An exploration of the role of Sertoli cells on fetal testis development using cell ablation strategy.

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh-Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α-smooth muscle actin-labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3ß-HSD double-positive fetal LCs could be observed in Amh-Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3ß-HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs' central role in fetal testis development.

Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

As the major somatic cell type, Sertoli cells undergo active proliferation and play essential roles to establish testis cord at fetal stage. They also function to maintain germ cell development throughout the life of testicular development. However, the significance of Sertoli cell number for testis cord development and gonocyte fate is still unclear. Nuclear protein ataxia-telangiectasia (NPAT, also known as p220), a substrate of cyclin E/cyclin-dependent kinase 2, is well known as a regulator of cell proliferation through regulating histone expression. To study the role of NPAT during Sertoli cell development, we generated a mouse strain carrying conditional floxed Npat alleles, when crossing with anti-Müllerian hormone-cre, leading to the specific deletion of Npat in Sertoli cells. Npat disruption in Sertoli cells inhibited the programmed proliferation of fetal Sertoli cells resulting in disruption of developing testis cords, and subsequent postnatal mutant testes were severely hypoplastic. Germ cells, which are presumed to be in quiescent status during perinatal stage, exited G0 phase arrest and re-enter mitotic cell cycle prematurely. Of particular note, some germ cells possessed the meiotic signal in Npat-deficient testes. Our data thus indicates that the function of Npat-dependent Sertoli cells is essential at multiple steps in testis development, and this study also identifies Sertoli cells as a major regulator of germ cell development, which are required to maintain a local growth niche to repress premature mitosis and meiosis of gonocytes.-Jiang, X., Yin, S., Fan, S., Bao, J., Jiao, Y., Ali, A., Iqbal, F., Xu, J., Zhang, Y., Shi, Q. Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

Study of Tnp1, Tekt1, and Plzf Genes Expression During an in vitro Three-Dimensional Neonatal Male Mice Testis Culture

Background: In vitro spermatogenesis has a long research history beginning in the early 20th century. This organ culture method was therefore abandoned, and alternative cell culture methods were chosen by many researchers. Here, whether Tnp1, Tekt1, and Plzf, which play a crucial role in spermatogenesis, can be expressed during testis organ culture was assessed. Methods: Testes of 10 mouse pups were first removed, and the testis tissue was then separated into smaller pieces of seminiferous tubules. The size of the pieces was arbitrary; approximately 1 mg in weight or 1 mm3 in size when compacted. Afterwards, the testis tissue fragments (1­3) were transferred to the hexahedrons, incubated in a culture incubator and cultured for 12 weeks. Histological assessment and molecular evaluation were carried out at the end of the study. Results: The results showed that the expression of Tekt1 as a mitotic gene in mouse pups decreased significantly (p ≤ 0.05) in comparison to adult mouse testis. Meanwhile, the expression of Tnp1 as a meiotic gene increased significantly (p ≤ 0.05) as compared to neonate mouse testis at the beginning of the culture. The expression of Plzf showed no significant difference during the 12 weeks of culture (p ≥ 0.05). Based on histological study, different types of spermatocytes and post-meiotic stages of germ cells could not be detected. Conclusion: This kind of three-dimensional culture can induce expression of post-meiotic gene, Tnp1, but only at the molecular level and not beyond meiosis.

Biology of the Sertoli Cell in the Fetal, Pubertal, and Adult Mammalian Testis.

A healthy man typically produces between 50 × 10(6) and 200 × 10(6) spermatozoa per day by spermatogenesis; in the absence of Sertoli cells in the male gonad, this individual would be infertile. In the adult testis, Sertoli cells are sustentacular cells that support germ cell development by secreting proteins and other important biomolecules that are essential for germ cell survival and maturation, establishing the blood-testis barrier, and facilitating spermatozoa detachment at spermiation. In the fetal testis, on the other hand, pre-Sertoli cells form the testis cords, the future seminiferous tubules. However, the role of pre-Sertoli cells in this process is much less clear than the function of Sertoli cells in the adult testis. Within this framework, we provide an overview of the biology of the fetal, pubertal, and adult Sertoli cell, highlighting relevant cell biology studies that have expanded our understanding of mammalian spermatogenesis.

Testis Cord Maintenance in Mouse Embryos: Genes and Signaling.

