Functions of TAM RTKs in regulating spermatogenesis and male fertility in mice

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.

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Gas6 and the Tyro 3 receptor tyrosine kinase subfamily regulate the phagocytic function of Sertoli cells

Reproduction ◽

10.1530/rep-07-0287 ◽

2008 ◽

Vol 135 (1) ◽

pp. 77-87 ◽

Cited By ~ 54

Author(s):

Weipeng Xiong ◽

Yongmei Chen ◽

Huizhen Wang ◽

Haikun Wang ◽

Hui Wu ◽

...

Keyword(s):

Germ Cells ◽

Receptor Tyrosine Kinase ◽

Sertoli Cells ◽

Tyrosine Kinases ◽

Phagocytic Function ◽

Spermatogenic Cells ◽

Wild Type ◽

Triple Mutant ◽

Dramatic Decrease ◽

Phagocytic Ability

The apoptotic spermatogenic cells and residual bodies are phagocytosed and degraded by Sertoli cells during spermatogenesis. The mechanisms of this process are largely unknown. Here, we demonstrate that Gas6 and its receptors, the Tyro 3 subfamily of receptor tyrosine kinases (RTKs; Tyro 3, Axl, and Mer), regulate the phagocytic function of Sertoli cells. The phagocytic ability of Sertoli cells increased by five times in the presence of Gas6 in serum-free medium when compared with controls. The Sertoli cells lacking Mer showed a 35% reduction in phagocytosis of apoptotic spermatogenic cells when compared with wild-type (WT) controls, whereas the Sertoli cells lacking Tyro 3 or Axl exhibited phagocytic activity comparable with the controls. Notably, the Sertoli cells lacking all three members of the Tyro 3 RTK subfamily showed a dramatic decrease in phagocytic ability of 7.6-fold when compared with WT Sertoli cells. The deficiency in phagocytosis by the triple-mutant Sertoli cells was due to the deficit in binding of the Sertoli cells to apoptotic germ cells. These findings suggest that Mer is responsible for triggering phagocytosis of apoptotic spermatogenic cells by Sertoli cells and that Tyro 3, Axl, and Mer participate in recognizing and binding apoptotic germ cells by Sertoli cells in a redundant manner. Gas6 is a functional ligand of the Tyro 3 RTK subfamily in mediating phagocytic ability of Sertoli cells.

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Minireview: Transcriptional Regulation of Gonadal Development and Differentiation

Endocrinology ◽

10.1210/en.2004-1454 ◽

2005 ◽

Vol 146 (3) ◽

pp. 1035-1042 ◽

Cited By ~ 60

Author(s):

Susan Y. Park ◽

J. Larry Jameson

Keyword(s):

Germ Cells ◽

Sertoli Cells ◽

Leydig Cells ◽

Cell Types ◽

Gonadal Development ◽

Targeted Mutagenesis ◽

Seminiferous Tubules ◽

Molecular Pathways ◽

Theca Cells ◽

Main Cell

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.

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Fertility and spermatogenesis are altered in α1b-adrenergic receptor knockout male mice

Journal of Endocrinology ◽

10.1677/joe-07-0071 ◽

2007 ◽

Vol 195 (2) ◽

pp. 281-292 ◽

Cited By ~ 18

Author(s):

Sakina Mhaouty-Kodja ◽

Anne Lozach ◽

René Habert ◽

Magali Tanneux ◽

Céline Guigon ◽

...

Keyword(s):

