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Spermiation is a multiple-step process involving profound cellular changes in both spermatids and Sertoli cells. We have observed spermiation defects, including abnormalities in spermatid orientation, translocation and release, in mice deficient in the retinoic acid receptor alpha (RARA) and upon treatment with a pan-RAR antagonist. To elucidate the role of retinoid signaling in regulating spermiation, we first characterized the time course of appearance of spermiogenic defects in response to treatment with the pan-RAR antagonist. The results revealed that defects in spermiation are indeed among the earliest abnormalities in spermatogenesis observed upon inhibition of retinoid signaling. Using fluorescent dye-conjugated phalloidin to label the ectoplasmic specialization (ES), we showed for the first time that these defects involved improper formation of filamentous actin (F-actin) bundles in step 8–9 spermatids and a failure of the actin-surrounded spermatids to move apically to the lumen and to disassemble the ES. The aberrant F-actin organization is associated with diminished nectin-3 expression in both RARA-deficient and pan-RAR antagonist-treated testes. An abnormal localization of both tyrosinated and detyrosinated tubulins was also observed during spermatid translocation in the seminiferous epithelium in drug-treated testes. These results highlight a crucial role of RAR receptor-mediated retinoid signaling in regulating microtubules and actin dynamics in the cytoskeleton rearrangements, required for proper spermiation. This is critical to understand in light of ongoing efforts to inhibit retinoid signaling as a novel approach for male contraception and may reveal spermiation components that could also be considered as new targets for male contraception., A crucial role of RAR receptor-mediated retinoid signaling in regulating microtubule and actin dynamics in cytoskeleton rearrangements during spermiogenesis is demonstrated.

Retinoic acid receptor alpha (RARA), a nuclear receptor protein, has been validated as a target for male contraception by gene knockout studies and also pharmacologically using a pan-retinoic acid receptor antagonist. Retinoic acid receptor alpha activity is indispensable for the spermatogenic process, and therefore its antagonists have potential as male contraceptive agents. This review discusses the effects of systematic dosing regimen modifications of the orally bioavailable and reversible pan-antagonist BMS-189453 as well as studies with the alpha-selective antagonists BMS-189532 and BMS-189614 in a murine model. We also provide an overview of structure–activity studies of retinoic acid receptor alpha antagonists that provide insight for the design of novel alpha-selective ligands., Fertility studies with retinoic acid receptor antagonists in male mice and the current state of RARA-selective antagonist development are reviewed.

We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication.

Idiopathic generalized epilepsy represents about 30–35% of all epilepsies in humans. The bromodomain BRD2 gene has been repeatedly associated with the subsyndrome of juvenile myoclonic epilepsy. Our previous work determined that mice haploinsufficient in Brd2 (Brd2+/−) have increased susceptibility to provoked seizures, develop spontaneous seizures and have significantly decreased GABA markers in the direct basal ganglia pathway as well as in the neocortex and superior colliculus. Here we tested male and female Brd2+/− and wild type littermate mice in a battery of behavioral tests (open field, tube dominance test, elevated plus maze, Morris water maze and Barnes maze) to identify whether Brd2 haploinsufficiency is associated with the human behavioral patterns, so-called juvenile myoclonic epilepsy personality. Brd2+/− females but not males consistently displayed decreased anxiety. Further, we found a highly significant dominance trait (aggression) in the Brd2+/− mice compared to the wild type, more pronounced in females. Brd2+/− mice of either sex did not differ from wild type mice in spatial learning and memory tests. Compared to wild type littermates, we found decreased numbers GABA neurons in the basolateral amygdala, which is consistent with the increase in aggressive behavior. Our results indicate that Brd2+/− haploinsufficient mice show no cognitive impairment but have behavioral traits similar to those found in patients with juvenile myoclonic epilepsy (recklessness, aggression). This suggests that either the BRD2 gene is directly responsible for influencing many traits of juvenile myoclonic epilepsy or it controls upstream regulators of individual phenotypes.

This article provides evidence that basic science research and education should be key priorities for global health training, capacity building, and practice. Currently, there are tremendous gaps between strong science education and research in developed countries (the North) as compared to developing countries (the South). In addition, science research and education appear as low priorities in many developing countries. The need to stress basic science research beyond the typical investment of infectious disease basic service and research laboratories in developing areas is significant in terms of the benefits, not only to education, but also for economic strengthening and development of human resources. There are some indications that appreciation of basic science research education and training is increasing, but this still needs to be applied more rigorously and strengthened systematically in developing countries.

Brdt is a testis-specific member of the distinctive BET sub-family of bromodomain motif-containing proteins, a motif that binds acetylated lysines and is implicated in chromatin remodeling. Its expression is restricted to the germ line, specifically to pachytene and diplotene spermatocytes and early spermatids. Targeted mutagenesis was used to generate mice carrying a mutant allele of Brdt, Brdt(Delta)(BD1), which lacks only the first of the two bromodomains that uniquely characterize BET proteins. Homozygous Brdt(Delta)(BD1/)(Delta)(BD1) mice were viable but males were sterile, producing fewer and morphologically abnormal sperm. Aberrant morphogenesis was first detected in step 9 elongating spermatids, and those elongated spermatids that were formed lacked the distinctive foci of heterochromatin at the peri-nuclear envelope. Quantitative reverse transcription (RT)-PCR showed threefold increased levels of histone H1t (Hist1h1t) in Brdt(Delta)(BD1/)(Delta)(BD1) testes and chromatin immunoprecipitation revealed that Brdt protein, but not Brdt(DeltaBD1) protein, was associated with the promoter of H1t. Intracytoplasmic sperm injection suggested that the DNA in the Brdt(Delta)(BD1) mutant sperm could support early embryonic development and yield functional embryonic stem cells. This is the first demonstration that deletion of just one of the two bromodomains in members of the BET sub-family of bromodomain-containing proteins has profound effects on in vivo differentiation.