Testis cords, embryonic precursors of the seminiferous tubules, are fundamental for testis structure and function. Delay or disruption of testis cord formation could result in gonadal dysgenesis. Although mechanisms regulating testis cord formation during sex determination have been well-studied, the genes and signaling pathways involving in testis cord maintenance after the cords have formed are not well characterized. It is now clear that the maintenance of cord structure is an active process. In this review, we summarize the recent findings regarding the regulation of testis cord integrity by a series of Sertoli cell transcription factors, including the WT1-SOX8/SOX9-beta-CATENIN-DHH network, GPR56, STIM1, and NR0B1 (also known as DAX1). In particularly, we emphasize the underappreciated role of peritubular myoid cells in testis cord maintenance and their cooperation with Sertoli cells. The regulation of the size, shape, and number of testis cords by Sertoli cell proliferation (e.g., SMAD4, GATA4, and TGF-beta signaling), Leydig cell products (e.g., ACTIVIN A), vascular development (a lesson learned from PDGF signaling), and available gonad space (as observed in Ift144 mutant mice) is also addressed. Further efforts and new genetic models are needed to unveil the gene networks and underlying mechanisms regulating testis cord integrity and morphology after sex determination.

The Type 3 Deiodinase Is a Critical Determinant of Appropriate Thyroid Hormone Action in the Developing Testis.

Timely and appropriate levels of thyroid hormone (TH) signaling are necessary to ensure normal developmental outcomes in many tissues. Studies using pharmacological models of altered TH status have revealed an influence of these hormones on testis development and size, but little is known about the role of endogenous determinants of TH action in the developing male gonads. Using a genetic approach, we demonstrate that the type 3 deiodinase (D3), which inactivates TH and protects developing tissues from undue TH action, is a key factor. D3 is highly expressed in the developing testis, and D3-deficient (D3KO) mice exhibit thyrotoxicosis and cell proliferation arrest in the neonatal testis, resulting in an approximately 75% reduction in testis size. This is accompanied by larger seminiferous tubules, impaired spermatogenesis, and a hormonal profile indicative of primary hypogonadism. A deficiency in the TH receptor-α fully normalizes testis size and adult testis gene expression in D3KO mice, indicating that the effects of D3 deficiency are mediated through this type of receptor. Similarly, genetic deficiencies in the D2 or in the monocarboxylate transporter 8 partially rescue the abnormalities in testis size and gonadal axis gene expression featured in the D3KO mice. Our study highlights the testis as an important tissue in which determinants of TH action coordinately converge to ensure normal development and identifies D3 as a critical factor in testis development and in testicular protection from thyrotoxicosis.

Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development.

A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head.

Androgen Receptor Coactivator ARID4B Is Required for the Function of Sertoli Cells in Spermatogenesis.

Defects in spermatogenesis, a process that produces spermatozoa inside seminiferous tubules of the testis, result in male infertility. Spermatogenic progression is highly dependent on a microenvironment provided by Sertoli cells, the only somatic cells and epithelium of seminiferous tubules. However, genes that regulate such an important activity of Sertoli cells are poorly understood. Here, we found that AT-rich interactive domain 4B (ARID4B), is essential for the function of Sertoli cells to regulate spermatogenesis. Specifically, we generated Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, and showed that the Arid4bSCKO male mice were completely infertile with impaired testis development and significantly reduced testis size. Importantly, severe structural defects accompanied by loss of germ cells and Sertoli cell-only phenotype were found in many seminiferous tubules of the Arid4bSCKO testes. In addition, maturation of Sertoli cells was significantly delayed in the Arid4bSCKO mice, associated with delayed onset of spermatogenesis. Spermatogenic progression was also defective, showing an arrest at the round spermatid stage in the Arid4bSCKO testes. Interestingly, we showed that ARID4B functions as a "coactivator" of androgen receptor and is required for optimal transcriptional activation of reproductive homeobox 5, an androgen receptor target gene specifically expressed in Sertoli cells and critical for spermatogenesis. Together, our study identified ARID4B to be a key regulator of Sertoli cell function important for male germ cell development.

Steroidogenic factor 1 differentially regulates fetal and adult leydig cell development in male mice.

