scholarly journals Expression of Atp8b3 in murine testis and its characterization as a testis specific P-type ATPase

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 345-351 ◽  
Author(s):  
Eun-Yeung Gong ◽  
Eunsook Park ◽  
Hyun Joo Lee ◽  
Keesook Lee
Keyword(s):  
Germ Cells ◽  
Testicular Development ◽  
Sperm Function ◽  
Developmentally Regulated ◽  
Primary Amino Acid Sequence ◽  
Biochemical Characteristics ◽  
Cellular Processes ◽  
Complex Process ◽  
Primary Amino ◽  
P Type

Spermatogenesis is a complex process that produces haploid motile sperms from diploid spermatogonia through dramatic morphological and biochemical changes. P-type ATPases, which support a variety of cellular processes, have been shown to play a role in the functioning of sperm. In this study, we isolated one putative androgen-regulated gene, which is the previously reported sperm-specific aminophospholipid transporter (Atp8b3, previously known asSaplt), and explored its expression pattern in murine testis and its biochemical characteristics as a P-type ATPase.Atp8b3is exclusively expressed in the testis and its expression is developmentally regulated during testicular development. Immunohistochemistry of the testis reveals thatAtp8b3is expressed only in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. As expected, from its primary amino acid sequence, ATP8B3 has an ATPase activity and is phosphorylated by an ATP-producing acylphosphate intermediate, which is a signature property of the P-Type ATPases. Together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization.

scholarly journals Human Cdc34 and Rad6B Ubiquitin-Conjugating Enzymes Target Repressors of Cyclic AMP-Induced Transcription for Proteolysis

1999 ◽  
Vol 19 (7) ◽  
pp. 5001-5013 ◽  
Author(s):  
Debananda Pati ◽  
Marvin L. Meistrich ◽  
Sharon E. Plon
Keyword(s):  
Cyclic Amp ◽  
Germ Cells ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Dominant Negative ◽  
Testicular Development ◽  
Developmentally Regulated ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

ABSTRACT Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIγ, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIγ and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIγ- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B −/−) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1–Cullin–F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

scholarly journals On the Need to Tell Apart Fraternal Twins eEF1A1 and eEF1A2, and Their Respective Outfits

2021 ◽  
Vol 22 (13) ◽  
pp. 6973
Author(s):  
Alberto Mills ◽  
Federico Gago
Keyword(s):  
De Novo ◽  
Elongation Factor ◽  
Missense Mutations ◽  
Developmentally Regulated ◽  
High Sequence Identity ◽  
80S Ribosome ◽  
Cellular Effects ◽  
Cellular Processes ◽  
Eukaryotic Elongation Factor ◽  
A Site

eEF1A1 and eEF1A2 are paralogous proteins whose presence in most normal eukaryotic cells is mutually exclusive and developmentally regulated. Often described in the scientific literature under the collective name eEF1A, which stands for eukaryotic elongation factor 1A, their best known activity (in a monomeric, GTP-bound conformation) is to bind aminoacyl-tRNAs and deliver them to the A-site of the 80S ribosome. However, both eEF1A1 and eEF1A2 are endowed with multitasking abilities (sometimes performed by homo- and heterodimers) and can be located in different subcellular compartments, from the plasma membrane to the nucleus. Given the high sequence identity of these two sister proteins and the large number of post-translational modifications they can undergo, we are often confronted with the dilemma of discerning which is the particular proteoform that is actually responsible for the ascribed biochemical or cellular effects. We argue in this review that acquiring this knowledge is essential to help clarify, in molecular and structural terms, the mechanistic involvement of these two ancestral and abundant G proteins in a variety of fundamental cellular processes other than translation elongation. Of particular importance for this special issue is the fact that several de novo heterozygous missense mutations in the human EEF1A2 gene are associated with a subset of rare but severe neurological syndromes and cardiomyopathies.

Aromatase and 11β-hydroxysteroid dehydrogenase 2 localisation in the testes of pigs from birth to puberty linked to changes of hormone pattern and testicular morphology

2008 ◽  
Vol 20 (4) ◽  
pp. 505 ◽  
Author(s):  
A. Wagner ◽  
R. Claus
Keyword(s):  
Blood Plasma ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Blood Collection ◽  
Testicular Development ◽  
Post Mortem ◽  
Cell Numbers ◽  
Testicular Morphology