Mammalian myocardial infarction is typically followed by scar formation with eventual ventricular dilation and heart failure. Here we present a novel model system in which mice constitutively expressing cyclin A2 in the myocardium elicit a regenerative response after infarction and exhibit significantly limited ventricular dilation with sustained and remarkably enhanced cardiac function. New cardiomyocyte formation was noted in the infarcted zones as well as cell cycle reentry of periinfarct myocardium with an increase in DNA synthesis and mitotic indices. The enhanced cardiac function was serially assessed over time by MRI. Furthermore, the constitutive expression of cyclin A2 appears to augment endogenous regenerative mechanisms via induction of side population cells with enhanced proliferative capacity. The ability of cultured transgenic cardiomyocytes to undergo cytokinesis provides mechanistic support for the regenerative capacity of cyclin A2.

The severe degeneration of the germinal epithelium and subsequent male sterility observed in mice null for the retinoic acid receptor alpha (RARalpha) gene suggested its critical role in spermatogenesis, although the etiology and progression of these abnormalities remain to be determined. Previous studies have revealed that elongated spermatids in RARalpha(-/-) testes were improperly aligned at the tubular lumen and did not undergo spermiation at stage VIII(*). We now report a distinctive failure of step 8-9 spermatids to orient properly with regard to the basal aspect of Sertoli cells, resulting in stage VIII(*)-IX(*) tubules with randomly oriented spermatids. By in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL), we noted that elongating spermatids frequently underwent apoptosis. Immunohistochemical analysis revealed that while activated caspase-3, the primary effector caspase in the apoptotic cell death machinery, was detected in the nuclei of primary spermatocytes in the first wave of spermatogenesis and occasionally in spermatogonia of both normal and mutant testes, it was not involved in the death of elongating spermatids in RARalpha(-/-) testes. Thus, sterility in RARalpha(-/-) males was associated with specific defects in spermiogenesis, which may correlate with a failure in both spermatid release and spermatid orientation to the basal aspect of Sertoli cells at stage VIII(*) in young adult RARalpha(-/-) testis. Further, the resulting apoptosis in elongating spermatids appears to involve pathways other than that mediated by activated caspase-3.

The bromodomain, an evolutionarily conserved motif that binds acetyl-lysine on histones, is found in many chromatin-associated proteins, transcription factors, and in nearly all known histone acetyltransferases. The BET subclass of bromodomain-containing proteins contains two bromodomains and one ET domain and consists of at least four members in mouse and human, Brd2, Brd3, Brd4, and Brdt. We isolated mouse cDNAs for these genes and studied their expression patterns with particular focus on the testis. Northern hybridization revealed that Brd3 is most abundant in testis, ovary, placenta, uterus, and brain; that Brd4 is rather ubiquitously expressed but is most abundant in mid-gestation embryo, testis, ovary, and brain; and that Brdt is specifically expressed in testis. In situ hybridization and immunostaining on histological sections of mouse testes revealed a strikingly specific and dynamic change of cellular specificity in the germ line during the progression of spermatogenesis. Brd4 is expressed in spermatogonia, Brdt is only expressed in mid- to late-spermatocytes, Brd2 is expressed in diplotene spermatocytes and round spermatids and at low levels in spermatogonia, and Brd3 is expressed in round spermatids. This unique expression pattern suggests that genes in this subclass are not simply redundant. Rather, their expression is tightly regulated in the male germ cell lineage, suggesting that they likely have specific roles in different developmental stages and/or cell types.

Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes. Therefore, the concept of stimulating myocyte mitotic divisions has dramatic implications for cardiomyocyte regeneration and hence, cardiovascular disease. Previous reports describing manipulation of cell cycle proteins have not shown induction of cardiomyocyte mitosis after birth. We now report that cyclin A2, normally silenced in the postnatal heart, induces cardiac enlargement because of cardiomyocyte hyperplasia when constitutively expressed from embryonic day 8 into adulthood. Cardiomyocyte hyperplasia during adulthood was coupled with an increase in cardiomyocyte mitosis, noted in transgenic hearts at all time points examined, particularly during postnatal development. Several stages of mitosis were observed within cardiomyocytes and correlated with the nuclear localization of cyclin A2. Magnetic resonance analysis confirmed cardiac enlargement. These results reveal a previously unrecognized critical role for cyclin A2 in mediating cardiomyocyte mitosis, a role that may significantly impact upon clinical treatment of damaged myocardium.

Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm. Here we show that Zfp145, encoding the transcriptional repressor Plzf, has a crucial role in spermatogenesis. Zfp145 expression was restricted to gonocytes and undifferentiated spermatogonia and was absent in tubules of W/W(v) mutants that lack these cells. Mice lacking Zfp145 underwent a progressive loss of spermatogonia with age, associated with increases in apoptosis and subsequent loss of tubule structure but without overt differentiation defects or loss of the supporting Sertoli cells. Spermatogonial transplantation experiments revealed a depletion of spermatogonial stem cells in the adult. Microarray analysis of isolated spermatogonia from Zfp145-null mice before testis degeneration showed alterations in the expression profile of genes associated with spermatogenesis. These results identify Plzf as a spermatogonia-specific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

We previously observed high levels of Brd2 (also known as female sterile homeotic related gene-1, Fsrg1) expression in several hormonally responsive tissues, including the ovary. Here, we report distinct localization patterns of Brd2 transcripts throughout ovarian folliculogenesis in normal mice as well as in two strains of mice with aberrant folliculogenesis: mice with mutated growth differentiation factor 9 (Gdf9) and follicle stimulating hormone β (Fshb) genes. The highest level of expression was seen in granulosa cells of growing follicles. Within the oocyte, three patterns of Brd2 RNA localization were observed: diffuse distribution in both the cytoplasm and nucleus, then intense nuclear expression, followed by an absence of Brd2 transcripts from the nucleus. The transition from intense nuclear localization to nuclear exclusion was found to correlate with oocyte maturation and meiotic competence, as determined by nuclear chromatin patterns. These same expression patterns were also seen in oocytes from Gdf9−/− and Fshb−/− mice. Thus, Brd2 expression appears to correlate with stages of oocyte maturation, independent of FSH or GDF9 action and the subsequent disruption in normal follicle development in these models. The distinct patterns of Brd2 localization within the adult ovary supports a role for Brd2 in mitotic and possibly meiotic cell cycle regulation. Mol. Reprod. Dev. 68: 261–268, 2004. © 2004 Wiley-Liss, Inc.

Aberrant expression of several key regulators controlling the G1/S phase of the cell cycle has been implicated in human male germ cell tumorigenesis. Given the critical role of cyclin A2 at both the G1/S and G2/M transitions and the essential role for cyclin A1 in male germ cell development, our present study focused on the involvement of the A-type cyclins in the transformation and progression of male germ cell tumors (GCTs). The expression of the A-type cyclins and their catalytic partners Cdk1 and Cdk2 was examined in all types and stages of human male GCTs, including carcinoma in situ(CIS), seminoma and non-seminoma GCTs, along with normal testis samples. Elevated levels of cyclin A2, Cdk1 and Cdk2 were detected in the majority of GCTs and were correlated with the invasiveness of the tumors (p < 0.05). Cyclin A1 expression was virtually undetectable in CIS and seminoma, but was aberrantly expressed in all non-seminomatous GCTs. Cyclin A2 expression was strongly correlated with that of its catalytic partners Cdk1 and Cdk2 in all types of testicular tumors examined (p < 0.05), whereas a strong correlation between cyclin A1 and Cdk1 or Cdk2 was only seen in non-seminomatous GCTs (p < 0.05). Histone kinase activities of cyclin A1/Cdks and cyclin A2/Cdks were found to be elevated in tumors. Our data suggest that aberrant expression of A-type cyclins and their Cdks is a significant factor in male germ cell tumorigenesis. The abundant ectopic expression of cyclin A1 in non-seminomatous GCTs and its absence in CIS and seminomas is likely linked to the tumor transformation and progression and may be relevant to clinical prognosis.

Targeted mutagenesis of the retinoic acid receptor alpha (RAR alpha) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RAR alpha(-/-) testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8-9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RAR alpha(-/-) testes and confirmed the arrest at step 8-9 spermatids. Retinoid signaling through RAR alpha, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations.

Mammalian gametogenesis provides a unique system in which to study cell-cycle regulation. Furthermore, understanding the genetic program controlling the mitotic and meiotic divisions of the germ line will provide insight into understanding infertility and new directions for contraception. Male and female germ cells have stages of cell-cycle regulation in common, including a mitotic proliferative stage, entry into meiosis, completion of a reductive division, and entry into a quiescent state awaiting signals at fertilization. However, the timing of these events - and, indeed, even the stage of development at which these events occurs - differs in the two sexes. The genes involved in controlling these specialized mitotic and meiotic cycles of mammalian germ cell differentiation are only now being identified. They include a complex array of kinases, phosphatases, regulatory proteins (e.g., cyclins), and an equally complex array of substrates, including components of the nuclear and cytoplasmic structures involved in cell division. This chapter provides an overview of our current understanding of cell-cycle regulation in mammalian mitotic cells and the importance of restriction points. A summary of observations regarding the expression of various cell-cycle regulatory genes in mouse gametes is provided, along with comments on interesting differences between mitotic and meiotic cells. Finally, the role of the novel A-type cyclin, cyclin A1, during male meiosis is discussed in depth.

Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system.

