Carla Boitani

The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes.

Bv8, a protein from skin secretions of Bombina variegata, reacts with receptors present in mammalian brain and intestine (Mollay et al. (1999) Eur. J. Pharmacol. 374, 189–196). As deduced from cloned cDNAs, the murine and human Bv8 homologues have identical amino‐terminal sequences and also contain 10 cysteines. From mouse testes, two forms of Bv8 mRNA have been characterized, of which one contains an additional exon which codes for 21 mostly basic amino acids. The mouse Bv8 gene is most active in mid‐late pachytene spermatocytes. In mouse testes, Bv8 mRNA can first be detected at the end of the second week post partum.

Identification of Spermatogonial Stem Cell Subsets by Morphological Analysis and Prospective Isolation

Spermatogenesis is maintained by a pool of spermatogonial stem cells (SSCs). Analyses of the molecular profile of SSCs have revealed the existence of subsets, indicating that the stem cell population is more heterogeneous than previously believed. However, SSC subsets are poorly characterized. In rodents, the first steps in spermatogenesis have been extensively investigated, both under physiological conditions and during the regenerative phase that follows germ cell damage. In the widely accepted model, the SSCs are type Asingle (As) spermatogonia. Here, we tested the hypothesis that As spermatogonia are phenotypically heterogeneous by analyzing glial cell line‐derived neurotrophic factor (GDNF) family receptor α1 (GFRA1) expression in whole‐mounted seminiferous tubules, via cytofluorimetric analysis and in vivo colonogenic assays. GFRA1 is a coreceptor for GDNF, a Sertoli cell‐derived factor essential for SSC self‐renewal and proliferation. Morphometric analysis demonstrated that 10% of As spermatogonia did not express GFRA1 but were colonogenic, as shown by germ cell transplantation assay. In contrast, cells selected for GFRA1 expression were not colonogenic in vivo. In human testes, GFRA1 was also heterogeneously expressed in Adark and in Apale spermatogonia, the earliest spermatogonia. In vivo 5‐bromo‐2′‐deoxyuridine administration showed that both GFRA1+ and GFRA1− As spermatogonia were engaged in the cell cycle, a finding supported by the lack of long‐term label‐retaining As spermatogonia. GFRA1 expression was asymmetric in 5% of paired cells, suggesting that As subsets may be generated by asymmetric cell division. Our data support the hypothesis of the existence of SSC subsets and reveal a previously unrecognized heterogeneity in the expression profile of As spermatogonia in vivo. STEM CELLS 2009;27:3043–3052

Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self‐maintain and to produce a transit‐amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil‐sensitive Hoechst dye‐effluxing properties, which define the characteristic “side population” (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a “side‐population” (T‐SP), which is Sca‐1pos, Ep‐CAMpos, EE2 pos, α6‐integrin pos, and αv‐integrin neg. A 13‐fold enrichment in SSC activity was observed when sorted T‐SP cells from ROSA 26 mice were transplanted in busulfan‐treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T‐SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.

Interleukin-1 is a potent growth factor for immature rat Sertoli cells

Testes from rats of different maturational ages were explored for presence of paracrine sertoli cell growth factors. Pubertal and adult testes contained a 17 kDa protein, with potent stimulatory effect on immature Sertoli cell multiplication in vitro. The bioactivity of this protein was mimicked by rat interleukin-1 (IL-1) and neutralized by IL-1 receptor antagonist. A receptor-mediated action was further supported by the demonstration of IL-1 receptor type I mRNA and protein expression in the cultured sertoli cells and in intact immature rat testes. IL-1alpha showed higher efficacy in stimulating proliferation than IL-1beta and follicle-stimulating hormone (FSH), and displayed synergistic action in combination with FSH. As IL-1alpha is constitutively produced by the rat testis and IL-1beta readily inducible by proinflammatory stimuli, our results suggest that IL-1 may serve as a growth factor for Sertoli cells under physiological and pathophysiological conditions.

A rapid method of Sertoli cell isolation by DSA lectin, allowing mitotic analyses

We have developed a rapid and convenient method of Sertoli cell preparation for studying the growth kinetics of these cells in in vitro culture. Datura Stramonium agglutinin (DSA)-coated dishes were used to rapidly purify single Sertoli cells from immature rat testis. We have monitored by immunohistochemical markers the degree of contamination of our Sertoli cell preparation by other cell types. The cell preparation is essentially free of germ cells and interstitial cells and contains a minimal percentage of myoid cells. Sertoli cells isolated with this method retain functional activities such as the FSH responsiveness in terms of cAMP production. In addition, we have studied the proliferative activity of Sertoli cells isolated by lectin binding from rats of different ages. Sertoli cells exhibited a characteristic pattern of proliferation which was a function of the donor animal age. The proliferative activity of isolated Sertoli cells decreased with age, being much higher in 3 day-old rats than in older animals. A similar pattern was observed when the mitotic activity of Sertoli cells in response to mitogens present in the testicular extracts from 5 day-old rats was evaluated. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro.