The nuclear receptor steroidogenic factor 1 (SF-1, AD4BP, NR5A1) is a key regulator of the endocrine axes and is essential for adrenal and gonad development. Partial rescue of Nr5a1(-/-) mice with an SF-1-expressing transgene caused a hypomorphic phenotype that revealed its roles in Leydig cell development. In contrast to controls, all male rescue mice (Nr5a1(-/-);tg(+/0)) showed varying signs of androgen deficiency, including spermatogenic arrest, cryptorchidism, and poor virilization. Expression of various Leydig cell markers measured by immunohistochemistry, Western blot analysis, and RT-PCR indicated fetal and adult Leydig cell development were differentially impaired. Whereas fetal Leydig cell development was delayed in Nr5a1(-/-);tg(+/0) embryos, it recovered to control levels by birth. In contrast, Sult1e1, Vcam1, and Hsd3b6 transcript levels in adult rescue testes indicated complete blockage in adult Leydig cell development. In addition, between Postnatal Days 8 and 12, peritubular cells expressing PTCH1, SF-1, and CYP11A1 were observed in control testes but not in rescue testes, indicating SF-1 is needed for either survival or differentiation of adult Leydig cell progenitors. Cultured prepubertal rat peritubular cells also expressed SF-1 and PTCH1, but Cyp11a1 was expressed only after treatment with cAMP and retinoic acid. Together, data show SF-1 is needed for proper development of fetal and adult Leydig cells but with distinct primary functions; in fetal Leydig cells, it regulates differentiation, whereas in adult Leydig cells it regulates progenitor cell formation and/or survival.

Phthalate esters affect maturation and function of primate testis tissue ectopically grafted in mice.

Di-n-Butyl (DBP) and Di-(2-EthylHexyl) (DEHP) phthalates can leach from daily-use products resulting in environmental exposure. In male rodents, phthalate exposure results in reproductive effects. To evaluate effects on the immature primate testis, testis fragments from 6-month-old rhesus macaques were grafted subcutaneously to immune-deficient mice, which were exposed to 0, 10, or 500 mg/kg of DBP or DEHP for 14 weeks or 28 weeks (DBP only). DBP exposure reduced the expression of key steroidogenic genes, indicating that Leydig cell function was compromised. Exposure to 500 mg/kg impaired tubule formation and germ cell differentiation and reduced numbers of spermatogonia. Exposure to 10 mg/kg did not affect development, but reduced Sertoli cell number and resulted in increased expression of inhibin B. Exposure to DEHP for 14 week also affected steroidogenic genes expression. Therefore, long-term exposure to phthalate esters affected development and function of the primate testis in a time and dosage dependent manner.

ErbB4, a receptor tyrosine kinase, coordinates organization of the seminiferous tubules in the developing testis.

Although close to every fifth couple nowadays has difficulty conceiving, the molecular mechanisms behind the decline in human reproduction remain poorly understood. We report here that the receptor tyrosine kinase Erbb4 is a candidate causal gene, because it is expressed in a sexually dimorphic manner and is abundant in the developing and adult testes in the mouse. Sertoli cell-specific Erbb4-knockout mice have a compromised 3-dimensional organization of the testicular seminiferous tubules that affects their fertility. More specifically, adhesion defects are observed in the absence of Erbb4, which are characterized by changes in the expression of laminin-1, N-cadherin, claudin-3, and certain cell-cell junction components between the Sertoli and germ cells. Interestingly, Erbb4 knockout also had an effect on the Leydig cells, which suggests a paracrine influence of Sertoli cells expressing ErbB4. Many of the defects observed in Erbb4-knockout mice are rescued in targeted ERBB4 gain-of-function mice, pointing to a coordination role for ErbB4 in the developing testis. Thus, the ErbB4 receptor tyrosine kinase promotes seminiferous tubule development by controlling Sertoli cell and germ cell adhesion.

Study of spermatogenesis fetal testis exposed noise stress during and after natal period in rat.