Oestrogens and glucocorticoids are important for spermatogenesis and are regulated via aromatase for oestradiol synthesis and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD 2) as an inactivator of cortisol. In the present study postnatal changes of these two enzymes were monitored together with testicular development and hormone concentrations. Pigs were assigned to three periods: Weeks 0–5, Weeks 5–11 or Weeks 11–17. In Period 1, groups of four piglets were killed after each week. Blood plasma and testes were sampled immediately post mortem. For Periods 2 and 3, groups of six pigs were fitted with vein catheters for daily blood collection. Testes from all pigs were obtained after killing. Levels of testosterone, oestradiol, LH, FSH and cortisol were determined radioimmunologically. The 11β-HSD 2- and aromatase-expressing cells were stained immunocytochemically. All hormones were maximal 2 weeks after birth. A rise of LH, testosterone and oestradiol occurred again at Week 17. FSH and cortisol remained basal. Parallel to the first postnatal rise, the presence of aromatase and 11β-HSD 2 in Leydig cells increased, together with germ and Sertoli cell numbers. Expression was low from 3 to 5 weeks, was resumed after Week 5 and was maximal at Week 17. The amount of 11β-HSD 2 in germ cells was greatest at birth, decreased thereafter and was absent after Week 3.

Inhibitory Effect of Tributyltin on Expression of Steroidogenic Enzymes in Mouse Testis

2008 ◽  
Vol 27 (2) ◽  
pp. 175-182 ◽  
Author(s):  
Suel-Kee Kim ◽  
Jong-Hoon Kim ◽  
Jung Ho Han ◽  
Yong-Dal Yoon
Keyword(s):  
Germ Cells ◽  
Leydig Cells ◽  
Serum Testosterone ◽  
Hydroxysteroid Dehydrogenase ◽  
Reproductive Organs ◽  
Inhibitory Effect ◽  
Testicular Development ◽  
Steroidogenic Enzymes ◽  
Hormone Production ◽  
Induced Apoptosis

Tributyltin (TBT) is known to disrupt the development of reproductive organs, thereby reducing fertility. The aim of this study was to evaluate the acute toxicity of TBT on the testicular development and steroid hormone production. Immature (3-week-old) male mice were given a single administration of 25, 50, or 100 mg/kg of TBT by oral gavage. Lumen formation in seminiferous tubule was remarkably delayed, and the number of apoptotic germ cells found inside the tubules was increased in the TBT-exposed animals, whereas no apoptotic signal was observed in interstitial Leydig cells. Reduced serum testosterone concentration and down-regulated expressions of the mRNAs for cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase/C17–20 lyase (P45017α), 3β-hydroxysteroid-dehydrogenase (3β-HSD), and 17β-hydroxysteroid-dehydrogenase (17β-HSD) were also observed after TBT exposure. Altogether, these findings demonstrate that exposure to TBT is associated with induced apoptosis of testicular germ cells and inhibition of steroidogenesis by reduction in the expression of steroidogenic enzymes in interstitial Leydig cells. These adverse effects of TBT would cause serious defects in testicular development and function.

scholarly journals Intracellular Retention of the Corticotrophin-releasing Hormone (CRH) Precursor Within COS-7 Cells

1998 ◽  
Vol 46 (10) ◽  
pp. 1193-1197 ◽  
Author(s):  
Marcelo J. Perone ◽  
Simon Windeatt ◽  
Ewan Morrison ◽  
Andy Shering ◽  
Peter Tomasec ◽  
...  
Keyword(s):  
Amino Acid ◽  
Golgi Apparatus ◽  
Amino Acid Sequence ◽  
Protein Secretion ◽  
Intracellular Trafficking ◽  
Secretory Pathway ◽  
Intracellular Localization ◽  
Primary Amino Acid Sequence ◽  
Releasing Hormone ◽  
Primary Amino

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.

scholarly journals Developmentally regulated GTPases: structure, function and roles in disease

2021 ◽  
Author(s):  
Christian A. E. Westrip ◽  
Qinqin Zhuang ◽  
Charlotte Hall ◽  
Charlotte D. Eaton ◽  
Mathew L. Coleman
Keyword(s):  
Cell Proliferation ◽  
Structural Biology ◽  
Regulatory Protein ◽  
Growth Control ◽  
Developmentally Regulated ◽  
Binding Partner ◽  
Cellular Processes ◽  
Gtp Binding ◽  
Evolutionarily Conserved ◽  
Cell Growth Control

AbstractGTPases are a large superfamily of evolutionarily conserved proteins involved in a variety of fundamental cellular processes. The developmentally regulated GTP-binding protein (DRG) subfamily of GTPases consists of two highly conserved paralogs, DRG1 and DRG2, both of which have been implicated in the regulation of cell proliferation, translation and microtubules. Furthermore, DRG1 and 2 proteins both have a conserved binding partner, DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively, that prevents them from being degraded. Similar to DRGs, the DFRP proteins have also been studied in the context of cell growth control and translation. Despite these proteins having been implicated in several fundamental cellular processes they remain relatively poorly characterized, however. In this review, we provide an overview of the structural biology and biochemistry of DRG GTPases and discuss current understanding of DRGs and DFRPs in normal physiology, as well as their emerging roles in diseases such as cancer.

