Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas.

Activin is a pleiotropic growth factor belonging to the transforming growth factor-beta (TGFB) superfamily of signaling molecules. Regulated activin signaling is known to influence several steps in rodent male gamete differentiation. TGFB ligand isoforms, TGFB1-B3, also influence germ cell survival in the rodent testis at the onset of spermatogenesis and around the time of puberty. Given the importance of regulated activin and TGFB signaling in testis development and function, we sought to investigate the cellular production sites of activin/TGFB-signaling modulators in normal and dysfunctional adult human testes samples. Signaling transducers phosphorylated SMAD2/3, and signaling modulators SMAD6, MAN-1, inhibin alpha (INHA), and beta-glycan were detected in Bouins fixed, paraffin-embedded adult human testis sections using immunohistochemistry. Additional samples examined were from testicular cancer patients and from normal men subjected to gonadotropin suppression with androgen-based contraceptives. Our findings identify distinct differences between normal and gonadotropin-deprived human testis in the expression and cellular localization of activin/TGFB-signaling modulators. The presence of a nuclear phosphorylated SMAD2/3 signal in all analyzed seminoma specimens indicated active activin/TGFB signaling. Moreover, a subset of seminoma specimens exhibited selective enhanced expression of beta-glycan (4 out of 28 seminoma tumors), INHA (6 out of 28), and MAN-1 (6 out of 28), highlighting potential functional differences between individual tumors in their capacity to regulate activin/TGFB signaling. Within the heterogenous nonseminomas, expression of signaling modulators was variable and reflected the degree of somatic differentiation. Thus, synthesis of activin and TGFB-signaling modulators may be affected by spermatogenic disruption and altered hormone levels in the testis.

Nuclear transport of parathyroid hormone (PTH)-related protein is dependent on microtubules.

PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin beta1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin beta1, but not importin alpha, enhanced the microtubular association of PTHrP with microtubules. Significantly, the dependence of PTHrP nuclear import on microtubules was shown by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. These results indicate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity and are consistent with a direct role for the cytoskeleton in protein transport to the nucleus.

Expression of Bambi is widespread in juvenile and adult rat tissues and is regulated in male germ cells.

Members of the TGF beta superfamily may compete for receptor occupancy and intracellular signaling molecules in specific developmental circumstances. We explored the potential importance of the TGF beta family inhibitor, Bambi (Bmp and activin membrane-bound inhibitor) by examining its pattern of mRNA expression in juvenile and adult rat tissues, with a focus on reproductive organs. The 1.8-kb transcript was ubiquitous, whereas a 3-kb transcript was unique to enriched spermatocyte and spermatid cell fractions and adult testis. The full-length rat cDNA is 89% (nucleic acid) and 95% (amino acid) identical to its human homolog, hnma. Using in situ hybridization, Bambi mRNA was detected in granulosa and thecal cells of adult ovaries and in spermatogonia, spermatocytes, round spermatids, and Sertoli cells of adult testes. In addition to a persistent signal in Sertoli cells in juvenile testes, this mRNA within germ cells appeared dramatically increased as gonocytes matured into spermatogonia immediately after birth. These data indicate that TGF beta superfamily signaling within male germ cells is down-regulated at the onset of spermatogenesis. The addition of exogenous activin A to 24-h cultures of newborn rat testis fragments decreased the Bambi mRNA level. Regulated Bambi mRNA synthesis may contribute to TGF beta superfamily signaling modulation in several organs, as suggested by its discrete expression switch in male germ cells.

Regulated production of SnoN2 is a feature of testicular differentiation.

Transforming growth factor betas (TGF beta s) and activins are key regulators of male fertility, affecting somatic and germ cell proliferation and differentiation in the developing and adult testis. Several studies have shown that these ligands influence discrete developmental stages, suggesting that temporal expression of modifying factors may determine their specific signaling outcomes. Upon binding to cell surface receptors, TGFbeta and activin signals are transduced intracellularly by the phosphorylation and nuclear accumulation of SMAD2 and SMAD3 transcription factors. The objective of this study was to determine the cellular localization of phosphorylated SMAD2/3 and the transcriptional repressor SnoN (Ski-like), a modifier of SMAD2/3 transcriptional activity, in mouse testes. Western blot established that only the smaller SnoN isoform, SnoN2, is produced in the testis. By immunohistochemistry, widespread phospho-SMAD2/3 distribution was observed in somatic and germ cells at all ages. In contrast, SnoN2 production was highly regulated, being detected only in gonocytes and interstitial cells at birth and in pachytene spermatocytes at puberty. In the adult, SnoN2 expression differed to that during the first wave, being ubiquitously expressed but exhibiting regulated nuclear localization. In another model of spermatogenic differentiation, the irradiated rat testis, widespread phospho-SMAD2/3 contrasted with restricted SnoN2 expression. SnoN2 was limited to interstitial cells, with reduced staining intensity observed associated with the timing of spermatogenesis resumption. We conclude that somatic and germ cells at all differentiation stages are actively transducing TGFbeta superfamily signals but that responses to these ligands may be selectively modulated by controlled production and nuclear localization of SnoN2.

SMAD expression in the testis: an insight into BMP regulation of spermatogenesis.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, extensively influence events that establish male fertility, affecting germ cells and somatic cells throughout fetal and postnatal life. BMP signals are relayed by SMAD proteins, transcription factors that translocate to the nucleus upon ligand stimulation. We show that BMP signaling in the testis may be regulated by selective expression of BMP-responsive and inhibitory SMADs, with expression differing between the first wave and adult spermatogenesis. Smad1, Smad5, Smad8, Smad4, Smad6, and Smad7 expression is ubiquitous during testis development but becomes cell-specific in the adult. Furthermore, regulated SMAD6 protein expression at the onset of spermatogenesis suggests differential responsiveness of spermatogonial subpopulations to ligands. In vitro, immature Sertoli cells and spermatogonia transduce BMP2 and BMP4 signals by means of SMAD1, SMAD5, and SMAD8. Based on these findings, we extrapolate these data to interpret BMP mutant testis phenotypes in terms of SMAD availability for signal transduction.

All in the family: TGF-beta family action in testis development.

To achieve and maintain fertility, the adult mammalian testis produces many generations of sperm. While testicular integrity is established in the fetus and develops further in juvenile life, sperm production does not ensue until much later in life, following the onset of puberty. Signals from the transforming growth factor-beta superfamily of proteins are vital for governance of testis development and spermatogenesis, and this review discusses our current understanding of the mechanisms and processes in which they have been implicated with a focus on the fetal and juvenile testis.

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis.

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.

TGFbeta superfamily members in spermatogenesis: setting the stage for fertility in mouse and Drosophila.

Male germ cell development involves a tightly controlled sequence of differentiation switches, from the time that this lineage is specified in the embryo to the moment of sperm release for transport from the testis. Recent research findings and technological advances have allowed key mediators of developmental switches to be identified, and several members of the TGFbeta superfamily of ligands have been implicated at distinct points of male gamete formation and spermatogenesis. This paper reviews the multiple points of control mediated by TGFbeta superfamily signalling molecules during differentiation of the mammalian male germ cell. Comparisons have been made with the Drosophila testis for which genetic analysis has yielded new information concerning the roles of TGFbeta signalling in early germ cell differentiation.