Noise stress is dangerous natural contaminant that produces harmful physiological, psychological and morphological outcomes to the body. So this study was conducted in order to investigate the effects of noise stress on the parenchyma of testis. Healthy mature females rats (n = 20) were mated with the mature male rats and then randomly allocated equally either to experimental or control groups. Experimental group has given daily noise stress up to birth their child. In the second step, the child's pregnant rats of experimental group were distributed to three subgroups as follow: group I (without exposure to noise stress), group II (exposure to noise for 8 weeks) and group III (exposure to noise for 14 weeks) for morphometric analysis of their child's testicles by sacrificing of them at weeks 14. In general, the testes of non-exposed group were grown larger than ones in the noise exposed groups. Moreover, the testes of the experimental group 1 were larger than the other experimental groups. Indeed, the rate of atrophic seminiferous tubules and jumbled appearance of the interstitial space were more observed in the noise stress exposed group than non-exposed ones. In addition, seminiferous tubules analysis revealed that the characteristics of interstitial space cells and epithelial germinative cells of the seminiferous tubules in the control group were better than the noise exposed groups. It seems that the noise stress has negative influences on the fertility of male based on enhancing of the apoptotic process induced by pathogenesis stress and suppressing the kinetics spermatogenesis.

Morphometry of seminiferous tubules of human testes in different age groups in Bangladeshi cadavers.

This cross sectional descriptive study was done to observe the mean diameter of seminiferous tubules and to determine its differences between different age groups in Bangladeshi male. Thirty human testes of different age groups were collected by purposive sampling technique. Among them 22 specimens were collected from cadavers during routine postmortem examination and 8 specimens were from dead fetuses from Gynaecology & Obstetrics Department. The specimens were grouped into three categories Group A (28 to 42 weeks of gestational age), Group B (Up to 14 years) and Group C (15 to 70 years). The mean diameter of seminiferous tubules was measured and significant differences of the dimensions between different age groups were determined. The mean±SD diameter was 85.37±15.51 µm in Group A, 144.04±63.34 µm in Group B and 227.92±22.47 µm in Group C. Statistically, differences between age groups were calculated by using Unpaired Students 't' test. The present study revealed that the diameter increased with age and mean differences were statistically significant between Groups A&C, B&C and A&B.

Seminiferous cord formation is regulated by hedgehog signaling in the marsupial.

The signaling molecule DHH, secreted by Sertoli cells, has essential regulatory functions in testicular differentiation. DHH is required for the differentiation of peritubular myoid cells that line the seminiferous cords and steroidogenic Leydig cells. The testicular cords in Dhh-null male mice lack a basal lamina and develop abnormally. To date, the DHH-signaling pathway has never been examined outside of any eutherian mammals. This study examined the effects of inhibition of DHH signaling in a marsupial mammal, the tammar wallaby, by culturing gonads in vitro in the presence of the hedgehog-signaling inhibitors cyclopamine and forskolin. Disruption of hedgehog signaling in the tammar testes caused highly disorganized cord formation. SOX9 protein remained strongly expressed in Sertoli cells, laminin distribution was highly fragmented, and germ cells were distributed around the cortical regions of treated testes in an ovarianlike morphology. This suggests that hedgehog signaling regulates cord formation in the tammar wallaby testis as it does in eutherian mammals. These data demonstrate that the hedgehog pathway has been highly conserved in mammals for at least 160 million years.

Maternal protein restriction in pregnancy and/or lactation affects seminiferous tubule organization in male rat offspring.

Maternal protein restriction (MPR) during pregnancy impaired the reproduction of male offspring. We investigated, during the first wave of spermatogenesis, whether MPR exerts deleterious effects on germ cell proliferation and differentiation, as well as androgen receptor (AR) protein expression, which was used as a marker for Sertoli cell (SC) maturation. At the beginning of pregnancy (day 0), dams were fed a control diet (C: 20% casein) or a restricted isocaloric diet (R: 10% casein). After birth, four groups were established: CC, RR, CR and RC (first letter diet during pregnancy and second during lactation). Male offspring were studied at postnatal days 14, 21 and 36. At birth, pup body weight was unchanged. Body weight and testis weight were reduced in RR and CR groups at all ages evaluated. MPR delayed the germinal epithelium development at all ages evaluated. On performing Western blot and immunohistochemistry, AR expression was found to be lower in the three restricted groups. The results suggest that MPR during pregnancy and/or lactation delays SC maturation and germ cell differentiation, and affects intratubular organization. These changes might be responsible for the lower fertility rate at older ages.

