324 TESTIS-SPECIFIC EXPRESSION OF Oct4-EGFP TRANSGENE IN PIG

2015 ◽  
Vol 27 (1) ◽  
pp. 251
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann

Oct4 is a transcription factor essential for establishment and maintenance of pluripotency in mammalian stem cells. Oct4 expression was found in early embryos and germ cells throughout fetal development. In male mice, Oct4 expression is found in mitotically arrested prospermatogonia until birth. After onset of spermatogenesis, expression is maintained in type A spermatogonia, but is downregulated in type B spermatogonia and in spermatocytes (Pesce et al. 1998 Mech. Dev). Previously, we successfully generated Oct4-EGFP reporter pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev.). This animal model is unique because it allows in vivo and in vitro visualisation of Oct4-positive cells. Germ line specific Oct4-EGFP expression was analysed in testis isolated from young (<1 week) and adult (>7 months) pigs. Squash preparation of testicular tissue isolated from adult transgenic boars revealed high amounts of EGFP-positive cells compared to young piglets. We confirmed Oct4 and EGFP expression in the testis from young and adult transgenic animals using Northern blot analysis. Specific expression of Oct4 and EGFP in testis could be observed in blots as a single band of 1.5 kb. As a loading control, the blot was rehybridized with a β-actin probe. Mammalian testes contain different cell types, including germ cells, Sertoli cells, Leydig cells, and peritubular cells. To define the cellular origin of EGFP-expressing cells, we isolated these cells from adult transgenic testis using fluorescence-activated cell sorting (FACS)-based techniques. Analysis of isolated EGFP positive cells with qRT-PCR demonstrated the presence of marker genes specific for undifferentiated (Oct4, UTF1, FGFR3, PGP 9.5, THY-1, SALL4, and GFRα1) and differentiated (BOLL and PRM2) germ cells. Markers specific for Sertoli cells (vimentin) and Leydig cells (LHCGR) were not observed. To verify the localization of EGFP-positive cells in seminiferous tubules, we performed immunohistochemical detection of GFP in adult pig testis. Unlike the Oct4-EGFP reporter mouse model, GFP protein was not found in spermatogonia attached to the basement membrane of seminiferous tubules, but instead were found in differentiated germ cells, including spermatocytes and spermatids. These results show that the Oct4-EGFP expression in testis differs between mouse and porcine Oct4-EGFP transgenic models. To verify that the EGFP expression driven by the mouse Oct4 promoter in porcine testis reflects the endogenous Oct4 expression profile, Western blot and histochemical analyses are currently underway.

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1035-1042 ◽  
Author(s):  
Susan Y. Park ◽  
J. Larry Jameson

The embryonic gonad is undifferentiated in males and females until a critical stage when the sex chromosomes dictate its development as a testis or ovary. This binary developmental process provides a unique opportunity to delineate the molecular pathways that lead to distinctly different tissues. The testis comprises three main cell types: Sertoli cells, Leydig cells, and germ cells. The Sertoli cells and germ cells reside in seminiferous tubules where spermatogenesis occurs. The Leydig cells populate the interstitial compartment and produce testosterone. The ovary also comprises three main cell types: granulosa cells, theca cells, and oocytes. The oocytes are surrounded by granulosa and theca cells in follicles that grow and differentiate during characteristic reproductive cycles. In this review, we summarize the molecular pathways that regulate the distinct differentiation of these cell types in the developing testis and ovary. In particular, we focus on the transcription factors that initiate these cascades. Although most of the early insights into the sex determination pathway were based on human mutations, targeted mutagenesis in mouse models has revealed key roles for genes not anticipated to regulate gonadal development. Defining these molecular pathways provides the foundation for understanding this critical developmental event and provides new insight into the causes of gonadal dysgenesis.


Development ◽  
1994 ◽  
Vol 120 (7) ◽  
pp. 1759-1766 ◽  
Author(s):  
K. Yomogida ◽  
H. Ohtani ◽  
H. Harigae ◽  
E. Ito ◽  
Y. Nishimune ◽  
...  

GATA-1 is an essential factor for the transcriptional activation of erythroid-specific genes, and is also abundantly expressed in a discrete subset of cells bordering the seminiferous epithelium in tubules of the murine testis. In examining normal and germ-line defective mutant mice, we show here that GATA-1 is expressed only in the Sertoli cell lineage in mouse testis. GATA-1 expression in Sertoli cells is induced concomitantly with the first wave of spermatogenesis, and GATA-1-positive cells are uniformly distributed among all tubules during prepubertal testis development. However, the number of GATA-1-positive cells declines thereafter and were found only in the peripheral zone of seminiferous tubules in stages VII, VIII and IX of spermatogenesis in the adult mouse testis. In contrast, virtually every Sertoli cell in mutant W/Wv, jsd/jsd or cryptorchid mice (all of which lack significant numbers of germ cells) expresses GATA-1, thus showing that the expression of this transcription factor is negatively controlled by the maturing germ cells. These observations suggest that transcription factor GATA-1 is a developmental stage- and spermatogenic cycle-specific regulator of gene expression in Sertoli cells.


