Expression of proenkephalin messenger RNA by mouse spermatogenic cells.

Abstract

The presence of proenkephalin mRNA in germ cells purified from adult mouse testis was examined using RNA gel-and dot-blot analyses. Both pachytene spermatocytes and round spermatids were shown to contain concentrations of this transcript that are 2-to 3-fold greater than that found for whole mouse testis, on a per ug ofpolyadenylylated RNA basis. spermatocytes, round spermatids, and residual bodies were isolated on linear gradients of bovine serum albumin using a modification (14) of the method of Romrell et al. (15). These three fractions represent the most highly purified germ-cell components recovered from dissociated seminiferous epithelium with this procedure as assessed by Nomarski and phase microscopy (15). Mature sperm were isolated from the vas ductus of the mouse and rat as described by Wolfe et al. (16). In three separate experiments, the purity of pachytene spermatocyte preparations ranged from 88 to 91%, with binucleated spermatids being the major contaminants. The round spermatid fractions were between 91 and 94% pure, with large condensing spermatids and residual bodies as the main contaminants. In both fractions, contamination by somatic cells (Leydig and Sertoli cells) was generally less than 1%. Residual bodies were 88-91% pure, with condensing spermatids being the major contamination.Analysis of Proenkephalin mRNA Content. Total RNA was prepared from isolated germ cells, whole testis, and liver from adult mice by the guanidinium thiocyanate/cesium chloride gradient procedure (17). Polyadenylylated RNA was prepared by oligo(dT) chromatography (18). RNA samples were separated on 1.5% agarose/methyl mercury hydroxide gels (19) and then transferred to nylon membranes (GeneScreenPlus, New England Nuclear) (20). Membranes were prehybridized for 4-16 hr at 420C, hybridized with nicktranslated rat proenkephalin cDNA, pRPE-1 (165-600) (21) (2-4 x 108 cpm/,ug) and then washed as described (12,21).Autoradiography was performed at -80'C for 16-24 hr. RNA blots were as described (12

Regulation of Male Fertility by the Opioid System

“…With regard to EOPs, the genes encoding the opioid precursors PENK, POMC and PDYN were found to be expressed in the rat and mouse testes (38,39,46), supporting the idea that testicular function could be regulated through local synthesis of EOPs. The finding that transgenic mice who overexpress PENK in the testes have impaired fertility, morphologically abnormal testicles and low sperm motility has provided further evidence suggesting that the opioid system participates in an important way in the regulation of testicular function (47).…”

“…In addition, binding studies have revealed the presence of all three types of opioid receptors (DOR, MOR and KOR) in rat testes. Moreover, the genes encoding the opioid precursors PENK, PDYN and POMC have also been found to be expressed in the testes of the rat and mouse (38,39), providing further evidence that the opioid system may play a role in regulating testicular function.…”

A new species of enkephalin precursor mRNA with a distinct 5′‐untranslated region in haploid germ cells

“…Recently, mRNA encoding Metenkephalin precursor, preproenkephalin (ppEnk), has been found not only in the nervous systems but also in various non-neuronal tissues such as lymphocytes [1], heart [2], and reproductive systems [3]. In the testis, spermatogenic germ cells [4,5] and Sertoli cells [6] contain ppEnk mRNA. The size of ppEnk mRNA in the germ cells is 1.7-1.9 kb, which is larger than those found in other tissues (1.45-1.5 kb) [4,5].…”

“…In the testis, spermatogenic germ cells [4,5] and Sertoli cells [6] contain ppEnk mRNA. The size of ppEnk mRNA in the germ cells is 1.7-1.9 kb, which is larger than those found in other tissues (1.45-1.5 kb) [4,5]. This specific type of ppEnk mRNA can be translatable into a precursor because Met-enkephalin immunoreactive peptides are generated by limited proteolysis of the The nucleotide sequence presented here has been submitted to the EMBL/GenBank database under the accession no.Y07503 extracts of haploid germ cells [5].…”

Localization of Kex2‐like processing endoproteases, furin and PC4, within mouse testis by in situ hybridization

“…Although many peptides which are synthesized from precursors through cleavage at basic sites have been reported to be present in the testis, most of them are produced in the somatic cells, such as Leydig and Sertoli cells [18]. One of the candidate substrates is proenkephalin, since its mRNA is detected in fractionated spe~atogenic cell populations [21,22], and since one of its processed products, Met-enkephalin-Arg-Phe, is detectable in the sperm acrosome [22]. However, the distribution of proenkephalin transcript in individual cells within the intact testis Antisense Sense has not been examined.…”