Objective: To gain insight into the function of D1Pas1 in spermatogenesis. Design: The cellular and subcellular distribution of D1Pas1 protein were examined. Setting: Academic research laboratory. Animals: Swiss Webster and C57B1/6J mice. Intervention(s): Antibodies were generated against a D1Pas1 fusion protein. Immunoblot analysis was performed on lysates of testicular cells separated into enriched populations of spermatogenic cells and fractionated into nuclear and cytoplasmic compartments. Immunohistochemistry was performed on histological sections of testis from adult and postnatal day 17 mice. Main Outcome Measure(s): D1Pas1 protein distribution. Result(s): D1Pas1 was expressed in germ cells, and its expression was developmentally regulated because it was detected specifically in the meiotic and postmeiotic haploid stages of spermatogenesis. D1Pas1 protein was predominantly localized in the nucleus, with weak cytoplasmic staining. Conclusion(s): Nuclear localization of D1Pas1 in the testis and its sequence homology to putative RNA helicases suggests a role of D1Pas1 in pre-mRNA processing during spermatogenesis. Germ cell expression of D1Pas1 and homology to the Y chromosome gene DBY, which is located in an area deleted in azoospermia, suggests a potential role for an autosomal gene in the regulation of spermatogenesis.

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary. Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos.

We have identified a retinol dehydrogenase (cRDH) that catalyzes the oxidation of 9- cis - but not all- trans -retinol and proposed that this enzyme plays an impor- tant role in synthesis of the transcriptionally active retinoid, 9- cis- retinoic acid. There is little information regarding either the biochemical properties of cRDH or how its 9- cis - retinol substrate is formed. We now report studies of the properties and expression of human and mouse cRDH and of the characteristics and location of the murine cRDH gene. Additionally, we report mouse hepatic 9- cis -retinol concentrations and demonstrate that 9- cis -retinol is formed in a time- and protein-dependent manner upon incubation of all- trans -retinol with cell homogenate. Human and mouse cRDH display similar substrate specificities for cis -isomers of retinol and retinaldehyde. Moreover, human and mouse cRDH show marked sensitivity to inhibition by 13- cis -retinoic acid, with both being inhibited by approximately 50% by 0.15 m M 13- cis -retinoic acid (for substrate concentrations of 10 m M ). Lesser inhibition is seen for 9- cis - or all- trans -retinoic acids. Immunoblot analysis using antiserum directed against human cRDH demonstrates cRDH expression in several tis- sues from first trimester human fetuses, indicating that cRDH is expressed early in embryogenesis. Adult mouse brain, liver, kidney, and to a lesser extent small intestine and placenta express cRDH. The murine cRDH gene consists of at least 5 exons and spans approximately 6 kb of genomic DNA. Backcross analysis mapped the mouse cRDH gene to the most distal region of chromosome 10. Taken to- gether, these data extend our understanding of the proper- ties of cRDH and provide additional support for our hy- pothesis that cRDH may play an important role in 9- cis - retinoic acid formation. —Gamble, M. V., E. Shang, R. P. Zott, J. R. Mertz, D. J. Wolgemuth, and W. S. Blaner. Bio- chemical properties, tissue expression, and gene structure of a short chain dehydrogenase/reductase able to catalyze cis -retinol oxidation. J. Lipid Res. 1999. 40: 2279-2292.

Megacolon occurs in neonatal and adult transgenic mice that overexpress the Hoxa-4 gene. Abnormalities, which are restricted to the terminal colon of these mice, include a hypoganglionosis, abnormal enteric ganglia with a structure appropriate for extra-enteric peripheral nerve and not the enteric nervous system (ENS), and gaps in the longitudinal muscle occupied by ganglia. To investigate the developmental origin of these abnormalities, we analyzed the development of the pelvis and terminal colon in Hoxa-4 transgenic mice. Morphological abnormalities were detected as early as E13. These included an enlargement of the mucosa and the bowel wall, a thickening of the enteric mesenchyme, and the ectopic location of pelvic ganglion cells, which initially clustered on the dorsolateral wall of the hindgut. As the bowel enlarged, these ectopic cells become ventrolateral and, between days E17 and E18.5, appeared to become incorporated into the gut, leaving neuron-filled gaps in the longitudinal muscle layer. The ectopic ganglia retained extra-enteric characteristics, including the presence of capillaries, basal laminae, collagen fibers, and catecholaminergic neurons, even after their incorporation into the bowel. It is proposed that the abnormal and ectopic expression of the Hoxa-4 transgene in the colon causes signalling molecule(s) of the enteric mesenchyme to be overproduced and that the overabundance of these signals leads to mucosal enlargement and misdirection of migrating pelvic neuronal progenitors.

STAT (signal transducer and activator of transcription) proteins have been shown to be essential transcription factors which mediate biological effects of cytokines. Although most of the STATs have been shown to be widely expressed, Stat4 mRNA has been detected in only a few tissues, including the testis. In the present study, immunoblot analysis confirmed that the presence of Stat4 protein was similarly restricted, with the highest level observed in testis. In situ hybridization, immunoblot, and immunohistochemistry analyses revealed that in the testis, Stat4 was abundantly and exclusively expressed in male germ cells which have completed meiosis, at the round and elongating spermatid stages. Cytolocalization at various times of spermatid differentiation showed that the level of Stat4 protein increased in parallel in both cytoplasm and nuclei. No specific nuclear translocation that would have been an indicator of Stat4 activation was observed at any stage of spermatogenic differentiation. Interestingly, the Stat4 transcription factor was localized to the condensing perinuclear theca of spermatids, a localization that was confirmed by selective biochemical extraction of thecal proteins. Since the theca is known to depolymerize in the cytoplasm of the oocyte during the hours following fertilization, we hypothesized that sperm Stat4 would represent an original paternal contribution to the fertilized egg which may be involved in the onset of zygotic transcription.