2014 ◽  
Vol 26 (1) ◽  
pp. 210
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.


1962 ◽  
Vol 13 (3) ◽  
pp. 487 ◽  
Author(s):  
CS Sapsford

In the ram, as in other mammals, the sex cords are made up of two types of cell: indifferent cells (derivatives of the coelomic epithelium) and primordial germ cells. In the cords, each type pursues a separate and independent line of development to become respectively the Sertoli cells and the stem cells (type A spermatogonia) of the adult testis. The principal changes taking place in the primordial germ cells (gonocytes) are a reduction in the size and number of the Feulgen-positive particles in the nuclei, the appearance and subsequent fusion of the nucleoli, and, finally, an increase in the size of the nuclei. While these changes are taking place, the cytoplasm increases in volume and inclusions become more numerous. Cells which have undergone all these transformations have been called prospermatogonia. The cells of the germ line are at first more centrally placed in the sex cords than the indifferent cells. Just before spermatogenesis begins, they migrate to the basement membrane of the seminiferous tubules. All germ cells in tubules in which spermatogenesis has been initiated are seen as prospermatogonia. These cells become flattened against the basement membrane, and their nuclei become more oval in shape. They thus become identical with the stem cells of the adult. Little change is evident in the nuclei of the indifferent cells until puberty. Feulgen-positive material is found in the form of coarse granules at earlier stages of development. At puberty, these granules become dispersed to give a much more homogeneous nucleus. Concurrently, nuclei increase in size, and single or double true nucleoli can be identified. During development, increases in cytoplasmic volume take place. Although cell boundaries between indifferent cells cannot be seen in fixed material, phase contrast observations of fresh material have demonstrated that some forms exist as mononucleate units. It could not be determined whether the same was true in the case of Sertoli cells. No striking change in the relative numbers of glandular interstitial cells could be observed at different stages of development.


Reproduction ◽  
2009 ◽  
Vol 138 (4) ◽  
pp. 655-666 ◽  
Author(s):  
Yongmei Chen ◽  
Huizhen Wang ◽  
Nan Qi ◽  
Hui Wu ◽  
Weipeng Xiong ◽  
...  

Mice lacking TYRO3, AXL and MER (TAM) receptor tyrosine kinases (RTKs) are male sterile. The mechanism of TAM RTKs in regulating male fertility remains unknown. In this study, we analyzed in more detail the testicular phenotype of TAM triple mutant (TAM−/−) mice with an effort to understand the mechanism. We demonstrate that the three TAM RTKs cooperatively regulate male fertility, and MER appears to be more important than AXL and TYRO3. TAM−/− testes showed a progressive loss of germ cells from elongated spermatids to spermatogonia. Young adult TAM−/− mice exhibited oligo-astheno-teratozoospermia and various morphological malformations of sperm cells. As the mice aged, the germ cells were eventually depleted from the seminiferous tubules. Furthermore, we found that TAM−/− Sertoli cells have an impaired phagocytic activity and a large number of differentially expressed genes compared to wild-type controls. By contrast, the function of Leydig cells was not apparently affected by the mutation of TAM RTKs. Therefore, we conclude that the suboptimal function of Sertoli cells leads to the impaired spermatogenesis in TAM−/− mice. The results provide novel insight into the mechanism of TAM RTKs in regulating male fertility.


2008 ◽  
Vol 20 (1) ◽  
pp. 223
Author(s):  
J. Luo ◽  
S. Megee ◽  
I. Dobrinski

During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells. This work was supported by 1R01 RR 17359-05.


Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 663-672 ◽  
Author(s):  
Tomoko Kato ◽  
Michiyo Esaki ◽  
Ayami Matsuzawa ◽  
Yayoi Ikeda

The orphan nuclear receptor steroidogenic factor 1 (NR5A1 (SF-1)) is expressed in both Sertoli and Leydig cells in the testes. This study investigates the postnatal development of the testes of a gonad-specific Nr5a1 knockout (KO) mouse, in which Nr5a1 was specifically inactivated. The KO testes appeared histologically normal from postnatal day 0 (P0) until P7. However, disorganized germ cells, vacuoles, and giant cells appeared by P14 in the seminiferous tubules of KO but not control mice. Expression of NR5A1 and various factors was examined by immunohistochemistry (IHC). The number of NR5A1-positive Sertoli cells in the KO testes was lower compared with controls at all the developmental stages and decreased to nearly undetectable levels by P21. IHC for anti-Müllerian hormone and p27, immature and mature Sertoli cell markers, respectively, indicated a delay in Sertoli cell maturation in the KO testes. The number of Sertoli cell-expressing factors involved in Sertoli cell differentiation including WT1, SOX9, GATA4, and androgen receptor were lower in the KO testes compared with controls. Furthermore, fewer proliferating cell nuclear antigen-positive proliferative germ cells were observed, and the number of TUNEL-labeled cells was significantly higher in the KO testes compared with controls at P14 and P21, indicating impaired spermatogenesis. IHC for CYP11A1 (SCC) indicated the presence of steroidogenic Leydig cells in the interstitium of the KO testes at all stages examined. These results suggest that NR5A1 is essential for Sertoli cell maturation and therefore spermatogenesis, during postnatal testis development.


