O-190 Comparison between effects of exposure to platinum-based chemotherapeutics (cisplatin and carboplatin) on Sertoli cell number and functions in immature human testicular tissues

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Matilionyte ◽  
M D Tharmalingam ◽  
I Sanou ◽  
F Lopes ◽  
R A Anderson ◽  
...  
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Cell Number ◽  
Chemotherapeutic Agents ◽  
Inhibin B ◽  
Hanging Drop ◽  
Platinum Drug ◽  
Term Survival ◽  
Post Exposure

Abstract Study question Does exposure to either cisplatin or carboplatin have a damaging effect on the Sertoli cell population in the immature human testicular tissues? Summary answer Exposure to cisplatin or carboplatin did not appear to have a major effect on Sertoli cell number or function in the immature human testicular tissues What is known already Long-term survival rates for children with cancer are more than 80%. However, childhood cancer treatment may result in subsequent infertility. Cisplatin is one of the most commonly used drugs for childhood cancers. Carboplatin, a second generation platinum drug, is administered at 10-times the dose of cisplatin and is believed to be less gonadotoxic. In our recent publication we have shown that exposure to both cisplatin and carboplatin acutely reduce the germ cell number in immature human testicular tissues. However, it is not known how cisplatin and carboplatin affect Sertoli cell number and function. Study design, size, duration In-vitro culture of human fetal and pre-pubertal testicular tissues was utilised. Tissue pieces were cultured for 1-3 days prior to exposure to clinically-relevant doses of chemotherapeutics or vehicle control for 24hrs in two sets of experiments: 1) 0.5 or 1 μg/ml cisplatin and culture ended at 24 and 96hrs post-exposure (fetal only); 2) 0.5 μg/ml cisplatin or 5 ­μg/ml carboplatin until 72 (both fetal and pre-pubertal) and 240hrs post-­exposure (fetal only). Participants/materials, setting, methods Testicular tissue fragments from second trimester human fetal (14-22 gestational weeks; n = 3-6) or pre-pubertal patients (1-8years old; n = 5) were cultured in a ‘hanging drop’ system.Quantification of Sertoli cell number (cells per cord/tubular area (mm2)) was performed on sections stained for expression of SOX9. Culture medium was collected to measure levels (ng/ml) of Anti-Mullerian hormone (AMH) and Inhibin B using ELISA. Statistical analysis was performed using two-way ANOVA to account for inter-individual variation between fetuses/patients. Main results and the role of chance Quantification of positively stained Sertoli cells showed that exposure to both doses of cisplatin had no effect on Sertoli cell number at 24 and 96hrs post-exposure. No changes in AMH and inhibin B levels were observed at these time-points. Comparison between cisplatin- or carboplatin-exposed human fetal testicular tissues showed no difference in Sertoli cell numbers at either 72hrs or 240hrs post-exposure. No difference in Sertoli cell number was observed in pre-pubertal testicular tissues exposed to either cisplatin or carboplatin at 72hrs post-exposure. Limitations, reasons for caution Human fetal and pre-pubertal testis tissue is of limited availability, thus, sample sizes used in this study were relatively low. ‘Hanging drop’ culture might not recapitulate all in-vivo aspects of immature testis microenvironment. Wider implications of the findings Exposure to cisplatin or carboplatin did not affect Sertoli cell number in the immature human testicular tissues. Taken together with our recent publication, this suggests that these two platinum-based chemotherapeutic agents cause direct damage to germ cells. Functionality of Sertoli cells in chemotherapy-exposed tissues need to be further investigated. Trial registration number not applicable

scholarly journals Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin beta B-subunit and inhibin B but not the inhibin alpha-subunit

2002 ◽  
Vol 172 (3) ◽  
pp. 565-574 ◽  
Author(s):  
RJ Clifton ◽  
L O'Donnell ◽  
DM Robertson
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Inhibin B ◽  
Alpha Subunit ◽  
Subunit Mrna ◽  
B Subunit ◽  
Subunit Expression ◽  
Pachytene Spermatocytes

This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P<0.01). A similar suppression in inhibin B was still observed (64% of Sertoli cells alone) when the pachytene spermatocytes were separated from Sertoli cells by a 0.45 microm pore membrane barrier in bicameral chambers. Pachytene spermatocytes also suppressed FSH-induced inhibin B levels in Sertoli cell co-cultures and this suppression was attributed to a decrease in basal inhibin B production rather than a change in FSH responsiveness. Quantitation of Sertoli cell inhibin alpha- and beta B-subunit mRNA by quantitative (real-time) PCR demonstrated that pachytene spermatocytes did not alter Sertoli cell alpha-subunit mRNA expression, but significantly (P<0.01) suppressed basal and FSH-induced beta B-subunit mRNA expression to a similar degree to that seen with inhibin B protein levels. It is concluded that pachytene spermatocytes in vitro suppress Sertoli cell inhibin B secretion via factor-mediated suppression of inhibin beta B-subunit expression. These findings support the hypothesis that specific germ cell types can influence inhibin B secretion by the testis independent of FSH regulation.

