scholarly journals Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Reproduction ◽  
2007 ◽  
Vol 133 (6) ◽  
pp. 1169-1179 ◽  
Author(s):  
Tu’uhevaha J Kaitu’u-Lino ◽  
Pavel Sluka ◽  
Caroline F H Foo ◽  
Peter G Stanton
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Cell Contacts ◽  
Barrier Formation ◽  
Protein Components ◽  
Blood Testis Barrier

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood–testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJsin vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P< 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesisin vivois partly via their effects on TJ proteins and regulation of the blood–testis barrier.

Hormonal regulation of spermatid binding

1991 ◽  
Vol 100 (3) ◽  
pp. 623-633
Author(s):  
D.F. Cameron ◽  
K.E. Muffly
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Unit Area ◽  
Junctional Complex ◽  
Cell Cytoplasm ◽  
Coculture Model ◽  
Ectoplasmic Specialization

A Sertoli-spermatid coculture model is described in which a large percentage (greater than 76%) of round spermatids remain viable for 48 h and bind to Sertoli cells. The effects of follicle-stimulating hormone (FSH) and testosterone on spermatid binding (expressed as the spermatid density; SD = the number of spermatids per unit area of Sertoli cell cytoplasm), ultrastructure of the Sertoli-spermatid junctional complex, and distribution in the Sertoli cell of junction-related F-actin and vinculin are described. Following 48 h of incubation, neither FSH alone nor testosterone alone affected spermatid binding to Sertoli cells beyond that observed in control cocultures. However, the combination of FSH and testosterone (FSH + testosterone) resulted in a significant increase in the density of spermatids bound to Sertoli cells. Junction-related structure of the Sertoli cell cytoskeleton between the Sertoli cell and the pre-step 8 spermatid was different than that observed between the Sertoli cell and the post-step 8 spermatid. The junction-related cytoskeletal modification of the Sertoli cell (JCMS) in the latter was similar in appearance to the well-described ‘Sertoli ectoplasmic specialization’ observed adjacent to post-step 8 spermatids in vivo. FSH + testosterone and FSH alone, but not testosterone alone, resulted in the peripheral distribution of actin and vinculin, which otherwise remained in stress fiber-like structures throughout the Sertoli cell. Results show that maximal spermatid binding to Sertoli cells in vitro requires FSH + testosterone and is associated with the peripheral distribution of actin and vinculin.

116. Contribution of claudin-11 to the inter-Sertoli cell tight junction, in vitro

2005 ◽  
Vol 17 (9) ◽  
pp. 72
Author(s):  
M. J. McCabe ◽  
P. G. Stanton
Keyword(s):  
Tight Junction ◽  
Mrna Expression ◽  
Sertoli Cell ◽  
Adhesion Molecule ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Adherens Junction Protein ◽  
Junction Protein

The inter-Sertoli cell tight junction (TJ) forms the blood testis barrier (BTB) between Sertoli cells and is composed of three major transmembrane proteins: claudin-11, occludin and junctional adhesion molecule. Formation of the BTB occurs during puberty associating with an increase in circulating gonadotrophins. Claudin-11 and occludin are hormonally regulated in vitro although their importance to the function of the TJ is unknown. The aim of this study was to investigate the contribution of claudin-11 to the inter-Sertoli cell TJ in vitro by blocking gene expression using RNA interference. Two claudin-11-specific siRNA fragments were designed for this purpose. Sertoli cells in primary culture formed stable TJs within 5 days as measured by transepithelial electrical resistance (TER). The addition of siRNA for 2 days resulted in a significant (P < 0.01) 55% (mean, SD, n = 4 cultures) decrease in TER along with a major reduction in claudin-11 localisation to the TJ as assessed by immunocytochemistry. The specificity of the siRNA was shown by the presence of extensive immunostaining of occludin and of the adherens junction protein β-catenin in the same treatments. Similarly, claudin-11 mRNA expression significantly (P < 0.01) decreased by 71% (mean, SD, n = 3 cultures) in response to both claudin-11 siRNA fragments. Occludin mRNA expression was not affected. It is concluded that claudin-11 contributes at least 55% to the function of the rat Sertoli cell TJ in vitro. It is hypothesised that the remaining 45% of TJ function can be attributed to other integral proteins, such as occludin and junctional adhesion molecule. It is expected that claudin-11 and other TJ proteins play a pivotal role in the function of the BTB in vivo with potential implications in fertility and contraception.

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.

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.

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 rpS6 Regulates Blood-Testis Barrier Dynamics Through Arp3-Mediated Actin Microfilament Organization in Rat Sertoli Cells. An In Vitro Study

Endocrinology ◽  
2015 ◽  
Vol 156 (5) ◽  
pp. 1900-1913 ◽  
Author(s):  
Ka-Wai Mok ◽  
Haiqi Chen ◽  
Will M. Lee ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cells ◽  
Protein Complexes ◽  
Mammalian Target Of Rapamycin ◽  
Attachment Site ◽  
In Vitro Study ◽  
Site Directed Mutagenesis ◽  
Actin Microfilaments ◽  
Blood Testis Barrier

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a “bundled” to an “unbundled/branched” configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

scholarly journals FSH regulates the formation of adherens junctions and ectoplasmic specialisations between rat Sertoli cells in vitro and in vivo

2006 ◽  
Vol 189 (2) ◽  
pp. 381-395 ◽  
Author(s):  
P Sluka ◽  
L O’Donnell ◽  
J R Bartles ◽  
P G Stanton
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Intermediate Filament Protein ◽  
Adult Rat