Germ Cells ◽

Adrenergic Receptor ◽

Male Fertility ◽

Leydig Cells ◽

Critical Role ◽

Wild Type ◽

Cell Death Pathway ◽

Testicular Weight ◽

Circulating Levels

To examine whether norepinephrine, through activation of α1b-adrenergic receptor, regulates male fertility and testicular functions, we used α1b-adrenergic receptor knockout (α1b-AR-KO) mice. In the adult stage (3–8 months), 73% of the homozygous males were hypofertile with relatively preserved spermatogenesis. Of the remaining males, 27% exhibited a complete infertility with a drastic reduction in testicular weight and spermatogenesis defect with germ cells entering a cell death pathway at meiotic stage. In both phenotypes, circulating levels of testosterone were highly reduced (−55 and −81% in hypofertile and infertile males respectively versus wild-type males). Consequently, circulating levels of LH were significantly elevated in α1b-AR-KO infertile mice. When incubated in vitro, the whole testes from infertile KO mice released significantly lower levels of testosterone (−40%). This, together with the fact that the mean absolute volume of Leydig cells per testis was not changed, suggests a compromised steroidogenic capacity of Leydig cells in infertile animals. In addition, RNA in situ hybridization study indicated an apparent higher expression of inhibin α- and βB-subunits in Sertoli cells of infertile α1b-AR-KO mice. This was correlated with a higher intra-testicular content of inhibin B (+220% above wild-type mice). Using specific primers, mRNA encoding α1b-AR was localized in early spermatocytes of wild-type testes. Our results indicate, for the first time, that α 1b-AR signaling plays a critical role in the control of male fertility, spermatogenesis, and steroidogenic capacityof Leydig cells. It is thus hypothesized that the absence of α1b-AR alters either directly germ cells or indirectly Sertoli cell/Leydig cell communications in infertile α1b-AR-KO mice.

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

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab324 ◽

2015 ◽

Vol 27 (1) ◽

pp. 251

Author(s):

M. Nowak-Imialek ◽

N. Lachmann ◽

D. Herrmann ◽

F. Jacob ◽

H. Niemann

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Sertoli Cells ◽

Leydig Cells ◽

Egfp Expression ◽

Seminiferous Tubules ◽

Marker Genes ◽

Specific Expression ◽

Oct4 Expression ◽

Egfp Reporter

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.

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NR5A1 is required for functional maturation of Sertoli cells during postnatal development

Reproduction ◽

10.1530/rep-11-0365 ◽

2012 ◽

Vol 143 (5) ◽

pp. 663-672 ◽

Cited By ~ 19

Author(s):

Tomoko Kato ◽

Michiyo Esaki ◽

Ayami Matsuzawa ◽

Yayoi Ikeda

Keyword(s):

Postnatal Development ◽

Sertoli Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Leydig Cells ◽

Developmental Stages ◽

Cell Maturation ◽

Nuclear Antigen ◽

Seminiferous Tubules ◽

Orphan Nuclear Receptor

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.

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Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration

eLife ◽

10.7554/elife.15635 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 32

Author(s):

Francisco J Barrionuevo ◽

Alicia Hurtado ◽

Gwang-Jin Kim ◽

Francisca M Real ◽

Mohammed Bakkali ◽

...

Keyword(s):

Sertoli Cells ◽

Male Fertility ◽

Leydig Cells ◽

Structural Proteins ◽

Seminiferous Tubules ◽

Adult Testis ◽

Key Genes ◽

Male To Female ◽

Mutant Cells ◽

Testis Regression

The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8-/- mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.

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NF-κB-repressed Sirt3 mediates testicular cholesterol metabolism and cytoskeleton assembly via P450scc/SOD2 deacetylation during spermatogenesis

10.1101/2021.02.22.432399 ◽

2021 ◽

Author(s):

Mei Wang ◽

Ling Zeng ◽

Yao Xiong ◽

Xiao-fei Wang ◽

Lin Cheng ◽

...

Keyword(s):

Male Infertility ◽

Germ Cells ◽

Sertoli Cells ◽

Male Fertility ◽

Leydig Cells ◽

Nuclear Translocation ◽

Cholesterol Metabolism ◽

Male Subfertility ◽

Upstream Regulator ◽

Testicular Cells

AbstractTesticular homeostasis requires the balanced interplay between specific molecules in Sertoli cells, Leydig cells, germ cells. Loss of this coordination can lead to the disruption of spermatogenesis, even male infertility. By operating the upregulation and downregulation of Sirt3 in our male subfertility rats model and two testicular cells models, we indicated that Sirt3 overexpression and activator ameliorated cholesterol metabolism via P450scc deacetylation in Leydig cells, and cytoskeleton assembly via PDLIM1 with SOD2 deacetylation in Sertoli cells and elongating spermatids. In terms of the upstream regulator of Sirt3, the phosphorylation of NF-κB p65Ser536 stimulated the nuclear translocation of NF-κB subunits (p50, p65, RelB), which bound to TFBS1 and TFBS2 synchronously in the promoter of Sirt3, repressing Sirt3 transcription. This study demonstrates that NF-κB-repressed SIRT3 acts directly on cholesterol metabolism of Leydig cells and cytoskeleton assembly of Sertoli cells via P450scc/SOD2 deacetylation to regulate sperm differentiation, influencing spermatogenesis, even male fertility.Research organism: Rat, mouse