Polyunsaturated fatty acids of germ cell membranes, glutathione and blutathione-dependent enzyme-PHGPx: From basic to clinic

The lipid metabolism in sperm cells is important both for energy production and for cell structure. A special composition of membrane phospholipids, rich in polyunsaturated fatty acids (PUFA), and the different composition of sperm and immature germ cell membrane are described and discussed. Testis germ cells as well as epididymal maturing spermatozoa are endowed with enzymatic and non-enzymatic scavenger systems to prevent lipoperoxidative damage. Catalase, superoxide dismutase, and glutathione-dependent oxidoreductases are present in variable amounts in the different developmental stages. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and roles in caput and cauda epididymal sperm cells are discussed. Also seminal plasma has a highly specialized scavenger system that defends the sperm membrane against lipoperoxidation and the degree of PUFA insaturation acts to achieve the same goal. Systemic predisposition and a number of pathologies can lead to an anti-oxidant/pro-oxidant disequilibrium. Scavengers, such as glutathione can be used to treat these cases as they can restore the physiological constitution of PUFA in the cell membrane.

Selenium, a Key Element in Spermatogenesis and Male Fertility

Selenium is essential for normal spermatogenesis of mammals and its critical role is mainly mediated by two selenoproteins, namely phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) and Selenoprotein P. PHGPx/GPx4 is the major selenoprotein expressed by germ cells in the testis, having multiple functions and representing the pivotal link between selenium, sperm quality and male fertility. Selenoprotein P is a plasma protein that is required for selenium supply to the testis. In the last years, nutritional studies and experimental animal models lacking/overexpressing a specific PHGPx isoform and selenoprotein P have highly expanded our understanding on how the male reproductive system depends on selenium. The focus of this review, is to report and discuss the most relevant and recent findings in this field. Clinical data have pointed to a correlation between abnormal PHGPx content in sperm and disturbance of human male fertility. However, additional evidence is still required to draw any definitive conclusions about therapeutical strategies for improving fertility by selenium administration.

Distribution of GFRA1-expressing spermatogonia in adult mouse testis

In mice and other mammals, spermatogenesis is maintained by spermatogonial stem cells (SSCs), a cell population belonging to undifferentiated type A spermatogonia. In the accepted model of SSC self-renewal, Asingle (As) spermatogonia are the stem cells, whereas paired (Apaired (Apr)) and chained (Aaligned (Aal)) undifferentiated spermatogonia are committed to differentiation. This model has been recently challenged by evidence that As and chained (Apr and Aal), undifferentiated spermatogonia are heterogeneous in terms of gene expression and function. The expression profile of several markers, such as GFRA1 (the GDNF co-receptor), is heterogeneous among As, Apr and Aal spermatogonia. In this study, we have analysed and quantified the distribution of GFRA1-expressing cells within the different stages of the seminiferous epithelial cycle. We show that in all stages, GFRA1+ chained spermatogonia (Apr to Aal) are more numerous than GFRA1+ As spermatogonia. Numbers of chained GFRA1+ spermatogonia are sharply reduced in stages VII-VIII when Aal differentiate into A1 spermatogonia. GFRA1 expression is regulated by GDNF and in cultures of isolated seminiferous tubules, we found that GDNF expression and secretion by Sertoli cells is stage-dependent, being maximal in stages II-VI and decreasing thereafter. Using qRT-PCR analysis, we found that GDNF regulates the expression of genes such as Tex14, Sohlh1 and Kit (c-Kit) known to be involved in spermatogonial differentiation. Expression of Kit was upregulated by GDNF in a stage-specific manner. Our data indicate that GDNF, besides its crucial role in the self-renewal of stem cells also functions in the differentiation of chained undifferentiated spermatogonia.

Essential role of citron kinase in cytokinesis of spermatogenic precursors

During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution, suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. We previously reported that Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In this report we show that, in addition, these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis revealed that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. Our data indicate that Citron kinase is specifically required for cytokinesis of the male germ line.

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.