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Using an anti-Fos family member antiserum, we previously described, in the testis of Rana esculenta, the presence of a nuclear 43-kDa protein that we hypothesized to be Fra-1. Using an antiserum against Fra-1, we here report on Fra-1 expression, localization, and putative activity in the R. esculenta testis during the annual reproductive cycle. Western blot analysis confirms that the nuclear 43-kDa protein is Fra-1. Immunocytochemistry demonstrates Fra-1 in peritubular myoid cells (PMC), efferent ducts, and blood vessels. We present, for the first time for a vertebrate, experimental evidence that the expression of Fra-1 in PMC is related to its activity during sperm transport from the tubular compartment to the efferent ducts.

MSJ-1 is member of the DnaJ/heat shock protein (Hsp) 40 chaperone protein family. It is present in mouse testis and spinal cord. In particular, MSJ-1 is localized in post-meiotic cells and in motoneurones of the ventral horns. To assess whether the role of this protein is evolutionarily conserved, we have investigated if msj-1 gene is expressed in the frog, Rana esculenta. Using reverse transcription-polymerase chain reaction (RT-PCR), a msj-1-like transcript was detected in testis, brain, and spinal cord. Homology ranging from 42.3 to 46.0% was found as compared with the mammalian counterparts. Muscle did not show any signal. By Western blot analysis, a signal of the predicted size of 30 kDa was evidenced in testis, brain, and spinal cord but not in ovary, heart, liver, kidney, and muscle. MSJ-1 fluctuations in the testis reveal that it appeared in concomitance with post-meiotic events during the annual sexual cycle, as shown in a previous study. The protein is localized in spermatids and is still retained in mature spermatozoa, where it has perinuclear and centriolar localization. MSJ-1 levels did not change in brain and spinal cord. Furthermore, in the brain MSJ-1 was mainly present in diencephalon and mesencephalon, while in spinal cord MSJ-1 was localized into several motoneurones of the cervical and thoracic tract. A putative role in vesicle trafficking is briefly discussed. Mol. Reprod. Dev. 68: 149–158, 2004. © 2004 Wiley-Liss, Inc.

The presence of c-jun like mRNA was assessed in the brain of the frog, Rana esculenta, during the annual sexual cycle. In parallel, Jun protein and GnRH molecular form (mammalian and chicken II also indicated as GnRH1 and GnRH2, respectively) activity was studied in order to establish possible relationships. Northern blot analysis of total RNA reveals the presence of a 2.7 kb c-jun-like mRNA. Western blots, carried out on cytoplasmic and nuclear protein extracts, show the presence of Jun immunoreactive band of 39 kDa in brain and pituitary. Fluctuations of c-jun-like mRNA and Jun immunoreactive protein (cytoplasmic and nuclear) levels in brains during the year indicate relationships among transcription, translation, and nuclear activity. In particular, mRNA levels increase gradually from September until November when Jun protein concentration peaks in cytosolic extracts. Conversely, the nuclear protein reaches highest concentration in July when the cytosolic level shows low values. Immunocytochemical studies confirm the presence of Jun immunoreactivity in both cytoplasmic and nuclear compartments of several brain areas, including those primarily involved in gonadotropin discharge (e.g., anterior preoptic area and preoptic nucleus). GnRH molecular forms and Jun are colocalized in anterior preoptic area and preoptic nucleus. Moreover, during the period characterized by GnRH release, Jun levels strongly decrease in nuclei. Finally, we show that treatments with a GnRH analog (buserelin, Hoechst, Frankfurt) increase Jun levels in brain nuclear extracts.

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.