2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 373-373
Author(s):  
Tatyana Kotova ◽  
Anastasia N Vetokh ◽  
Ludmila A Volkova ◽  
Natalia Volkova ◽  
Natalia A Zinovieva

Abstract The use of testicular stem cells (spermatogonia) is of most interest for obtaining individuals with predetermined traits and genome genetic modification and for conservation of poultry gene pool. A significant population of mature donor germ cells (sperm) is formed upon successful spermatogonia cells transplantation into the testes of male recipients. Obtained sperm can be used to produce offspring with the desired traits. A key step in this technology is the removal of own spermatogenic cells (inhibition of spermatogenesis) in male recipients. The aim of research was to develop and optimize methodological approaches to inhibit the spermatogenesis in quail using busulfan. This drug was injected directly into the testes parenchyma of mature males by multiple injection at the concentration from 20 to 100 mg per 1kg of body weight (n = 25). Histological preparations of testes from the experimental quails were obtained to study composition of spermatogenic cells in the seminiferous tubules after busulfan administration. The male peers who were not injected with busulfan were used as a control. Experimental quails showed a decrease in the number of spermatogenic cells in the seminiferous tubules 32, 75, 111, 119 and 118 times compared with the control when using busulfan in concentrations 20, 40, 60, 80 and 100 mg/kg of weight, respectively (P &lt; 0.001). The cells composition in the seminiferous tubules from experimental quails was represented mainly by Sertoli cells and spermatogonia. After busulfan introduction at the concentrations 20, 40, 60, 80 and 100 mg/kg, the percentage of spermatogonia was 55±5 %, 24±4 %, 6±2 %, 5±2 % and 4±1 %, respectively. The use of busulfan at the concentration of 80–100 mg/kg led to high mortality of quails. Thus, it was found that the optimal busulfan concentration for elimination of quail spermatogenic cells was 60 mg/kg. Supported by RFBR within Project №18-29-07079.


2021 ◽  
Vol 52 (6) ◽  
pp. 370-378
Author(s):  
A. Yu. Kulibin ◽  
E. A. Malolina

Abstract The rete testis connects seminiferous tubules in which germ cells develop to the efferent ducts and the epididymis, where gametes mature and gain mobility. Several recent studies have thoroughly explored the morphogenesis of this structure in mice during embryonic and postnatal periods. A part of the rete testis has been shown to derive from the precursors of gonad somatic cells before sex determination. The other part forms from embryonal Sertoli cells of testis cords adjacent to the mesonephros. The transformation of Sertoli cells into rete testis cells is apparently not limited to the embryonic stage of development and continues during postnatal testis development. Recently, it was found that the rete testis participates in the formation and maintenance of specialized Sertoli cells in terminal segments of seminiferous tubules, transitional zones. Current views suggest that the transitional zones of the seminiferous tubules may represent a niche for spermatogonial stem cells, the site of the prolonged proliferation of Sertoli cells in the pubertal and postpubertal periods of testis development, and also could be a generator of spermatogenic waves. To sum up, the rete testis transports gametes from the testis to the epididymis, maintains pressure within seminiferous tubules, regulates the composition of the testicular fluid, and impacts the spermatogenic process itself.


2003 ◽  
Vol 51 (3) ◽  
pp. 311-318 ◽  
Author(s):  
Herbert Sipahutar ◽  
Pascal Sourdaine ◽  
Safa Moslemi ◽  
Bruno Plainfossé ◽  
Gilles-Eric Séralini

High levels of plasma estrogens constitute an endocrine peculiarity of the adult stallion. This is mostly due to testicular cytochrome P450 aromatase, the only irreversible enzyme responsible for the bioconversion of androgens into estrogens. To identify more precisely the testicular aromatase synthesis sites in the stallion, testes from nine horses (2–5 years) were obtained during winter or spring. Paraplast-embedded sections were processed using rabbit anti-equine aromatase, followed by biotinylated goat anti-rabbit antibodies, and amplified with a streptavidin-peroxidase complex. Immunore-activity was detected with diaminobenzidine. Immunofluorescence detection, using fluoroisothiocyanate-conjugated goat anti-rabbit antibodies, was also applied. Specific aromatase immunoreactivity was observed intensely in Leydig cells but also for the first time, to a lesser extent, in the cytoplasm surrounding germ cells at the junction with Sertoli cells. Interestingly, the immunoreactivity in Sertoli cells appears to vary with the spermatogenic stages in the basal compartment (with spermatogonia) as well as in the adluminal one (with spermatids). Relative staining intensity in Leydig and Sertoli cells and testicular microsomal aromatase activity increased with age. The present study in stallions indicates that in addition to Leydig cells, Sertoli cells also appear to participate in estrogen synthesis, and this could play a paracrine role in the regulation of spermatogenesis.


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