibitory effect on both the gelatinolytic and proteolytic activity of prawn sperm. Meantime, it was found that the activity of a novel sperm gelatinase (MSG) was blocked by MRPINK specifically [11].…”