The double bromodomain-containing BET ( b romodomain and e xtra t erminal) family of proteins is highly conserved from yeast to humans and consists not just of transcriptional regulators but also histone-interacting chromatin remodelers. The four mammalian BET genes are each expressed at unique times during spermatogenesis, and the testis-specific gene Brdt is essential for spermatogenesis. Loss of the first bromodomain of BRDT results in improper/incomplete spermatid elongation and severely morphologically defective sperm. The elongation defects observed in mutant spermatids can be directly tied to altered postmeiotic chromatin architecture. BRDT is required for creation/maintenance of the chromocenter of round spermatids, a structure that forms just after completion of meiosis. The chromocenter creates a defined topology in spermatids, and the presence of multiple chromocenters rather than a single intact chromocenter correlates with loss of spermatid polarity, loss of heterochromatin foci at the nuclear envelope, and loss of proper spermatid elongation. BRDT is not only essential for proper chromatin organization but also involved in regulation of transcription and in cotranscriptional processing. That is, transcription and alternative splicing are altered in spermatocytes and spermatids that lack full-length BRDT. Additionally, the transcription of mRNAs with short 3′ UTRs, which is characteristic of round spermatids, is also altered. Examination of the genes regulated by BRDT yields many possible targets that could in part explain the morphologically abnormal sperm produced by the BRDT mutant testes. Thus, BRDT and possibly the other BET genes are required for proper spermatogenesis, which opens up the possibility that the recently discovered small molecule inhibitors of the BET family could be useful as reversible male contraceptives.

We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

The roles of the cyclin dependent kinase (Cdk) family in murine germ cell development have been examined by studying the expression of five Cdk family genes (Cdc2, Cdk2, Cdk4, Pctaire-1, and Pctaire-3) in mouse reproductive organs. Northern blot and in situ hybridization analyses revealed distinctive expression patterns of these genes with striking cellular, lineage, and developmental stage specificity. We observed Cdk expression in cell types with proliferative activity: Cdc2 and Cdk2 expression in premeiotic spermatocytes in the testis, and Cdc2, Cdk2, and Cdk4 expression in granulosa cells of ovarian follicles. Cdc2 transcripts were most abundant in late pachytene to diplotene spermatocytes, soon to undergo meiosis. Surprisingly, we also observed expression of Cdk family genes in non-proliferating cell types. All five Cdk family genes examined were expressed in Sertoli cells of the adult testis, which are no longer mitotically active. With regard to Pctaire-1 and Pctaire-3, the highest levels of expression were observed in postmeiotic spermatids. Immunoblot analysis also revealed the presence of high levels of Pctaire-1 in postmeiotic germ cells. These results suggest that Cdk family kinases may exhibit various functions in germinal and somatic cells during gametogenesis, not only in the cell cycle but also in other regulatory processes, including differentiation. © 1995 wiley-Liss, Inc.

International audience; Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic and post-meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential genes and repressing a ‘progenitor cells’ gene expression program. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.

Previous studies have shown that in adult male mice, expression of the meg1 gene is restricted to meiotic and early postmeiotic testicular germ cells. We have now analyzed the expression of meg1 during postnatal testicular development and the comparable meiotic stages in the female. The 0.75 kb transcript for meg1 begins to accumulate in testes at d8-9 of postnatal (pn) development, coincident with the entry of germ cells into meiosis, and is expressed most abundantly at pn d14 and subsequent stages, when the spermatocytes have entered pachytene. In situ hybridization analysis shows that meg1 is expressed at very low levels in leptotene cells and increases as the cells progress through zygotene and pachytene stages. In the embryonic ovary, meg1 is not detected until after day 15 of gestation when the cells have entered the pachytene stage of meiosis I. In situ hybridization analysis suggests that meg1 transcripts are expressed at higher levels in degenerating rather than in healthy pachytene stage oocytes; meg1 is not expressed in any cells of the adult ovary, regardless of the stage of follicular development. These results suggest that meg1 is indeed a meiosis-associated gene in both male and female germ cells through the pachytene stage of meiosis I and appears to exhibit sex-specific differences in its expression thereafter.

Here we investigated a pharmacological approach to inhibit spermatogenesis in the mouse model by manipulating retinoid signaling using low doses of the pan-retinoic acid receptor (RAR) antagonist BMS-189453. Spermatogenesis was disrupted, with a failure of spermatid alignment and sperm release and loss of germ cells into lumen, abnormalities that resembled those in vitamin A-deficient and RARα-knockout testes. Importantly, the induced sterility was reversible. Enhanced efficacy and a lengthened infertility period with full recovery of spermatogenesis were observed using systematically modified dosing regimens. Hematology, serum chemistry, and hormonal and pathological evaluations revealed no detectable abnormalities or adverse side effects except the distinct testicular pathology. Our results suggest that testes are exquisitely sensitive to disruption of retinoid signaling and that RAR antagonists may represent new lead molecules in developing nonsteroidal male contraceptives.

