Apoptosis Process in Mouse Leydig Cells during Postnatal Development

2003 ◽  
Vol 9 (1) ◽  
pp. 68-73 ◽  
Author(s):  
Maria José Salles Faria ◽  
Zilá Luz Paulino Simões ◽  
Laurelucia Orive Lunardi ◽  
Klaus Hartfelder
Keyword(s):  
Dna Fragmentation ◽  
Leydig Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Seminiferous Tubules ◽  
Biphasic Pattern ◽  
Puberty Stages ◽  
Apoptosis Process ◽  
Neonatal Testis ◽  
Fetal Stage

The development of Leydig cells in mammals has been widely described as a biphasic pattern with two temporally mature Leydig cell populations, fetal stage followed by the adult generation beginning at puberty. In the present study, mouse Leydig cells were examined for apoptosis during postnatal testis development using electron microscopy and in situ DNA fragmentation by terminal deoxynucleotidyl transferase staining (TdT). Both the morphological study and the DNA fragmentation analysis showed that cellular death by apoptosis did not occur in Leydig cells during the neonatal, prepubertal, puberty, and adult periods. From these results, we suggest that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes. In contrast, in the prepubertal and puberty stages, fragmentation of apoptotic DNA was detected in germ cells present in some seminiferous tubules.

Biochemical and immunocytochemical analysis of a histone H1 variant from the mouse testis

1989 ◽  
Vol 94 (1) ◽  
pp. 61-71
Author(s):  
B.K. Rasheed ◽  
E.C. Whisenant ◽  
R.D. Ghai ◽  
V.E. Papaioannou ◽  
Y.M. Bhatnagar
Keyword(s):  
Leydig Cells ◽  
Random Distribution ◽  
Primary Spermatocyte ◽  
Cell Types ◽  
Mouse Testis ◽  
Seminiferous Tubules ◽  
Developmental Studies ◽  
Somatic Tissues ◽  
Early Primary

An H1 histone variant, H1a, has been isolated and purified from the mouse testis. Biochemical and amino acid analyses indicate its similarity with the rat testis H1a. Specific antibodies against the purified H1a have been generated in rabbits and used to study its tissue and species distribution using protein blotting procedures. We have also used the immunocytochemical technique to determine in situ distribution of H1a in spermatogenic cells and somatic tissues of the mouse. A non-random distribution of H1a has been noted in the nuclei of certain somatic cell types such as Sertoli cells, Leydig cells and neurons. By contrast, hepatocyte nuclei lacked detectable levels of H1a. In adult seminiferous tubules, the early primary spermatocyte nuclei displayed a greater level of immunoreactivity relative to other cell types. Developmental studies indicate its initial expression in the 7-day-old mouse testis concomitant with the appearance of intermediate and type B spermatogonia.

scholarly journals Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

1992 ◽  
Vol 40 (7) ◽  
pp. 903-908 ◽  
Author(s):  
T Suzuki ◽  
H Sasano ◽  
T Sawai ◽  
J I Mason ◽  
H Nagura
Keyword(s):  
In Situ Hybridization ◽  
Leydig Cells ◽  
Cellular Localization ◽  
Cdna Probe ◽  
Simultaneous Detection ◽  
Seminiferous Tubules ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Theca Interna

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.

scholarly journals Expression of prostaglandin D synthetase during development in the mouse testis

Reproduction ◽  
2001 ◽  
pp. 553-559 ◽  
Author(s):  
PJ Baker ◽  
PJ O'Shaughnessy
Keyword(s):  
Leydig Cells ◽  
Developmental Process ◽  
Major Change ◽  
Mouse Testis ◽  
Seminiferous Tubules ◽  
Interstitial Tissue ◽  
Rt Pcr ◽  
Subsequent Increase ◽  
Splice Isoforms ◽  
Neonatal Testis

Prostaglandin D synthetase is expressed relatively highly in the testis and reproductive tract of a number of species, including the mouse. In adult mouse testis, expression is confined largely to the Leydig cells and in this study changes in the expression and localization of prostaglandin D synthetase mRNA during testis development were examined. Initial studies using RT-PCR and isolated testicular compartments indicated that prostaglandin D synthetase expression in the neonatal testis was predominantly within the seminiferous tubules. In situ hybridization studies confirmed that prostaglandin D synthetase mRNA appears to be expressed only in the tubules of neonatal mouse testes and only in the interstitial tissue of the adult testis. TaqMan real-time PCR was used to quantify prostaglandin D synthetase mRNA content during development using an exogenous mRNA as a control standard. Expression per testis decreased after birth to < 10% at day 15 before recovering again by days 25-30. After day 30, expression per testis increased 40-fold during final development to adulthood. Studies using RT-PCR showed that early expression before day 15 was restricted to the tubular compartment, whereas the subsequent increase in expression after day 30 was restricted to the interstitial compartment. Database analysis showed that the 3' end of the prostaglandin D synthetase transcript was subject to alternate splicing. Both splice isoforms were shown by RT-PCR to be present throughout development and without a major change in expression pattern. These results indicate that expression of prostaglandin D synthetase mRNA shifts during development from the tubular compartment of the fetal or neonatal testis to the developing adult Leydig cells, with expression in the Leydig cells increasing markedly after puberty. These changes are similar to those observed for 17beta-hydroxysteroid dehydrogenase type III and may indicate that this developmental process is not uncommon in the testis.

