Immunohistochemical analysis of cAMP response element-binding protein in mouse testis during postnatal development and spermatogenesis

Basal activity and cellular localization of cAMP response element-binding protein (CREB) was examined in mouse testis during postnatal development and spermatogenesis. Testes of ICR mice sampled on postnatal day (PND) 3, 7, 14, 21, 28, 35, 42, and 49 were analyzed using Western blotting. Basal CREB activity was significantly higher in early phase (PND 3–7) developing testes than in intermediate- and late-phase developing (PND 14–42) and adult testes (PND 49). Furthermore, immunohistochemical analysis demonstrated the change of CREB phosphorylation in various testicular cell types during postnatal development. In particular, CREB phosphorylation in seminiferous tubules of the adult testis varied according to the spermatogenic cycle, while phosphorylation was evident in spermatogonia during all stages. Phosphorylation was moderate in pachytene spermatocytes of stages I–III and intense in round and elongate spermatids of spermiogenesis in stages XII–IX. These results suggest that CREB plays an important role in cell proliferation and differentiation in the early phase of postnatal development and spermatogenesis of mouse testis.     

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley-Liss, Inc.     

Distribution of dynamins in testis and their possible relation to spermatogenesis

Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynamin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immunoreactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Neonatal exposure to endocrine active compounds or an ERbeta agonist increases adult anxiety and aggression in gonadally intact male rats

Endocrine active compounds (EACs) have been shown to influence a number of reproductive endpoints but less is known about how they might affect other hormone dependent behaviors including anxiety and aggression. Recent evidence suggests that these effects may be mediated through the beta form of the estrogen receptor (ERbeta). Using male Long Evans rats, we sought to determine how neonatal exposure to EACs affects anxiety and aggression in adulthood. Anxiety was assessed using the elevated plus maze and aggression was assessed 8 weeks later using the resident intruder test. To gain insight into which ER subtype (ERalpha vs ERbeta) might be mediating these effects we used agonists specific for ERalpha (1,3,5-tris(4-Hydroxyphenyl)-4-propyl-1H-pyrazole (PPT)) or ERbeta (Diarylpropionitrile (DPN)) as additional treatment groups. For these experiments the synthetic EAC bisphenol-A (BPA) and the phytoestrogen metabolite equol (EQ) were used. Male neonates were injected with either 0.05 ml sesame oil (control), 50 microg estradiol benzoate (EB), 1 mg/kg DPN, 1 mg/kg PPT, 50 microg/kg BPA, or 10 mg/kg EQ daily for 4 days beginning on the day of birth (PND 0). Compared to the oil treated controls, significantly fewer open arm entries were made by the males neonatally treated with DPN, EQ, or BPA. The DPN and EQ treated males were also more aggressive compared to the controls. These findings suggest that neonatal exposure to EACs with agonistic activity on ERbeta may influence affective behavior in adulthood, including anxiety and aggression.