MT1-MMP in rat testicular development and the control of Sertoli cell proMMP-2 activation

2001 ◽  
Vol 114 (11) ◽  
pp. 2125-2134 ◽  
Author(s):  
Juliette Longin ◽  
Patricia Guillaumot ◽  
Marie-Agnès Chauvin ◽  
Anne-Marie Morera ◽  
Brigitte Le Magueresse-Battistoni
Keyword(s):  
Germ Cells ◽  
Follicle Stimulating Hormone ◽  
Developmental Period ◽  
Testicular Development ◽  
Hormone Regulation ◽  
Cell Lineages ◽  
Apical Compartment ◽  
Membrane Type ◽  
Stimulating Hormone

Metalloproteases (MMPs) are likely to be involved in the restructuring events occurring in the testis throughout development. We here demonstrate that membrane-type 1 (MT1)-MMP, a physiological activator of proMMP-2 under TIMP-2 control, is present within the testis together with MMP-2 and TIMP-2. In the prepubertal testis MT1-MMP immunoreactivity was uniformly distributed, whereas in the adult it was confined to the apical compartment of the tubules, where meiosis and spermiogenesis occur. We further showed that the two cell lineages (somatic and germinal) expressed MT1-MMP and TIMP-2, whereas MMP-2 was of somatic origin. To get a better picture into proMMP-2 activation, use was made of a model of cultured Sertoli cells treated with FSH or co-cultured with germ cells to mimic an immature or a mature developmental period, respectively. We found that follicle-stimulating hormone enhanced the expression of MMP-2 and TIMP-2 but not of MT1-MMP, and promoted the activation of proMMP-2. In co-cultures, a tremendous elevation and activation of MMP-2 was observed, which might relate to the processed MT1-MMP form solely detected in germ cells. That MMP-2 synthesis and activation are under local (germ cells) and hormonal (follicle-stimulating hormone) regulation emphasizes the importance of MMPs in testicular physiology.

Stem cell factor protects germ cells from apoptosis in vitro

2000 ◽  
Vol 113 (1) ◽  
pp. 161-168 ◽  
Author(s):  
W. Yan ◽  
J. Suominen ◽  
J. Toppari
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Primordial Germ Cells ◽  
Survival Function ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Dna Laddering ◽  
Survival Effect

Stem cell factor (SCF) plays an important role in migration, adhesion, proliferation, and survival of primordial germ cells and spermatogonia during testicular development. However, the function of SCF in the adult testis is poorly described. We have previously shown that, in the presence of SCF, there were more type A spermatogonia incorporating thymidine at stage XII of rat seminiferous tubules cultured in vitro than in the absence of SCF, implying that the increased DNA synthesis might result from enhanced survival of spermatogonia. To explore the potential pro-survival function of SCF during spermatogenesis, the seminiferous tubules from stage XII were cultured in the presence or absence of SCF (100 ng/ml) for 8, 24, 48, and 72 hours, respectively, and apoptosis was analyzed by DNA laddering and in situ 3′-end labeling (ISEL) staining. Surprisingly, not only spermatogonia, but also spermatocytes and spermatids, were protected from apoptosis in the presence of SCF. Apoptosis took place much later and was less severe in the SCF-treated tubules than in the controls. Based on previous studies showing that FSH prevents germ cells from undergoing apoptosis in vitro, and that SCF level is increased dramatically in response to FSH stimulation, we also tested if the pro-survival effect of FSH is mediated through SCF by using a function-blocking monoclonal antibody, ACK-2, to block SCF/c-kit interaction. After 24 hours of blockade, the protective effect of FSH was partially abolished, as manifested by DNA laddering and ISEL analyses. The present study demonstrates that SCF acts as an important survival factor for germ cells in the adult rat testis and FSH pro-survival effect on germ cells is mediated partially through the SCF/c-kit pathway.

The fusome organizes the microtubule network during oocyte differentiation in Drosophila

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4253-4264 ◽  
Author(s):  
N.C. Grieder ◽  
M. de Cuevas ◽  
A.C. Spradling
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Meiotic Prophase ◽  
Crucial Role ◽  
Skeletal Structure ◽  
Network Organization ◽  
Microtubule Network ◽  
Complex Process ◽  
Gamma Tubulin ◽  
Oocyte Differentiation

Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.

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