Post-transcriptional block in oxytocin gene expression within the seminiferous tubules of the bovine testis

1994 ◽  
Vol 140 (1) ◽  
pp. 63-72 ◽  
Author(s):  
H Ungefroren ◽  
M Davidoff ◽  
R Ivell
Keyword(s):  
Gene Expression ◽  
Leydig Cells ◽  
Blot Hybridization ◽  
Seminiferous Tubules ◽  
Transcriptional Level ◽  
Gene Transcript ◽  
Northern Blot Hybridization ◽  
Rnase Protection ◽  
Low Level

Abstract Northern blot hybridization showed that bovine and sheep testis, unlike testes from other mammals, contain moderate levels of an apparently normal oxytocin gene transcript. In situ hybridization localized this mRNA to within the seminiferous tubules, possibly in the Sertoli cells. Conflicting with this result, immunohistochemistry showed that both oxytocin and the syngeneic neurophysin I epitopes are both clearly restricted to the Leydig cells, being expressed here at a low level. Since illegitimate transcription from spurious start sites can lead to a lack of translation product, the integrity of the major ruminant testicular transcripts of the oxytocin gene was checked using differential hybridization, RNase protection and multiple polymerase chain reaction assays. All tests showed the transcripts to have a normal, translatable composition and to be transcribed from the conventional 5' initiation site. Therefore, the block in oxytocin gene expression within the tubules is probably due to a lesion at the post-transcriptional level. The low level peptide expression in the Leydig cells can probably be attributed to the presence of functional transcripts in these cells, which are below the level of significant detection for the in situ hybridization assay. Journal of Endocrinology (1994) 140, 63–72

In situ end labeling of fragmented DNA in induced ovarian atresia

1994 ◽  
Vol 72 (11-12) ◽  
pp. 573-579 ◽  
Author(s):  
Katharina D'herde ◽  
Guido De Pestel ◽  
Frank Roels
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Granulosa Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Fragments ◽  
Dna Polymerase I ◽  
Late Event ◽  
Chromatin Configuration ◽  
Polymerase I

Apoptosis is studied in a model of induced follicular atresia in the ovary of Japanese quail (Coturnix coturnix japonica) by in situ end labeling of DNA fragments in granulosa cells using two different techniques (incorporation of labeled nucleotides by DNA polymerase I or terminal deoxynucleotidyl transferase). The most remarkable observation related to apoptosis in this model is the predominant cytoplasmic localization of labeled DNA fragments, while DNA fragmentation appears to be absent from compacted chromatin masses of apoptotic nuclei and apoptotic nuclear fragments. Unstained apoptotic bodies are present adjacent to stained ones, so that their detection rate on hematoxylin + eosin stained sections is better than on the in situ end-labeled sections. This suggests that DNA fragmentation is a late event or not obligatory in apoptotic granulosa cell death. In contrast to similar studies on atretic granulosa in mammalian models, the process of apoptosis is asynchronous in the granulosal epithelium, with a majority of nuclei with normal chromatin configuration remaining negative for DNA fragmentation. Finally it is shown that the techniques used are not specific for apoptosis, as DNA fragmentation in necrotic granulosa cells is detected as well.Key words: programmed cell death, apoptosis, in situ DNA end labeling, endonuclease, necrosis.

scholarly journals Localization of 17β-Hydroxysteroid Dehydrogenase/17-Ketosteroid Reductase Isoform Expression in the Developing Mouse Testis—Androstenedione Is the Major Androgen Secreted by Fetal/Neonatal Leydig Cells*

Endocrinology ◽  
2000 ◽  
Vol 141 (7) ◽  
pp. 2631-2637 ◽  
Author(s):  
P. J. O’Shaughnessy ◽  
P. J. Baker ◽  
M. Heikkilä ◽  
S. Vainio ◽  
A. P. McMahon
Keyword(s):  
Enzyme Activity ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Mouse Testis ◽  
Seminiferous Tubules ◽  
Interstitial Tissue ◽  
Androgen Synthesis ◽  
Isoform Expression ◽  
Neonatal Testis ◽  
Type 3

