Unknown Regulatory Factors Research Articles

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation.

The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.

PAH Pathogenic Variants and Clinical Correlations in a Group of Hyperphenylalaninemia Patients from North-Western Romania.

Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase (PAH) gene and is characterized by altered amino acid metabolism. More than 1500 known PAH variants intricately determine a spectrum of metabolic phenotypes. We aim to report on clinical presentation and PAH variants identified in 23 hyperphenylalaninemia (HPA)/PKU Romanian patients. Our cohort exhibited classic PKU (73.9%, 17/23), mild PKU (17.4%, 4/23), and mild HPA (8.7%, 2/23). Severe central nervous system sequelae are frequent in our cohort in late-diagnosis symptomatic patients, which highlights yet again the significance of an early dietary treatment, neonatal screening and diagnosis, and facilitated access to treatment. Next-generation sequencing (NGS) identified a total of 11 PAH pathogenic variants, all previously reported, mostly missense changes (7/11) in important catalytic domains. c.1222C>T p.Arg408Trp was the most frequent variant, with an allele frequency of 56.5%. Twelve distinct genotypes were identified, the most frequent of which was p.Arg408Trp/p.Arg408Trp (34.8%, 8/23). Compound heterozygous genotypes were common (13/23), three of which had not been previously reported to the best of our knowledge; two correlated with cPKU and one showed an mPKU phenotype. Generally, there are genotype-phenotype correlation overlaps with the public data reported in BIOPKUdb; as our study shows, clinical correlates are subject to variation, in part due to uncontrolled or unknown epigenetic or environmental regulatory factors. We highlight the importance of establishing the genotype on top of using blood phenylalanine levels.

Genetic regulation of nitrogen use efficiency in Gossypium spp.

Cotton (Gossypium spp.) is the most important fibre crop, with desirable characteristics preferred for textile production. Cotton fibre output relies heavily on nitrate as the most important source of inorganic nitrogen (N). However, nitrogen dynamics in extreme environments limit plant growth and lead to yield loss and pollution. Therefore, nitrogen use efficiency (NUE), which involves the utilisation of the 'right rate', 'right source', 'right time', and 'right place' (4Rs), is key for efficient N management. Recent omics techniques have genetically improved NUE in crops. We herein highlight the mechanisms of N uptake and assimilation in the vegetative and reproductive branches of the cotton plant while considering the known and unknown regulatory factors. The phylogenetic relationships among N transporters in four Gossypium spp. have been reviewed. Further, the N regulatory genes that participate in xylem transport and phloem loading are also discussed. In addition, the functions of microRNAs and transcription factors in modulating the expression of target N regulatory genes are highlighted. Overall, this review provides a detailed perspective on the complex N regulatory mechanism in cotton, which would accelerate the research toward improving NUE in crops.

The regulation and potential roles of m6A modifications in early embryonic development and immune tolerance at the maternal-fetal interface.

The immune microenvironment at the maternal-fetal interface was determined by the crosstalk between the trophoblast and maternal-derived cells, which dynamically changed during the whole gestation. Trophoblasts act as innate immune cells and dialogue with maternal-derived cells to ensure early embryonic development, depending on the local immune microenvironment. Therefore, dysfunctions in trophoblasts and maternal decidual cells contribute to pregnancy complications, especially recurrent pregnancy loss in early pregnancy. Since many unknown regulatory factors still affect the complex immune status, exploring new potential aspects that could influence early pregnancy is essential. RNA methylation plays an important role in contributing to the transcriptional regulation of various cells. Sufficient studies have shown the crucial roles of N6-methyladenosine (m6A)- and m6A-associated- regulators in embryogenesis during implantation. They are also essential in regulating innate and adaptive immune cells and the immune response and shaping the local and systemic immune microenvironment. However, the function of m6A modifications at the maternal-fetal interface still lacks wide research. This review highlights the critical functions of m6A in early embryonic development, summarizes the reported research on m6A in regulating immune cells and tumor immune microenvironment, and identifies the potential value of m6A modifications in shaping trophoblasts, decidual immune cells, and the microenvironment at the maternal-fetal interface. The m6A modifications are more likely to contribute to embryogenesis, placentation and shape the immune microenvironment at the maternal-fetal interface. Uncovering these crucial regulatory mechanisms could provide novel therapeutic targets for RNA methylation in early pregnancy.

Combining transposon mutagenesis and reporter genes to identify novel regulators of the topA promoter in Streptomyces

BackgroundIdentifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest in Streptomyces.ResultsA method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria.ConclusionsOur approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.