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Immunolocalization of Aromatase in Stallion Leydig Cells and Seminiferous Tubules

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100306 ◽

2003 ◽

Vol 51 (3) ◽

pp. 311-318 ◽

Cited By ~ 21

Author(s):

Herbert Sipahutar ◽

Pascal Sourdaine ◽

Safa Moslemi ◽

Bruno Plainfossé ◽

Gilles-Eric Séralini

Keyword(s):

Germ Cells ◽

Sertoli Cells ◽

Leydig Cells ◽

Staining Intensity ◽

Seminiferous Tubules ◽

Aromatase Activity ◽

Estrogen Synthesis ◽

Cytochrome P450 Aromatase ◽

Streptavidin Peroxidase ◽

First Time

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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Male Reproductive Function

Textbook of Endocrine Physiology ◽

10.1093/oso/9780199744121.003.0012 ◽

2011 ◽

Author(s):

William J. Kovacs

Keyword(s):

Tight Junctions ◽

Sertoli Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Leydig Cells ◽

Reproductive Function ◽

Rete Testis ◽

Seminiferous Tubules ◽

Male Reproductive Function ◽

Peritubular Tissue

The testes are the source of both germ cells and hormones essential for male reproductive function. The production of both sperm and steroid hormones is under complex feedback control by the hypothalamic-pituitary system. The testis consists of a network of tubules for the production and transport of sperm to the excretory ducts and a system of interstitial cells (called Leydig cells) that express the enzymes required for the synthesis of androgens. The spermatogenic or seminiferous tubules are lined by a columnar epithelium composed of the germ cells themselves as well as supporting Sertoli cells surrounded by peritubular tissue made up of collagen, elastic fibers, and myofibrillar cells. Tight junctions between Sertoli cells at a site between the spermatogonia and the primary spermatocyte form a diffusion barrier that divides the testis into two functional compartments, basal and adluminal. The basal compartment consists of the Leydig cells surrounding the tubule, the peritubular tissue, and the outer layer of the tubule containing the spermatogonia. The adluminal compartment consists of the inner two-thirds of the tubules containing primary spermatocytes and germ cells in more advanced stages of development. The base of the Sertoli cell is adjacent to the basement membrane of the spermatogenic tubule, with the inner portion of the cell engulfing the developing germ cells so that spermatogenesis actually takes place within a network of Sertoli cell cytoplasm. The mechanism by which spermatogonia pass through the tight junctions between Sertoli cells to begin spermatogenesis is unknown. The close proximity of the Leydig cell to the Sertoli cell with its embedded germ cells is thought to be critical for normal male reproductive function. The seminiferous tubules empty into a network of ducts termed the rete testis. Sperm are then transported into a single duct, the epididymis. Anatomically, the epididymis can be divided into the caput, the corpus, and the cauda regions. The caput epididymidis consists of 8 to 12 ductuli efferentes, which have a larger lumen tapering to a narrower diameter at the junction of the ductus epididymidis.

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Sex chimaerism, fertility and sex determination in the mouse

Development ◽

10.1242/dev.113.1.311 ◽

1991 ◽

Vol 113 (1) ◽

pp. 311-325 ◽

Cited By ~ 4

Author(s):

C.E. Patek ◽

J.B. Kerr ◽

R.G. Gosden ◽

K.W. Jones ◽

K. Hardy ◽

...

Keyword(s):

Sex Determination ◽

Sertoli Cells ◽

Leydig Cells ◽

Sex Chromosome ◽

Tunica Albuginea ◽

In Situ Hybridisation ◽

Ovarian Surface Epithelium ◽

Surface Epithelium ◽

Seminiferous Tubules ◽

Coelomic Epithelium

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

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