Spermatogenesis is dependent on the ability of Sertoli cells to form mature junctions that maintain a unique environment within the seminiferous epithelium. Adjacent Sertoli cells form a junctional complex that includes classical adherens junctions and testis-specific ectoplasmic specialisations (ES). The regulation of inter-Sertoli cell junctions by the two main endocrine regulators of spermatogenesis, FSH and testosterone, is unclear. This study aimed to investigate the effects of FSH and testosterone on inter-Sertoli cell adherens junctions (as determined by immunolocalisation of cadherin, catenin and actin) and ES junctions (as determined by immunolocalisation of espin, actin and vinculin) in cultured immature Sertoli cells and GnRH-immunised adult rat testes given FSH or testosterone replacement in vivo. When hormones were absent in vitro, adherens junctions formed as discrete puncta between interdigitating, finger-like projections of Sertoli cells, but ES junctions were not present. The adherens junction puncta included actin filaments that were oriented perpendicularly to the Sertoli cell plasma membrane, but were not associated with the intermediate filament protein vimentin. When FSH was added in vitro, ES junctions formed, and adjacent adherens junction puncta fused into extensive adherens junction belts. After hormone suppression in vivo, ES junctions were absent, while FSH replacement restored ES junctions, as confirmed by electron microscopy and confocal analysis of ES-associated proteins. Testosterone alone did not affect adherens junctions or ES in vitro or in vivo. We conclude that FSH can regulate the formation of ES junctions and stimulate the organisation and orientation of extensive adherens junctions in Sertoli cells.

scholarly journals Regulation of the blood-testis barrier by coxsackievirus and adenovirus receptor

2012 ◽  
Vol 303 (8) ◽  
pp. C843-C853 ◽  
Author(s):  
Linlin Su ◽  
Dolores D. Mruk ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Regulatory Protein ◽  
Integral Membrane Protein ◽  
Permeability Barrier ◽  
Structural Protein ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Adenovirus Receptor ◽  
Blood Testis Barrier

The blood-testis barrier (BTB) divides the seminiferous epithelium into the basal and the adluminal compartment. It restricts paracellular diffusion of molecules between Sertoli cells, confers cell polarity, and creates a unique microenvironment in the adluminal compartment for spermatid development. However, it undergoes restructuring during the epithelial cycle so that preleptotene spermatocytes differentiated from type B spermatogonia residing in the basal compartment can traverse the BTB at stage VIII of the cycle, while the immunological barrier is maintained. Herein, coxsackievirus and adenovirus receptor (CAR), a tight junction (TJ) integral membrane protein in the testis and multiple epithelia and endothelia, was found to act as a regulatory protein at the BTB, besides serving as a structural adhesion protein. RNAi-mediated knockdown of CAR in a Sertoli cell epithelium with an established TJ-permeability barrier that mimicked the BTB in vivo resulted in a disruption of the TJ barrier and an increase in endocytosis of the TJ-protein occludin. Furthermore, such an enhancement in occludin endocytosis was accompanied by a downregulation of Thr-phosphorylation in occludin and an increase in the association of endocytosed occludin with early endosome antigen-1. These findings were confirmed by overexpressing CAR in Sertoli cells, which was found to “tighten” the Sertoli cell TJ barrier, promoting BTB function. These findings support the emerging concept that CAR is not only a structural protein, it is involved in conferring the phosphorylation status of other adhesion proteins at the BTB (e.g., occludin) possibly mediated via its structural interactions with nonreceptor protein kinases, thereby modulating endocytic vesicle-mediated protein trafficking.

scholarly journals Liver-regulating protein (LRP) is a plasma membrane protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes

1995 ◽  
Vol 108 (3) ◽  
pp. 917-925
Author(s):  
N. Gerard ◽  
A. Corlu ◽  
B. Kneip ◽  
H. Kercret ◽  
M. Rissel ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cell ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Cell Function ◽  
Cell Contact ◽  
Dependent Manner ◽  
Biliary Epithelial Cell

We have identified a liver-regulating protein involved in cell contact-mediated regulation of Sertoli cell function by primary spermatocytes in rat testis. Liver-regulating protein was studied using monoclonal antibody L8 prepared from rat primitive biliary epithelial cells. This molecule was located in vivo at the interface of Sertoli cells and spermatocytes, and expressed in a stage-dependent manner (expression peaked on leptotene-zygotene spermatocytes). In vitro, the liver-regulating protein was found on Sertoli cell, spermatocyte and early spermatid membranes. Immunoaffinity procedures revealed two peptides of 85 and 73 kDa for Sertoli cells, while spermatocytes and spermatids displayed a single smaller peptide of 56 kDa. The involvement of the liver-regulating protein in cell interaction-mediated regulation of Sertoli cell was assessed in vitro by tracing Sertoli cell transferrin and inhibin secretion, as well as mRNA synthesis in spermatocyte-Sertoli cell cocultures and in rat liver biliary epithelial cell-Sertoli cell cocultures, performed in the presence or absence of monoclonal antibody L8. Inhibition of the spermatocyte- and liver biliary epithelial cell-stimulated secretion of transferrin and inhibin by Sertoli cells was observed in the presence of antibody, whereas spermatocyte adhesiveness was unchanged. Using northern blot analysis, the steady state levels of transferrin mRNA decreased when the anti-liver-regulating protein antibody was added to the Sertoli cell-spermatocyte cocultures or to the Sertoli cell-liver biliary epithelial cell cocultures. The data demonstrate the role of the liver-regulating protein in cell-cell contact-mediated regulation of Sertoli function by primary spermatocytes and the important implications of this cell contact-dependent control in testicular activity.

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