Histogenesis of testicular parenchyma during prenatal life in buffalo / Histogénesis del parénquima testicular durante la vida intrauterina en el búfalo

The study was conducted on the testes of 18 buffalo foetii to reveal histogenesis and differentiation of different cells of testicular parenchyma. At 8.0 cm CVR (65 days) the seminiferous tubules were present at gonadal periphery and a network of polygonal mesenchymal cells was seen in the centre of testis. These tubules were surrounded by a distinct basement membrane and a single layer of peritubular cells at 10 cm CVR (74 days), which became double layered at 88.0 cm CVR (272 days). The testicular parenchyma at 12.0 cm CVR had two zones; outer zone having longitudinal tubules and inner zone having rounded tubules. But a reverse pattern of their arrangement was observed at 14.0 cm CVR (92 days). The pre-Sertoli cells were first observed in buffalo foetii of 8.0 cm CVR (65 days) in the periphery of seminiferous tubular epithelium whereas the gonocytes were demonstrable in the centre of tubules at 10.6 cm CVR (76 days). The fetal Leydig cells were also reported at 8.0 cm CVR (65 days) but at 14.0 cm CVR (92 days), the interstitium had considerably expanded due to the differentiation of mesenchymal cells into the Leydig cells.

El estudio fue realizado en los testículos de 18 fetos de búfalos, para revelar la histogénesis y diferenciación de las diferentes células de parénquima testicular. A los 8,0 cm de longitud corona-rabadilla (LCR) (65 días) los túbulos seminíferos estuvieron presentes en la periferia de la gónada y una red poligonal de células mesenquimales se observó en el centro del testículo. Estos túbulos estaban rodeados por una membrana basal y una sola capa de células peritubular a los 10 cm LCR (74 días), la cual se convirtió en una doble capa a los 88,0 cm LCR (272 días). El parénquima testicular a 12,0 cm LCR tenía dos zonas, zona exterior con túbulos longitudinales y zona interior con los túbulos redondeados transversalmente. Sin embargo, un patrón inverso en su disposición se observó a los 14,0 cm LCR (92 días). Las células pre-Sertoli se observaron primero en fetos de búfalos de 8,0 cm LCR (65 días) en la periferia del epitelio seminífero tubular, mientras que los gonocitos fueron visibles en el centro de los túbulos a 10,6 cm LCR (76 días). Las células de Leydig fetales también se observaron a los 8,0 cm LCR (65 días), pero a los 14,0 cm LCR (92 días), el intersticio tuvo una considerable expansión debido a la diferenciación de células mesenquimales en células de Leydig.

Histological development of human testicular cords from 70 to 90 days of gestation.

The development of the testicular cord structure was investigated in 4 human fetuses between 70 and 90 days of gestation, in which the testicular cords are differentiating into the seminiferous tubules. Histological examinations were performed using stains with haematoxylin-eosin (HE), Masson's trichrome (MT), periodic acid schiff (PAS), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibodies, and TUNEL methods. It was found that the testicular cords structures were indefinitely observed in HE-stained sections of four fetuses. However, the basement membranes of the testicular cord were clearly stained with MT, showing the tubular structure. Furthermore, cells in the testicular cords were positive with PAS, but the interstitial tissues outside the testicular cords were negative. PCNA-positive cells were detected not only inside but also outside the testicular cords, however, TUNEL positive cells are not detected throughout all testicular tissues.

Dazl promotes germ cell differentiation from embryonic stem cells.

It has been demonstrated that through the formation of embryoid bodies (EBs) germ cells can be derived from embryonic stem (ES) cells. Here, we describe a transgene expression approach to derive germ cells directly from ES cells in vitro without EB formation. Through the ectopic expression of Deleted in Azoospermia-Like (Dazl), a germ cell-specific RNA-binding protein, both motile tailed-sperm and oocytes were induced from mouse ES (mES) cells in culture. Furthermore, transient overexpression of Dazl led to suppression of Nanog but induced germ cell nuclear antigen in mES cells. Dazl knockdown resulted in reduction in the expression of germ cell markers including Stella, MVH and Prdm1. Our study indicates that Dazl is a master gene controlling germ cell differentiation and that ectopic expression of Dazl promotes the dynamic differentiation of mouse ES cells into gametes in vitro.

A deficiency of lunatic fringe is associated with cystic dilation of the rete testis.

Lunatic fringe belongs to a family of beta1-3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.