scholarly journals Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation

2005 ◽  
Vol 186 (3) ◽  
pp. 429-446 ◽  
Author(s):  
Sarah J Meachem ◽  
Saleela M Ruwanpura ◽  
Jessica Ziolkowski ◽  
Jacquelyn M Ague ◽  
Michael K Skinner ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Cathepsin L ◽  
Passive Immunisation ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
The Impact

The critical influence of follicle stimulating hormone (FSH) on male fertility relates both to its impact on Sertoli cell proliferation in perinatal life and to its influence on the synthesis of Sertoli cell-derived products essential for germ cell survival and function in the developing adult testis. The nature and timing of this shift of germ cells to their reliance on specific Sertoli cell-derived products are not defined. Based on existing data, it is apparent that the dominant function of FSH shifts between 9 and 18 day postpartum (dpp) during the first wave of spermatogenesis from driving Sertoli cell proliferation to support germ cells. To enable comprehensive analysis of the impact of acute in vivo FSH suppression on Sertoli and germ cell development, FSH was selectively suppressed in Sprague–Dawley rats by passive immunisation for 2 days and/or 4 days prior to testis collection at 3, 9 and 18 dpp. The 3 dpp samples displayed no measurable changes, while 4 days of FSH suppression decreased Sertoli cell proliferation and numbers in 9 dpp, but not 18 dpp, animals. In contrast, germ cell numbers were unaffected at 9 dpp but decreased at 18 dpp following FSH suppression, with a corresponding increase in germ cell apoptosis measured at 18 dpp. Sixty transcripts were measured as changed at 18 dpp in response to 4 days of FSH suppression, as assessed using Affymetrix microarrays. Some of these are known as Sertoli cell-derived FSH-responsive genes (e.g. StAR, cathepsin L, insulin-like growth factor binding protein-3), while others encode proteins involved in cell cycle and survival regulation (e.g. cyclin D1, scavenger receptor class B 1). These data demonstrate that FSH differentially affects Sertoli and germ cells in an age-dependent manner in vivo, promoting Sertoli cell mitosis at day 9, and supporting germ cell viability at day 18. This model has enabled identification of candidate genes that contribute to the FSH-mediated pathway by which Sertoli cells support germ cells.

scholarly journals Overexpression of Bcl-w in the Testis Disrupts Spermatogenesis: Revelation of a Role of BCL-W in Male Germ Cell Cycle Control

2003 ◽  
Vol 17 (9) ◽  
pp. 1868-1879 ◽  
Author(s):  
Wei Yan ◽  
Jun-Xing Huang ◽  
Anna-Stina Lax ◽  
Lauri Pelliniemi ◽  
Eeva Salminen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Testicular Development ◽  
Dna Ladder

Abstract To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken β-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5′-bromo-2′deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.

scholarly journals Formation of organotypic testicular organoids in microwell culture†

2019 ◽  
Vol 100 (6) ◽  
pp. 1648-1660 ◽  
Author(s):  
Sadman Sakib ◽  
Aya Uchida ◽  
Paula Valenzuela-Leon ◽  
Yang Yu ◽  
Hanna Valli-Pulaski ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Cell Interactions ◽  
Dependent Manner ◽  
Tissue Architecture ◽  
Cell Cell

Abstract Three-dimensional (3D) organoids can serve as an in vitro platform to study cell–cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell–cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.

scholarly journals HMGA1B/2 transcriptionally activated-POU1F1 facilitates gastric carcinoma metastasis via CXCL12/CXCR4 axis-mediated macrophage polarization

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Cheng Tang ◽  
Xiong Lei ◽  
Lingqiang Xiong ◽  
Zhigao Hu ◽  
Bo Tang
Keyword(s):  
Cell Proliferation ◽  
Tumor Growth ◽  
Poor Prognosis ◽  
Macrophage Polarization ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
The Impact ◽  
Cxcr4 Axis

AbstractTumor-associated macrophages (TAMs) in the tumor microenvironment contribute to poor prognosis in gastric cancer (GC). However, the underlying mechanism by which TAMs promote GC progression and metastasis remains elusive. Expression of POU1F1 was detected in 60 matched GC-normal tissue pairs using qRT-PCR and immunohistochemistry (IHC) analysis. The correlation between POU1F1 and the clinical-pathological factors of GC patients were further assessed. Cell proliferation was monitored by CCK-8, colony formation, and 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assays. Cell migration and invasion were assessed by transwell assays. The impact on angiogenesis was evaluated by tube formation assay. Xenograft model was generated to investigate the role of POU1F1 on tumor growth and lung metastasis in vivo. GST pull-down and Co-immunoprecipitation (Co-IP) were used to study the interaction between HMGA1B/2 and POU1F1. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were performed to investigate the transcriptional regulation of POU1F1. Flow cytometry was performed to detect the surface expression of macrophage markers. Upregulated POU1F1 observed both in GC tissues and cell lines was positively correlated with poor prognosis. Knockdown of POU1F1 inhibited cell proliferation, migration, invasion, and angiogenesis in vitro, and suppressed tumor growth in vivo. HMGA1B/2 transcriptionally activated-POU1F1. POU1F1 promoted GC progression via regulating macrophage proliferation, migration, polarization, and angiogenesis in a CXCL12/CXCR4-dependent manner. POU1F1 also promoted GC metastasis in lung by modulating macrophage polarization through CXCL12/CXCR4 axis in vivo. HMGA1B/2-upregulated POU1F1 promoted GC metastasis via regulating macrophage polarization in a CXCL12/CXCR4-dependent manner.

scholarly journals Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro.

