Localization of pituitary adenylate cyclase-activating polypeptide and its messenger ribonucleic acid in the rat testis by light and electron microscopic immunocytochemistry and in situ hybridization.

Abstract

Pituitary adenylate cyclase-activating-polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide family of peptides; it occurs as two amidated forms with 38 (PACAP38) and 27 (PACAP27) amino acids. Rabbit antisera against synthetic PACAP27 were characterized by enzyme-linked immunosorbent assay. One of the antisera, using a high antibody titer, recognized both PACAP27 and PACAP38 and was found useful for immunohistochemistry. The distribution and ultrastructural localization of PACAP-like immunoreactivity (PACAP-LI) in the rat testes at different stages of spermatogenesis were studied with this antiserum. Four oligonucleotide probes, each complementary to a different region covering a different intron-exon junction, were chosen to maximize hybridization based on the predicted secondary structure of PACAP messenger RNA. PACAP-LI was detected in the developing germ cells but not in either Sertoli or Leydig cells. Intense PACAP-LI was found in spermatids situated near the lumen of the seminiferous tubules. Lower levels of PACAP-LI were detected in spermatogonia and primary spermatocytes, but no PACAP-LI was found in mature spermatids, testicular spermatozoa, or epididymal spermatozoa. In spermatids, PACAP-LI was detected during the cap phase and acrosome phase but not in the maturation phase. At the ultrastructural level, numerous gold particles representing PACAP-LI were found in both acrosomal granules and acrosomal caps of spermatids, while a few particles were found in the Golgi complex. Very few gold particles were seen in the acrosome of mature spermatids and spermatozoa. PACAP-LI decreased and finally disappeared from spermatids during the late developmental stages. In situ hybridization indicated that most of the signal was detected near the perimeter of seminiferous tubules in early developing germ cells, especially in spermatogonia and primary spermatocytes, suggesting that transcription of the PACAP gene occurs in spermatogonia and primary spermatocytes. The processing of the prohormone appears to be slow, and mature PACAP only appears in spermatids. These morphological findings suggest that PACAP-like substances, synthesized by germ cells, participate in spermatogenesis, particularly spermiogenesis, probably by an autocrine and paracrine mechanism. However, the possibility that PACAP acts on the Sertoli and/or Leydig cells cannot be excluded.