The final step in the biosynthesis of testosterone is reduction of androstenedione by the enzyme 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17βHSD/17KSR). In this study, we have examined expression of the four known reductive isoforms of 17βHSD/17KSR (types 1, 3, 5, and 7) in the developing mouse testis and have determined changes in the localization of isoform expression and testosterone secretion during development. Using RT-PCR isoforms 1, 3, and 7 were shown to be expressed in the seminiferous tubules of neonatal testis, whereas isoforms 3 and 7 were expressed in the interstitial tissue of the adult testis. The type 7 isoform is unlikely to be involved in androgen synthesis and further study concentrated on the type 3 isoform. Developmentally, isoform type 3 was expressed in the seminiferous tubules up to day 10, showed little or no expression on day 20 and from day 30 was confined to the interstitial tissue. In situ hybridization confirmed that the type 3 isoform was expressed only in the seminiferous tubules in fetal testes and in the interstitial tissue in adult testes. In accordance with the localization of enzyme messenger RNA expression 17-ketosteroid reductase enzyme activity was very low in isolated interstitial tissue from neonatal testes while interstitial tissue from adult testes showed high activity. Seminiferous tubules from both neonatal and adult testes showed high levels of enzyme activity. The major androgen secreted by the interstitial tissue of prepubertal animals was androstenedione up to day 20 while 5α-androstanediol and/or testosterone were the major androgens secreted from day 30 onwards. These results show that fetal Leydig cells do not express significant levels of a reductive isoform of 17βHSD/17KSR and that androstenedione is the major androgen secreted by these cells. Production of testosterone up until puberty is dependent upon 17βHSD/17KSR activity in the seminiferous tubules—a“ two cell” requirement for testosterone synthesis. Expression of the 17βHSD/17KSR type 3 isoform (the main reductive isoform in the testis) declines in the seminiferous tubules before puberty but then reappears in the developing adult Leydig cell population.

scholarly journals Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes

2016 ◽  
Vol 113 (10) ◽  
pp. 2666-2671 ◽  
Author(s):  
Xiaoheng Li ◽  
Zhao Wang ◽  
Zhenming Jiang ◽  
Jingjing Guo ◽  
Yuxi Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Lineage ◽  
Seminiferous Tubules ◽  
Paracrine Factors ◽  
Adult Testis ◽  
Adult Leydig Cells ◽  
Neonatal Testis

Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor β (TGF-β). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-β, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry.

Cellular changes in the hamster testicular interstitium with ageing and after exposure to short photoperiod

2016 ◽  
Vol 28 (6) ◽  
pp. 838 ◽  
Author(s):  
E. Beltrán-Frutos ◽  
V. Seco-Rovira ◽  
C. Ferrer ◽  
J. F. Madrid ◽  
F. J. Sáez ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Light And Electron Microscopy ◽  
Short Photoperiod ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Ultrastructural Changes ◽  
Synthetic Activity ◽  
Normal Morphology ◽  
Cellular Changes ◽  
Irreversible Nature

The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an ‘age group’ with three subgroups – young, adult and old animals – and a ‘regressed group’ with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.

scholarly journals Effect of Phitofert® on testicular function of vasectomized mature mice

2018 ◽  
Vol 11 (2) ◽  
pp. 9-16
Author(s):  
Muna Yousif ◽  
saad Al-Dujal ◽  
Jawad Arrak
Keyword(s):  
Dna Fragmentation ◽  
Adult Male ◽  
Leydig Cells ◽  
Treated Group ◽  
Reproductive Hormones ◽  
Current Data ◽  
Seminiferous Tubules ◽  
Control Group ◽  
Adult Mice ◽  
Serum Lh

This study was designed to evaluate the role of Phitofert® in the improvement of testes function and attenuating DNA fragmentation in vasectomized and healthy adult mice. Twenty four adult male breed such Albino mice were randomly and equally divided into four groups (G1, G2, G3 and G4). Mice were treated for 35days as follows: the G1 mice were non-vasectomized and given DW and served as controls, mice in G2 were non-vasectomized and given Phitofert® daily with a dose of 0.035mg/kg BW, mice in G3 were vasectomized without treatment while the mice in G4 were vasectomized and given same dose of Phitofert®. At the end of the experiment, fasting blood samples were collected by cardiac puncture for measuring LH and FSH hormones concentrations and section from testes were taken tomeasure the number of Leydig cells and diameter of semniferous tubules. The results of current data showed significant increase of serum LH and FSH concentrations in G2 healthy treated group as compared with control and other groups. Also, the treatment of G4 vasectomized mice with Phitofert® caused significant increase in serum concentration of the above hormones as compared with G3 vasectomized non-treated. The study showed that DNA fragmentation that resulted from the G2 healthy treated mice were lower than that obtained from the control group and other vasectomized mice (G3, G4). The diameters of the seminiferous tubules and the numbers of Leydig cells showed significant increase in healthy and vasectomized treated group (G1, G4) as compared with G3 vasectomized non-treated group. It was concluded that healthy and vasectomized treated of adult male mice with Phitofert® lead to clear improvement of level of reproductive hormones.

Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽  
2000 ◽  
pp. 325-335 ◽  
Author(s):  
A Calvo ◽  
LM Pastor ◽  
S Bonet ◽  
E Pinart ◽  
M Ventura
Keyword(s):  
Ricinus Communis ◽  
Lectin Histochemistry ◽  
Apical Part ◽  
Seminiferous Tubules ◽  
In Situ Characterization ◽  
Intense Staining ◽  
Terminal Galactose ◽  
Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

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