A possible correlation between the structural organization of homeobox-containing genes and their cell-specific expression was examined in studies determining similarities and differences in the expression patterns of orthologues and paralogues of the murine Hox-1.4 gene. We first compared the expression pattern of members of the Hox-1.4 subfamily, Hox-2.6 and Hox-4.2, in the adult mouse testis. Although these three evolutionarily related genes exhibited similar anterior limits of expression in the embryonic central nervous system, their cellular specificity of expression was very different in the adult testis. Hox-1.4 was abundantly expressed only in the germ cells; Hox-2.6 was expressed at very low levels in both spermatogenic cells and somatic cells; and Hox-4.2 transcripts appeared to be restricted to the somatic cells. We next analyzed the expression of several of the Hox-1.4 orthologues in the mouse testis to determine if other members of the Hox-1 cluster are involved in the male germ cell differentiation pathway. We showed that the two adjacent genes of Hox-1.4, Hox-1.3 and Hox-1.5, are expressed in the germ line but at lower levels. Further, both Hox-1.3 and Hox-1.4 produced unique, germ line-specific transcripts as compared to other adult tissues and the Day 12.5 embryo.

Objective: We investigated the patterns of expression of HOXB5, cyclin D1 and proliferating cell nuclear antigen (PCNA) proteins in human congenital cystic adenomatoid malformation (CCAM) to establish the molecular basis of its etiology. Methods: Immunohistochemistry was performed on frozen archival specimens of CCAM and normal lung tissue as controls using antibodies against HOXB5, cyclin D1 and PCNA proteins. Results: Immunohistochemistry revealed a higher level of expression of HOXB5, cyclin D1 and PCNA predominantly in mesenchymal cells of the CCAM tissues as compared to normal adjacent control lung tissues. Conclusion: Elevated levels of immunohistochemical detection of HOXB5, cyclin D1 and PCNA were characteristic properties of lung tissue cells in CCAM. This elevated HOXB5 expression may correlate with the aberrant cellular differentiation observed in the CCAM disorder. Elevated expression of cyclin D1 and PCNA further suggests that increased cellular proliferation contributes to the overgrowth of lung tissue in CCAM.

Key components of the cell cycle machinery are the regulatory subunits, the cyclins, and their catalytic partners the cyclin-dependent kinases. Regulating the cell cycle in the male germ line cells represents unique challenges for this machinery given the constant renewal of gametes throughout the reproductive lifespan and the induction of the unique process of meiosis, a highly specialized kind of cell division. With challenges come opportunities to the critical eye, recognizing that understanding these specialized modes of regulation will provide considerable insight into both normal differentiation as well as disease conditions, including infertility and oncogenesis.

Physiological states of insulin resistance such as obesity and diabetes have been linked to abnormalities in female reproductive function. However, it is difficult to distinguish the direct effects of impaired insulin signaling from those of adiposity or hyperglycemia because these conditions often coexist in human syndromes and animal models of insulin resistance. In this study, we used lean, normoglycemic mouse lines with differing degrees of hyperinsulinemia and insulin receptor (Insr) expression to dissect the effects of altered insulin signaling on female reproduction. All three mouse lines [Ttr-Insr−/−, Insr+/−, and Insr+/+ (wild type)] are able to maintain fertility. However, the insulin-resistant and hyperinsulinemic mice demonstrate altered duration of estrous cycles as well as aberrant distribution and morphology of ovarian follicles. These effects appear to be independent of hyperandrogenism in the mice. Pregnancy studies indicate decreased success in early progression of gestation. In successful pregnancies, decreased embryo weights and increased placental calcification also implicate altered insulin signaling in later gestational effects. Thus, abnormal insulin signaling, independent of adipose tissue mass, adipokine expression levels, and hyperglycemia, can affect parameters of the female hypothalamic-pituitary-gonadal axis and pregnancy outcomes.

Signaling through vitamin A metabolites is indispensable for spermatogenesis, and disruption of retinoic acid receptor alpha (RARalpha) function resulted in male sterility and aberrant spermatogenesis, which resembled vitamin A deficiency. Here we investigated the lineage- and cell-specific role of RARalpha-mediated signaling during spermatogenesis using germ-cell transplantation and genetically manipulated mouse models. We demonstrated that RARalpha-deficient germ-cell stem cells were able to repopulate germ-cell-depleted wild-type testes and initiate spermatogenesis; however, improper cellular associations and abnormal sperm formation were observed. We further generated RARalpha-deficient mice that expressed RARalpha-EGFP fusion protein uniquely in haploid germ cells. Strikingly, spermatid orientation, alignment and release, as well as sperm morphology, were normal and there was a partial rescue of sterility. These data provide the first direct evidence for a distinct requirement of RARalpha-mediated retinoid signaling specifically in germ cells.

The specialized cell cycles that characterize various aspects of the differentiation of germ cells provide a unique opportunity to understand heretofore elusive aspects of the in vivo function of cell cycle regulators. Key components of the cell cycle machinery are the regulatory sub-units, the cyclins, and their catalytic partners, the cyclin-dependent kinases. Some of the cyclins exhibit unique patterns of expression in germ cells that suggest possible concomitant distinct functions, predictions that are being explored by targeted mutagenesis in mouse models. A novel, meiosis-specific function has been shown for one of the A-type cyclins, cyclin A1. Embryonic lethality has obviated understanding of the germline functions of cyclin A2 and cyclin B1, while yet other cyclins, although expressed at specific stages of germ cell development, may have less essential function in the male germline.