scholarly journals Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures

2005 ◽  
Vol 187 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Y Okuma ◽  
A E O’Connor ◽  
J A Muir ◽  
P G Stanton ◽  
D M de Kretser ◽  
...  
Keyword(s):  
Sertoli Cell ◽  
Inflammatory Mediators ◽  
Sertoli Cells ◽  
Reproductive Function ◽  
Inhibitory Effect ◽  
Activin A ◽  
Inhibin B ◽  
Hormonal Stimulation ◽  
Adult Rat

The regulation of Sertoli cell activin A and inhibin B secretion during inflammation was investigated in vitro. Adult rat Sertoli cells were incubated with the inflammatory mediators, lipopolysaccharide (LPS), interleukin-1β (IL-1β), IL-6 and the IL-1 receptor antagonist (IL-1ra) over 48 h in culture. Activin A, inhibin B and IL-1α were measured in the culture medium by specific two-site ELISAs. Both IL-1β- and LPS-stimulated activin A and inhibited inhibin B secretion. LPS also stimulated the production of IL-1α in the cultures. In contrast to IL-1β, IL-6 had no effect on activin A, although it did have a significant inhibitory effect on inhibin B secretion. Ovine follicle-stimulating hormone (FSH) and the cAMP analogue dibutyryl cAMP opposed the actions of IL-1 and LPS by suppressing activin A and IL-1α secretion and by stimulating inhibin B. Blocking IL-1 activity in the cultures by addition of an excess of IL-1ra completely prevented the response of activin A to exogenous IL-1β, and reduced the response to LPS by 50%. In the presence of IL-1ra, basal secretion of inhibin B was increased, but IL-1ra was unable to reverse the suppression of inhibin B by LPS. These data indicate the importance of both IL-1 isoforms in regulating secretion of activin A and inhibin B by mature Sertoli cells during inflammation. The data also establish that inflammation exerts its effects on activin A and inhibin B secretion via other pathways in addition to those mediated by IL-1, and that hormonal stimulation by FSH and cAMP moderates the Sertoli cell response to inflammation. Interference with the complex interactions between these cytokines and hormones may contribute to the disruption of reproductive function that can accompany infection and illness in men.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

Age-related changes in human Leydig cell status

2020 ◽  
Vol 35 (12) ◽  
pp. 2663-2676
Author(s):  
Valentina Mularoni ◽  
Valentina Esposito ◽  
Sara Di Persio ◽  
Elena Vicini ◽  
Gustavo Spadetta ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Cell Number ◽  
Organ Donors ◽  
Steroidogenic Enzymes ◽  
Age Related ◽  
Age Range

Abstract STUDY QUESTION What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS Testis biopsies were obtained from 15 heart beating organ donors (age range: 19–85 years) and 24 patients (age range: 19–45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE Increasing age was negatively associated with Leydig cell number (R = −0.49; P &lt; 0.01) and concomitantly with the Sertoli cell population size (R= −0.55; P &lt; 0.001). A positive correlation (R = 0.57; P &lt; 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= −0.52; P &lt; 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. LIMITATIONS, REASONS FOR CAUTION In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

Trophic Effect of Porcine Sertoli Cells on Rat and Human Ventral Mesencephalic Cells and Hnt Neurons in Vitro

1998 ◽  
Vol 7 (2) ◽  
pp. 157-164 ◽  
Author(s):  
Agneta I. Othberg ◽  
Alison E. Willing ◽  
Don F. Cameron ◽  
Alex Anton ◽  
Samuel Saporta ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Islet Cells ◽  
Treated Group ◽  
Term Survival ◽  
Teratocarcinoma Cell ◽  
Trophic Effect ◽  
Soma Size ◽  
Ventral Mesencephalic

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

scholarly journals Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

1999 ◽  
Vol 340 (1) ◽  
pp. 309-320 ◽  
Author(s):  
Sikha Bettina MUKHERJEE ◽  
S. ARAVINDA ◽  
B. GOPALAKRISHNAN ◽  
Sushma NAGPAL ◽  
Dinakar M. SALUNKE ◽  
...  
Keyword(s):  
Cell Culture ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Culture Media ◽  
Glutathione S Transferase ◽  
Steroid Binding ◽  
Spent Media

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of ‘Sertoli cell only’ animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of < 9.8×10-7 M for testosterone and 9×10-6 M for oestradiol respectively.