ATAC-Seq Identifies Chromatin Landscapes Linked to the Regulation of Oxidative Stress in the Human Fungal Pathogen Candida albicans.

Human fungal pathogens often encounter fungicidal stress upon host invasion, but they can swiftly adapt by transcriptional reprogramming that enables pathogen survival. Fungal immune evasion is tightly connected to chromatin regulation. Hence, fungal chromatin modifiers pose alternative treatment options to combat fungal infections. Here, we present an assay for transposase-accessible chromatin using sequencing (ATAC-seq) protocol adapted for the opportunistic pathogen Candida albicans to gain further insight into the interplay of chromatin accessibility and gene expression mounted during fungal adaptation to oxidative stress. The ATAC-seq workflow not only facilitates the robust detection of genomic regions with accessible chromatin but also allows for the precise modeling of nucleosome positions in C. albicans. Importantly, the data reveal genes with altered chromatin accessibility in upstream regulatory regions, which correlate with transcriptional regulation during oxidative stress. Interestingly, many genes show increased chromatin accessibility without change in gene expression upon stress exposure. Such chromatin signatures could predict yet unknown regulatory factors under highly dynamic transcriptional control. Additionally, de novo motif analysis in genomic regions with increased chromatin accessibility upon H2O2 treatment shows significant enrichment for Cap1 binding sites, a major factor of oxidative stress responses in C. albicans. Taken together, the ATAC-seq workflow enables the identification of chromatin signatures and highlights the dynamics of regulatory mechanisms mediating environmental adaptation of C. albicans.

Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization

Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.

Chromatin Accessibility Identifies CTCF As a Gatekeeper of Stemness Functions in Human Hematopoietic Development

Hematopoietic stem cells (HSC), at the apex of the blood hierarchy, generate a series of increasingly committed progenitors and ultimately produce terminally differentiated cells at the bottom of the hierarchy. Although HSC and their immediate downstream, multipotent progenitors (MPP) have full multilineage differentiation capacity, only HSC have the capacity for long term self-renewal. Thus comparison of HSC with downstream progenitors that exhibit gradual fate restriction by assuming the identity of a mature blood cell is central to defining how the stemness state is established and how lineage commitment is executed. Interestingly, few genes are differentially expressed between human HSC and MPP, suggesting that some important stem cell mechanisms are in a permissive state and therefore difficult to identify simply by differential gene expression analysis. We hypothesized that these permissive genetic regions could be identified by chromatin analysis. To identify permissive genomic regions, we delineated regions of accessible chromatin through ATAC-seq assays in HSC, MPP, committed progenitors and seven mature lineages from human Cord Blood. DNA recognition motif analysis across accessible chromatin in each developmental stage identified both known and unknown candidate master regulatory transcription factors for each step of lineage development.The DNA recognition motif for CTCF, a crucial effector of chromatin interactions, is enriched in accessible chromatin associated with progenitors but not in HSC, even though CTCF expression is not significantly different among stem and progenitor populations. Knockdown of CTCF in HSC by lentiviral vector system increased the proportion of HSC in G0 cell cycle and consequently reduced the number of mature cells generated in both conventional and high sensitivity single cell assays. The development of differentiated lymphoid and megakaryocytic lineages was affected. Few effects were seen when CTCF was silenced in MPP or more downstream progenitors. Collectively, our results suggest that CTCF is a "gate keeper factor" that regulates transition from dormant to active stages in HSC, and might control stem cell pool throughout life.In parallel to normal blood hierarchies, AML is also composed of a hierarchy with leukemia stem cells and non-stem-like leukemia cells; LSC and non-LSC fractions sorted from 24 AML patients have also been subjected to ATAC-seq. Currently we are comparing HSC and non-HSC with LSC and non-LSC fractions to identify the similarity and differences between normal HSC and LSC. Overall, our study is revealing insights into the stemness state of normal and leukemic stem cells in primary human tissues. DisclosuresTakayanagi:Kyowa Hakko Kirin: Employment.

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy.

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.

BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma.

Large-scale detection and analysis of adenosine-to-inosine RNA editing during development in Plutella xylostella.