International audience; We report a cross-species expression profiling analysis of the human, mouse, and rat male meiotic transcriptional program, using enriched germ cell populations, whole gonads, and high-density oligonucleotide microarrays (GeneChips). Among 35% of the protein-coding genes present in rodent and human genomes that were found to be differentially expressed between germ cells and somatic controls, a key group of 357 conserved core loci was identified that displays highly similar meiotic and postmeiotic patterns of transcriptional induction across all three species. Genes known to be important for sexual reproduction are significantly enriched among differentially expressed core loci and a smaller group of conserved genes not detected in 17 nontesticular somatic tissues, correlating transcriptional activation and essential function in the male germ line. Some genes implicated in the etiology of cancer are found to be strongly transcribed in testis, suggesting that these genes may play unexpected roles in sexual reproduction. Expression profiling data further identified numerous conserved genes of biological and clinical interest previously unassociated with the mammalian male germ line.

Male mice lacking cyclin A1 protein are sterile. Their sterility results from an arrest in the meiotic cell cycle of spermatocytes, which we now identify as occurring at late diplotene, immediately before diakinesis. The stage of arrest in cyclin A1-deficient mice is distinct from the arrest seen in spermatocytes that are deficient in its putative catalytic partner Cdk2, which occurs much earlier in pachytene. The arrest in cyclin A1-deficient spermatocytes is also accompanied by an unusual clustering of centromeric heterochromatin. Consistent with a possible defect in the centromeric region, immunofluorescent staining of cyclin A1 protein shows localization in the region of the centromere. Phosphorylation of histone H3 at serine 10 in pericentromeric heterochromatin, which normally occurs in late diplotene, is reduced in spermatocytes from heterozygous Ccna1+/− testes and completely absent in spermatocytes with no cyclin A1 protein. Concomitantly, the levels of pericentromeric aurora B kinase, known to phosphorylate histone H3 during meiosis, are partially reduced in spermatocytes from testes of heterozygous mice and further reduced in homozygous null spermatocytes. These data suggest a critical and concentration-dependent function for cyclin A1 in the pericentromeric region in late diplotene of meiosis, perhaps in assembly or function of the passenger protein complex.

Objective We hypothesized that administration of alcohol during the second trimester of gestation at the pseudoglandular phase of lung development might lead to aberrant differentiation and growth, similar to that seen in congenital cystic adenomatoid malformation in human. We further hypothesized that these effects would be apparent morphologically and by altered Hoxb5 expression. Study Design C57BL/6J mice, exposed to ethanol at embryonic day (E) 11.5 to E13.5, which corresponds to the pseudoglandular stage of lung development, were examined at E18.5. The lungs were analyzed histologically by immunostaining. Results The average body and lung weight of alcohol-exposed (AE) fetuses were lower than those of control fetuses, the reduction in lung mass being more than the body weight. Histology showed that AE lungs were less developed and exhibited higher expression of Hoxb5 in AE lungs than controls. Conclusion Alcohol exposure at E13.5 affected fetal lung development, with delayed differentiation and increased Hoxb5.

We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V–VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.

The human Brd2 (Bromodomain-containing 2) gene codes for a double-bromodomain protein that associates with the cell cycle-driving transcription factors E2F-1 and E2F-2. Expression of mouse Brd2 has been shown previously to be expressed in specific patterns in proliferating cells in the developing alveoli in the mammary gland. In the present study, in situ hybridization and immunohistochemical analyses were used to examine expression of Brd2 in developing neural tissues. Brd2 mRNA was detected in brain vesicles, neural tube, spinal cord and dorsal root ganglia (DRG). Immunostaining proved that the message is translated in these tissues and further revealed that Brd2 protein localizes to the nucleus in proliferating cells, but is cytoplasmic in differentiated neurons that are no longer cycling. Brd2 protein in the nuclei of the proliferating neuronal precursors is excluded from the heterochromatin. These observations are consistent with our previous finding that nuclear localization of Brd2 protein correlates with an active cell cycle in mouse mammary alveoli during the reproductive cycle, and similar results from others in cultured fibroblasts. Our findings are also consistent with the cell cycle progression/transcription coactivator function suggested by the association of Brd2 with E2F-1 and E2F-2.

The tilted mutant mouse, which lacks otoconia in the inner ear, was used to study development of the mouse vestibular system in the absence of gravity perception. Otoconia are dense particles composed of proteins and calcium carbonate crystals suspended in the gelatinous macular membrane. They enhance, and are largely responsible for, sensitivity to gravity. Morphometric analysis of the vestibular ganglion showed that the mutant developed more slowly than the normal controls, both in rate of development and cell number, particularly during the first week of post-natal development. The mutant ganglia also exhibited a reduction of cells during the first 6 days of post-natal development.