148. HORMONALLY REGULATED miRNAS TARGET THE TUBULOBULBAR COMPLEX IN THE TESTIS

2010 ◽  
Vol 22 (9) ◽  
pp. 66
Author(s):  
P. K. Nicholls ◽  
P. G. Stanton ◽  
K. L. Walton ◽  
R. I. McLachlan ◽  
L. O'Donnell ◽  
...  
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Focal Adhesions ◽  
Intercellular Junctions ◽  
Protein Translation ◽  
Seminiferous Epithelium ◽  
Seminiferous Tubules ◽  
Micro Rnas

Spermatogenesis is absolutely dependent on follicle stimulating hormone (FSH) and androgens; acute suppression of these hormones inhibits germ cell development and thus sperm production. The removal of intercellular junctions and release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to acute hormone suppression(1). To define the molecular mechanisms that mediate FSH and androgen effects in the testis, we investigated the expression and hormonal regulation of micro-RNAs (miRNA), small non-coding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs that were upregulated >2-fold in stage VIII seminiferous tubules following hormone suppression, and in vitro in primary Sertoli cells. We subsequently validated the expression and hormonal regulation of several miRNAs, including miR-23b, -30d and -690 by quantitative PCR in primary Sertoli cells. Bioinformatic analysis of potential targets of hormonally-suppressed miRNAs identified genes associated with Focal adhesions (54 genes, P = –ln(17.97)) and the Regulation of the actin cytoskeleton (52 genes, P = –ln(10.16)), processes known to be intimately associated with adhesion of spermatids to Sertoli cells(2, 3). Furthermore, this analysis identified numerous components of the testicular tubulobulbar complex (TBC) as being targets of hormonally sensitive miRNAs. The TBC is a podosome-like structure between Sertoli and adjacent spermatids in the testis, which internalises intact inter-cellular junctions by endocytotic mechanisms prior to spermiation(4). We then demonstrate the hormonal regulation of predicted miRNA target proteins, and validate novel inhibitory miRNA interactions with Pten, nWASP, Eps15 and Picalm by luciferase knockdown in vitro. We hypothesise that hormonally suppressed miRNAs inhibit TBC function, and subsequently, endocytosis of intercellular junctions. In conclusion, we have demonstrated that hormonal suppression in the testis stimulates the expression of a subset of Sertoli cell miRNAs that are likely regulators of cell adhesion protein networks involved in spermiation. (1) Saito K, O’Donnell L, McLachlan RI, Robertson DM 2000 Spermiation failure is a major contributor to early spermatogenic suppression caused by hormone withdrawal in adult rats. Endocrinology 141: 2779–2.(2) O’Donnell L, Stanton PG, Bartles JR, Robertson DM 2000 Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat. Biol Reprod 63: 99–108.(3) Beardsley A, Robertson DM, O’Donnell L 2006 A complex containing alpha6beta1-integrin and phosphorylated focal adhesion kinase between Sertoli cells and elongated spermatids during spermatid release from the seminiferous epithelium. J Endocrinol 190(3): 759–70.(4) Young JS, Guttman JA, Vaid KS, Vogl AW 2009 Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis. Biol Reprod 80: 162–74.

Activin is produced by rat Sertoli cells in vitro and can act as an autocrine regulator of Sertoli cell function.

Endocrinology ◽  
1993 ◽  
Vol 132 (3) ◽  
pp. 975-982 ◽  
Author(s):  
J P de Winter ◽  
H M Vanderstichele ◽  
M A Timmerman ◽  
L J Blok ◽  
A P Themmen ◽  
...  
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Cell Function

A combination of insulin-like growth factor I (IGF-I) and FSH promotes proliferation of prepubertal bovine Sertoli cells isolated and cultured in vitro

2017 ◽  
Vol 29 (8) ◽  
pp. 1635 ◽  
Author(s):  
A. Dance ◽  
J. Kastelic ◽  
J. Thundathil
Keyword(s):  
Growth Factor ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Seminiferous Tubules ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Bull Calves ◽  
Igf I ◽  
Growth Factor I

Beef and dairy bull calves fed a low-nutrition diet during early life had decreased concentrations of circulating insulin-like growth factor I (IGF-I), delayed increases in testosterone, smaller testes and delayed puberty compared with those fed high-nutrition diets. Although IGF-1 has important roles in Sertoli cell function in rats and mice, this has not been well documented in bulls. The objectives of this study were to: (1) isolate Sertoli cells from bull calves at 8 weeks of age, (2) culture them in vitro and (3) determine the effects of IGF-I, FSH and a combination of both hormones on cell proliferation. For Sertoli cell isolation, minced testicular tissues were treated with collagenase followed by trypsin and hyaluronidase to digest seminiferous tubules and release Sertoli cells. In this study, Sertoli cells were successfully isolated from 8-week-old Holstein bull calves (n = 4) and these cells were cultured for up to 8 days. A combination of IGF-I and FSH increased proliferation (~18%) and therefore cell number (1.5-fold) of prepubertal bovine Sertoli cells in culture, providing clear evidence that IGF-I has a similar role in bovine Sertoli cells as reported in rodents.

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