Adenosine-to-inosine (A-to-I) RNA editing is site-specific modification of RNAs that increases the diversity of the transcriptome and proteome. Most insects undergo complete metamorphosis, including four life cycle stages: egg, larva, pupa and adult. Many previous studies have confirmed that RNA-editing events occur in a development-specific manner; in other words, RNA-editing levels change during metamorphosis. Here, we describe an effort to identify the developmental specificity of RNA-editing events using a large-scale computational analysis of RNA-seq data derived from four developmental stages of the diamondback moth, Plutella xylostella. One thousand one hundred and eighty-seven A-to-I RNA-editing sites were predicted to be developmental stage specific (false-discovery rate <0.01) and 1,094 of these sites were located in protein-coding regions. Editing of 152 sites resulted in an altered amino acid residue. A putative adult-specific A-to-I RNA-editing site was verified by comparing cDNA sequences with its corresponding genomic locus at different stages of the P. xylostella life cycle. Our findings will help elucidate the role of A-to-I RNA editing in the regulation of metamorphosis. Further studies detailing changes in the extent of editing are needed to establish how as yet unknown regulatory factors are involved in the editing mechanism and what biological functions' editing serves.

High-throughput screening of small interfering ribonucleic acid identifies important modulators in islet dysfunction and apoptosis.

High-throughput screening of small interfering ribonucleic acid identifies important modulators in islet dysfunction and apoptosis.

Mutant DnaAs of Escherichia coli that are refractory to negative control

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

New insights in the role of HtdA in the regulation of R27 conjugation

R27 is the prototype of the IncHI group of conjugative plasmids, which are associated with multidrug resistance in several relevant pathogens. The transfer of this plasmid is thermodependent and all transfer-related genes are encoded in six operons (tra operons). Very little is known about the factors involved in the regulation of the R27 conjugation. This report describes transcriptional studies of the six tra operons. Our results indicate that HtdA, encoded in the R27 plasmid, is involved in the transcriptional repression of four tra operons (F, H, AC and Z). Although HtdA plays a pivotal role in the transcriptional regulation of those operons, it does not exert its effect as a classical repressor. The data indicate the existence of a crosstalk between HtdA and other unknown regulatory factors. The HtdA-mediated regulation of conjugation is independent of the R27 H-NS protein.

Morphometry and Ultrastructure of Stage IX-Specific Effects on Rat Sertoli and Spermatogenic Cells Immediately After 7-Day Testosterone Treatment in One Group and the Same Treatment in Another Group Followed by 7-Day Non-Treatment

Purpose: In our previous study, morphometric and ultrastructural analysis of stage-specific effects of Sertoli and spermatogenic cells were seen immediately after 7-day testosterone treatment (T group) in rat testes. The results strongly suggested that significant regulatory factors in spermatogenesis remain to be discovered. The present study was conducted to determine the morphometric and ultrastructural analysis of rat sertoli and spermatogenic cells in stage IX T group in one group and the same treatment in another group followed by 7-day non treatment (AT group), and also to assess whether or not the suggested unknown regulatory factors exist in the AT group. Results: In the AT group, concentrations of testosterone decreased more prominently than in its counterpart in the T group. However, its concentration was lower than its counterpart in the control group of rats. Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the AT group increased significantly more than their counterparts in the T group. Further they were closer to their counterparts in the control group than their counterparts in the T group. The absolute volumes of seminiferous tubules in the AT group increased to levels closer to their counterparts in the control group than to their counterpart in the T group. In the cytoplasm of the Step 9 spermatid in the T group, disorganization of the microtubules in a manchette-like structure appeared. However, they assumed a normal manchette-like orientation in the AT group. The fine structures corresponding to the transverse section of tails in normal spermatozoa were detected in the adluminal region of the seminiferous epithelium in the AT group, and these findings suggest that normal spermatozoa were formed in the AT group. Conclusion: The present study strongly suggests that unknown regulatory factors remain to be discovered.

Wildervanck’s syndrome and mirror movements: a congenital disorder of axon migration?

Cell outgrowth and migration in the developing nervous system result from guidance cues, whose molecular bases and clinical correlates are only partly known. We describe a patient with brain stem malformation, paroxysmal left sided lacrimation when eating ("crocodile tears") and mirror movements in addition to Wildervanck's cervico-oculo-acusticus (COA) syndrome, which encompasses Klippel-Feil anomaly, congenital hearing loss and Duane's syndrome. The unique symptom constellation has not been reported in that combination before and can be discussed in the context of congenital disordered axonal migration based on dysfunction of signalling pathways. However, mutations in some recently discovered genes, associated with single findings also present in our patient, were not found. Therefore, we suppose that the disturbance of an as yet unknown regulatory factor may explain the congenital malformation syndrome of our patient. In general, only a few human disorders have yet been found to result from defects in axon guidance. Nevertheless, disorders of axon guidance can certainly be regarded as a new category of neurodevelopmental disorders.