It has been proposed that cis -retinol dehydroge- nase (cRDH) acts within the body to catalyze the oxidation of 9- cis -retinol, an oxidative step needed for 9- cis -retinoic acid synthesis, the oxidation of 11- cis- retinol (an oxidative step needed for 11- cis -retinal (visual chromophore) synthe- sis), and 3 � -hydroxysteroid transformations. To assess in vivo the physiological importance of each of these pro- posed actions of cRDH, we generated cRDH-deficient (cRDH � / � ) mice. The cRDH � / � mice reproduce normally and appear to be normal. However, the mutant mice do have a mild visual phenotype of impaired dark adaptation. This phenotype is evidenced by electroretinagram analysis of the mice and by biochemical measures of eye levels of retinoid intermediates during recovery from an intense photobleach. Although it is thought that cRDH is expressed in the eye almost solely in retinal pigment epithelial cells, we detected cRDH expression in other retinal cells, includ- ing ganglion cells, amacrine cells, horizontal cells, and the inner segments of the rod photoreceptor cells. Aside from the eye, there are no marked differences in retinoid levels in other tissues throughout the body for cRDH � / � compared with cRDH � / � mice. Moreover, we did not detect any non- visual phenotypic changes for cRDH � / � mice, suggesting that these mice do not have problems in metabolizing 3 � - hydroxysteroids. Thus, cRDH may act essentially in the visual cycle but is redundant for catalyzing 9- cis- retinoic acid formation and 3 � -hydroxysteroid metabolism. —Shang, E., K. Lai, A. I. Packer, J. Paik, W. S. Blaner, M. de Morais Vie- ira, P. Gouras, and D. J. Wolgemuth. Targeted disruption of the mouse cis -retinol dehydrogenase gene: visual and nonvi- sual functions. J. Lipid Res. 2002. 43: 590-597.

The p38 stress-activated protein kinase pathway is involved in regulation of phosphorylation of Hsp25, which in turn regulates actin filament dynamic in non-neuronal cells. We report that p38, Hsp25 and Akt signaling pathways were specifically activated in spinal motor neurons after sciatic nerve axotomy. The activation of the p38 kinase was required for induction of Hsp25 expression. Furthermore, Hsp25 formed a complex with Akt, a member of PI-3 kinase pathway that prevents neuronal cell death. Together, our observations implicate Hsp25 as a central player in a complex system of signaling that may both promote regeneration of nerve fibers and prevent neuronal cell death in the injured spinal cord.

A mammalian A-type cyclin, cyclin A1, is highly expressed in testes of both human and mouse and targeted mutagenesis in the mouse has revealed the unique requirement for cyclin A1 in the progression of male germ cells through the meiotic cell cycle. While very low levels of cyclin A1 have been reported in the human hematopoietic system and brain, the sites of elevated levels of expression of human cyclin A1 were several leukemia cell lines and blood samples from patients with hematopoietic malignances, notably acute myeloid leukemia. To evaluate whether cyclin A1 is directly involved with the development of myeloid leukemia, mouse cyclin A1 protein was overexpressed in the myeloid lineage of transgenic mice under the direction of the human cathepsin G (hCG) promoter. The resulting transgenic mice exhibited an increased proportion of immature myeloid cells in the peripheral blood, bone marrow, and spleen. The abnormal myelopoiesis developed within the first few months after birth and progressed to overt acute myeloid leukemia at a low frequency (≈15%) over the course of 7–14 months. Both the abnormalities in myelopoiesis and the leukemic state could be transplanted to irradiated SCID (severe combined immunodeficient) mice. The observations suggest that cyclin A1 overexpression results in abnormal myelopoiesis and is necessary, but not sufficient in the cooperative events inducing the transformed phenotype. The data further support an important role of cyclin A1 in hematopoiesis and the etiology of myeloid leukemia.

We have examined the expression of the Hoxa4 gene in embryonic vibrissae and developing and cycling postnatal pelage hair follicles by digoxigenin-based in situ hybridization. Hoxa4 expression is first seen in E13.5 vibrissae throughout the follicle placode. From E15.5 to E18.5 its expression is restricted to Henle's layer of the inner root sheath. Postnatally, Hoxa4 expression is observed at all stages of developing pelage follicles, from P0 to P4. Sites of expression include both inner and outer root sheaths, matrix cells, and the interfollicular epidermis. Hoxa4 is not expressed in hair follicles after P4. Hoxb4, however, is expressed both in developing follicles at P2 and in catagen at P19, suggesting differential expression of these two paralogous genes in the hair follicle cycle.

A number of genes are known to be expressed primarily in hematopoietic cells and testis and are thought to function in the control of both blood cell and male germ cell differentiation. We have recently identified a mouse gene, HemT, that encodes two alternatively spliced transcripts specific to hematopoietic cells (HemT-1 and HemT-2) and kidney (HemT-2). We have now isolated a third HemT transcript, HemT-3, that is found only in testis by Northern blot analysis and RT-PCR. HemT-3 is alternatively spliced and may be initiated differently from HemT-1 and HemT-2. RNA in-situ hybridization of testis from wild-type and germ-cell-deficient adult mice, as well as from mice at different developmental stages, indicates that HemT-3 is expressed only in early spermatocytes. HemT-3 cDNA has a major open reading frame related to a human glycosylphosphatidylinositol (GPI)-anchored protein, GML. Using an antibody generated against a peptide derived from the HemT-3 open reading frame, we have detected a testis-specific 22kDa protein by Western blot analysis.