Transient expression of the cholecystokinin gene in male germ cells and accumulation of the peptide in the acrosomal granule: possible role of cholecystokinin in fertilization.

Abstract

Expression of the gene encoding the neurotransmitter/neuromodulator cholecystokinin (CCK) was demonstrated in testis ofseveral different species. Two tcular CCK mRNA transcripts of different sizes were detected, and studies on the ontogeny of CCK gene expression indicated that the gene was expressed in male germ ceils. In situ hybridization revealed CCK mRNA-expressing cells in the peripheral parts of the seminiferous tubules. Biochemical identification showed that the majority of prepro-CCK products in the testis represented pro-CCK. Immunofluorescence studies revealed CCK-like peptides primarily in spermatocytes and spermatids of mouse, rat, and monkey. Immuno electron microscopy of monkey testis demonstrated CCK immunoreactivity in the acrosomal granule of spermatids. Hence, an interesting possibility is that CCK peptides can be released during the acrosome reaction and thus may be of importance in the fertilization process.Cholecystokinin (CCK) was first purified by Jorpes and Mutt (1, 2) as a 33-amino acid peptide (CCK-33) from pig gut. Several different forms of CCK have been described in central and peripheral tissues (3-6). The brain contains CCK-8 and several CCK desoctapeptides but no detectable CCK-33. In the gut, however, intact CCK-33, CCK-39, and CCK-58 have been found together with the forms also seen in the brain (3-6). CCK-8 has been detected in many regions of the vertebrate nervous system (7-14) and fulfills several of the criteria of a neurotransmitter/neuromodulator (for review, see ref. 15), although the overall role of CCK in the brain is poorly understood.Molecular cloning of cDNAs for both pig and rat CCK mRNA has established that all identified CCK peptides are derived by proteolytic processing from a 114-or 115 (rat)-amino acid precursor that is identical in brain and gut (16,17). The various molecular forms of CCK in the brain and gut are therefore generated by tissue-specific posttranslational events. In accordance with this an '=0.8-kilobase (kb) CCK mRNA has been demonstrated in rat brain and gut (16,17).In this study the distribution of CCK mRNA and CCK peptides was examined in several tissues, including the male reproductive system. CCK immunoreactivity was detected in spermatogenic cells, and its occurrence in the acrosome of male germ cells indicates a possible role of CCK in the fertilization process.MATERIALS AND METHODS RNA Preparations and Blot Analysis. Total and poly(A)+ RNAs (18) were prepared from the indicated tissues, electrophoresed in 1% agarose gels containing 0.7% formaldehyde, and transferred to nitrocellulose filters (19,20). The filters were hybridized to a 540-base-pair rat CCK cDNA insert (16) labeled with [a-32P]dCTP by nick-translation to a specific activity of =5 x 108 cpm/,ug. Filters containing RNA prepared from rat tissues were washed at high stringency (0.1 x SSC/0.1% SDS, 540C; 1 x SSC is 150 mM NaCI/15 mM sodium citrate, pH 7.0), whereas filters with RNA prepared from other species were washed at reduced stringency (0.2 x SSC/0.1% SDS, 450C)....

Expression and localization of gastrin messenger RNA and peptide in spermatogenic cells.

“…Polyadenylated RNA was prepared from human testis and duodenum, as well as from monkey antrum and testis. In agreement with a previous study (23), high ,Ievels of an 800-nucleotide-long CCK mRNA were found in monkey testis (Fig. 1 a).…”

“…The CCK peptides in spermatogenic cells are accumulated in the acrosome (22)(23)(24). CCK receptors have been detected on frog oocytes (25), and we have suggested that accumulated CCK peptides in the acrosome of spermatids and spermatozoa may be released during the acrosome reaction and participate in the fertilization process.…”

“…The gastrin gene is expressed mainly in the antroduodenal G cells, and to a minor extent in the neonatal pancreas (12), vagal neurons (13), pituitary cells (14), and bronchial mucosal cells (15). In addition to I cells of the small intestine, the CCK gene is expressed in multiple central (16)(17)(18)(19) and peripheral neurons (20), in pituitary corticotrophs (21) and, as recently reported, in spermatogenic cells of several mammalian species (22,23).…”

“…In agreement with this, progastrin was found in the human testis, and bioactive a-amidated gastrin was detected in ejaculated human sperm cells. Thus, human spermatogenic cells appear to express the gene encoding the CCK-related hormone gastrin, rather than CCK expressed in spermatogenic cells of other mammals (22,23).…”

Expression of the cholecystokinin gene in a human (small‐cell) lung carcinoma cell‐line

“…The peptides are synthesized in a cell-specific manner in the intestinal tract, the central nervous system [3-51, the pituitary [6] and in male germ cells [7]. The biogenesis of CCK and its regulation are difficult to investigate in tissues as complex as the brain or endocrine glands.…”