1985 ◽  
Vol 101 (4) ◽  
pp. 1511-1522 ◽  
Author(s):  
M A Hadley ◽  
S W Byers ◽  
C A Suárez-Quian ◽  
H K Kleinman ◽  
M Dym
Keyword(s):  
Extracellular Matrix ◽  
Cell Differentiation ◽  
Basement Membrane ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Type I ◽  
Extracellular Matrix Components

Sertoli cell preparations isolated from 10-day-old rats were cultured on three different substrates: plastic, a matrix deposited by co-culture of Sertoli and peritubular myoid cells, and a reconstituted basement membrane gel from the EHS tumor. When grown on plastic, Sertoli cells formed a squamous monolayer that did not retain contaminating germ cells. Grown on the matrix deposited by Sertoli-myoid cell co-cultures, Sertoli cells were more cuboidal and supported some germ cells but did not allow them to differentiate. After 3 wk however, the Sertoli cells flattened to resemble those grown on plastic. In contrast, the Sertoli cells grown on top of the reconstituted basement membrane formed polarized monolayers virtually identical to Sertoli cells in vivo. They were columnar with an elaborate cytoskeleton. In addition, they had characteristic basally located tight junctions and maintained germ cells for at least 5 wk in the basal aspect of the monolayer. However, germ cells did not differentiate. Total protein, androgen binding protein, transferrin, and type I collagen secretion were markedly greater when Sertoli cells were grown on the extracellular matrices than when they were grown on plastic. When Sertoli cells were cultured within rather than on top of reconstituted basement membrane gels they reorganized into cords. After one week, tight junctional complexes formed between adjacent Sertoli cells, functionally compartmentalizing the cords into central (adluminal) and peripheral (basal) compartments. Germ cells within the cords continued to differentiate. Thus, Sertoli cells cultured on top of extracellular matrix components assume a phenotype and morphology more characteristic of the in vivo, differentiated cells. Growing Sertoli cells within reconstituted basement membrane gels induces a morphogenesis of the cells into cords, which closely resemble the organ from which the cells were dissociated and which provide an environment permissive for germ cell differentiation.

Neonatal administration of FSH increases Sertoli cell numbers and spermatogenesis in gonadotropin-deficient (hpg) mice

1996 ◽  
Vol 151 (1) ◽  
pp. 37-48 ◽  
Author(s):  
J Singh ◽  
D J Handelsman
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Postnatal Life ◽  
Testis Size ◽  
Cell Numbers ◽  
Natural Time ◽  
Neonatal Administration

Abstract We previously demonstrated that androgens alone, in the complete absence of gonadotropins, initiated qualitatively complete spermatogenesis in hypogonadal (hpg) mice. Although germ cell to Sertoli cell ratios were normal in hpg mice with androgen-induced spermatogenesis, testicular size, Sertoli cell and germ cell numbers only reached 40% of those of non-hpg mice, and Sertoli cell numbers were unaffected by androgen treatment started at 21 days of age. We postulated that these observations were due to diminished gonadotropin-dependent Sertoli cell proliferation during perinatal life while the Sertoli cells still exhibited normal carrying capacity for mature germ cells. In order to test this hypothesis, we examined the effects of administering androgens and gonadotropins to hpg mice during the first 2 weeks of postnatal life when Sertoli cells normally continue to proliferate. The study end-points were Sertoli and germ cell numbers in hpg mice following induction of spermatogenesis by 8 weeks treatment with 1 cm subdermal silastic testosterone implants. Newborn pups (postnatal day 0–1) were injected s.c. with recombinant human FSH (rhFSH) (0·5 IU/20 μl) or saline once daily for 14 days, with or without a single dose of testosterone propionate (TP) (100 μg/20 μl arachis oil) or human chorionic gonadotropin (hCG) (1 IU/20 μl). Untreated hpg and phenotypically normal littermates were studied as concurrent controls. At 21 days of age, all treated weanling mice received a 1 cm silastic subdermal testosterone implant and, finally, 8 weeks after testosterone implantation, all mice were killed. As expected, qualitatively complete spermatogenesis was induced in all groups by testosterone despite undetectable circulating FSH levels. Exogenous rhFSH increased testis size by 43% (P<0·002) but a single neonatal dose of either TP or hCG reduced the FSH effect although neither TP nor hCG had any effect alone. In contrast, a single neonatal dose of TP or hCG increased final seminal vesicle size whereas FSH had no effect. FSH and TP treatment significantly increased absolute numbers of testicular spermatids compared with saline treatment, whereas hCG and TP significantly increased testicular sperm when expressed relative to testis size. Stereological evaluation of Sertoli and germ cell numbers demonstrated a rise in the absolute numbers of Sertoli and all germ cell populations induced by neonatal administration of hormones. When expressed per Sertoli cells the numbers of germ cells in the treated mice were between 85 and 90% of non-hpg controls. We conclude that exogenous FSH treatment during the first 2 weeks of postnatal life, coinciding with the natural time of Sertoli cell proliferation, increases Sertoli cell numbers and thereby the ultimate size of the mature testis and its germ cell production. Thus neonatal gonadotropin secretion may be a critical determinant of the sperm-producing capacity of the mature testis. In addition, neonatal exposure to androgens could be important for the imprinting of sex accessory organs in hpg mice, with the long-term effects of altering the sensitivity of the accessory organs to exogenous testosterone later in life. Journal of Endocrinology (1996) 151, 37–48