Regulation of DNA Topoisomerase IIβ through RNA-dependent Association with Heterogeneous Nuclear Ribonucleoprotein U (hnRNP U)

Recent studies suggest that DNA topoisomerase IIbeta (topo IIbeta) is involved in transcriptional activation of certain genes, which assumes accurate targeting of the enzyme to its action site. The target selection may be achieved by cooperation with unknown regulatory factors. To seek out such factors, we looked for proteins associated with the enzyme in differentiating cerebellar neurons. Antibody against topo IIbeta co-precipitated RNA-binding proteins including PSF, NonO/p54nrb, as well as hnRNP U/SAF-A/SP120. Reconstitution experiments with tag-purified proteins showed that topo IIbeta associates stoichiometrically with SP120 in the presence of RNA that was co-purified with SP120. The most effective RNA species for the complex formation was a subset of cellular polyadenylated RNAs. The C-terminal 187-residue domain of SP120 was necessary and sufficient for the association with both topo IIbeta and the endogenous RNA. The RNA isolated from the tag-purified SP120 inhibited the relaxation of supercoiled DNA by topo IIbeta. When the enzyme associates with SP120, however, the inhibition was abolished and the catalytic property was modulated to more processive mode, which may prolong its residence time at the genomic target site. Furthermore, the presence of SP120 was required for the stable expression of topo IIbeta in vivo. Thus, SP120 regulates the enzyme in dual ways.

Activity Determinants and Functional Specialization of Arabidopsis PEN1 Syntaxin in Innate Immunity

In eukaryotes, proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family are believed to have a general role for the fusion of intracellular transport vesicles with acceptor membranes. Arabidopsis thaliana PEN1 syntaxin resides in the plasma membrane and was previously shown to act together with its partner SNAREs, the adaptor protein SNAP33, and endomembrane-anchored VAMP721/722 in the execution of secretory immune responses against powdery mildew fungi. We conducted a structure-function analysis of PEN1 and show that N-terminal phospho-mimicking and non-phosphorylatable variants neither affected binary nor ternary SNARE complex formation with cognate partners in vitro. However, expression of these syntaxin variants at native protein levels in a pen1 mutant background suggests that phosphorylation is required for full resistance activity in planta. All tested site-directed substitutions of SNARE domain or "linker region" residues reduced PEN1 defense activity. Two of the variants failed to form ternary complexes with the partner SNAREs in vitro, possibly explaining their diminished in planta activity. However, impaired pathogen defense in plants expressing a linker region variant is likely because of PEN1 destabilization. Although Arabidopsis PEN1 and SYP122 syntaxins share overlapping functions in plant growth and development, PEN1 activity in disease resistance is apparently the result of a complete functional specialization. Our findings are consistent with the hypothesis that PEN1 acts in plant defense through the formation of ternary SNARE complexes and point to the existence of unknown regulatory factors. Our data indirectly support structural inferences that the four-helical coiled coil bundle in ternary SNARE complexes is formed in a sequential order from the N- to C-terminal direction.

Morphometric and ultrastructural analysis of stage-specific effects of Sertoli and spermatogenic cells seen after short-term testosterone treatment in young adult rat testes

The effects of testosterone treatment on spermatogenesis in the rat have been investigated by morphometric and structural analysis at the ultrastructural level in stages VII-IX. The aim has been to characterize the changes in Sertoli and spermatogenic cells to elucidate the mechanism of testosterone effects on spermatogenesis and to test the possibilities of developing male contraceptives. In stage VII, the morphometric parameters of volume and surface area in Sertoli cells (see abbreviations below): and the morphometric parameter of volume in the spermatogenic cells such as V(VPG,T), V(VPC,T), V(VrPT,T) and V(VelPT,T) decreased. In stage VIII, the respective values of Sertoli cells, VSN, and VSN/VSC decreased while SSJ increased, and the respective morphometric parameters in the spermatogenic cells, V(VPG,T), V(VPC,T), and V(VrPT,T) increased. In stage IX, in Sertoli cells VSC, VSN, VSN/VSC, and SSJ remained unchanged. In spermatogenic cells V(VPG,T), V(VPC,T), and V(VrPT,T) increased. Further, in all stages, a close apposition of mitochondria and rough endoplasmic reticulum in basolateral cytoplasm of Sertoli cells suggested active protein synthesis. In elongated spermatids in stage IX the microtubular manchette became disorganized. This disorganization and the unexpected shift after testosterone treatment from decrease in several morphometric parameters in stage VIII to increases in stage IX cannot be explained by alterations in testosterone (T), LH, FSH, and their respective receptors. Therefore, still unknown regulatory factors in spermatogenesis are apparently involved in the developmental interactions between Sertoli and spermatogenic cells.