scholarly journals Bioactive isoprenoids guide migrating germ cells to the embryonic gonad

2021 ◽  
Author(s):  
Lacy Barton ◽  
Justina Sanny ◽  
Emily P Dawson ◽  
Marcela Nouzova ◽  
Fernando G Noriega ◽  
...  
Keyword(s):  
Cell Migration ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Development ◽  
Germ Cell Development ◽  
Germ Cell Migration ◽  
The Impact

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in insects, impact the germline lifecycle from meiosis to gametogenesis. Emerging evidence suggests that these bioactive isoprenoids also influence embryonic reproductive development, though the precise functions remain unclear. Here, we investigated the specific molecular pathways by which JHs regulates embryonic germ cell development in Drosophila. With a newly generated in vivo reporter, we find that JH signaling is active in the vicinity of germ cells as they migrate to colonize the somatic gonad. Through a combination of in vivo and in vitro assays, we find that JHs are both necessary and sufficient for primordial germ cell migration through mechanisms independent of canonical nuclear receptor-mediated transcription. These findings reveal that JH is present during Drosophila embryogenesis and that bioactive isoprenoids impact germ cell development earlier than previously appreciated. Interestingly, we find that like JH in Drosophila, RA is sufficient for murine germ cell migration in vitro, suggesting that the impact of bioactive isoprenoids on embryonic germ cell development may be broadly conserved.

scholarly journals The impact of indole-3-lactic acid on immature intestinal innate immunity and development: a transcriptomic analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wuyang Huang ◽  
Ky Young Cho ◽  
Di Meng ◽  
W. Allan Walker
Keyword(s):  
Lactic Acid ◽  
Mechanistic Model ◽  
Transcriptomic Analysis ◽  
Dependent Manner ◽  
Protective Effects ◽  
Very Preterm Infants ◽  
Anti Inflammatory ◽  
The Impact

AbstractAn excessive intestinal inflammatory response may have a role in the pathogenesis of necrotizing enterocolitis (NEC) in very preterm infants. Indole-3-lactic acid (ILA) of breastmilk tryptophan was identified as the anti-inflammatory metabolite involved in probiotic conditioned media from Bifidobacteria longum subsp infantis. This study aimed to explore the molecular endocytic pathways involved in the protective ILA effect against inflammation. H4 cells, Caco-2 cells, C57BL/6 pup and adult mice were used to compare the anti-inflammatory mechanisms between immature and mature enterocytes in vitro and in vivo. The results show that ILA has pleiotropic protective effects on immature enterocytes including anti-inflammatory, anti-viral, and developmental regulatory potentials in a region-dependent and an age-dependent manner. Quantitative transcriptomic analysis revealed a new mechanistic model in which STAT1 pathways play an important role in IL-1β-induced inflammation and ILA has a regulatory effect on STAT1 pathways. These studies were validated by real-time RT-qPCR and STAT1 inhibitor experiments. Different protective reactions of ILA between immature and mature enterocytes indicated that ILA’s effects are developmentally regulated. These findings may be helpful in preventing NEC for premature infants.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

scholarly journals The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals

2021 ◽  
Vol 95 (3) ◽  
pp. 1103-1116
Author(s):  
Francesco Marchetti ◽  
Gu Zhou ◽  
Danielle LeBlanc ◽  
Paul A. White ◽  
Andrew Williams ◽  
...  
Keyword(s):  
Germ Cells ◽  
False Negative ◽  
Sampling Time ◽  
Male Germ Cells ◽  
Negative Results ◽  
False Negative Results ◽  
Detection Of Mutations ◽  
The Impact ◽  
Sampling Times

AbstractThe Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose–response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

scholarly journals A MYBL2 complex for RRM2 transactivation and the synthetic effect of MYBL2 knockdown with WEE1 inhibition against colorectal cancer

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Qian Liu ◽  
Lijuan Guo ◽  
Hongyan Qi ◽  
Meng Lou ◽  
Rui Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Dna Synthesis ◽  
Ectopic Expression ◽  
Building Blocks ◽  
Small Subunit ◽  
S Phase ◽  
Clinical Samples ◽  
Dependent Manner

AbstractRibonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

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