Sertoli Cell-enriched Cultures Research Articles

Rat and mouse testicular testin is different from the human tumor suppressor gene TESTIN (Tes)

In response to a “Letter to the Editor” from Dr. Shailendra Kapoor with regard to a paper published on testin in Spermatogenesis,1 we wish to clarify some misinformation on this protein in the literature. In the late 1980s, two papers were published, reporting a new testicular protein purified from rat Sertoli cell-enriched culture media. It was designated testin based on its unique N-terminal amino acid sequence.2,3 Specifically, testin is a testis-specific protein,4 composed of two isoforms of testin I (35 kDa) and testin II (37 kDa) where testin II has three extra N-terminal amino acids of Thr-Ala-Pro.3 The full-length cDNA encoding rat testicular testin was cloned, sequenced and published shortly thereafter (GenBank™ Accession Numbers: {type:entrez-nucleotide,attrs:{text:U16858,term_id:577429}}U16858; {type:entrez-nucleotide,attrs:{text:NM_173132,term_id:402693052}}NM_173132, {type:entrez-protein,attrs:{text:NP_775155,term_id:27465595}}NP_775155).5 Testin was subsequently cloned and sequenced in the mouse (GenBank™ Accession Numbers: {type:entrez-nucleotide,attrs:{text:NM_178098,term_id:31342510}}NM_178098, {type:entrez-protein,attrs:{text:NP_835199,term_id:30017423}}NP_835199) and shown to display 90.1% similarity with rat testin.6 Testin is also a gonad-specific protein,4 with its expression restricted largely to the testis and ovary in adult rats.7,8 Interestingly, its expression is upregulated during disruption of the testis-specific anchoring junction known as the apical ectoplasmic specialization (ES).8-10 The recent report published in Spermatogenesis has shown that testin is also an actin-binding protein at the ES.1 While the rat testin primary amino acid sequence contains an apparent His- and an Asn-active site, a characteristic feature of cysteine proteases, and is ~60% homologous with human cathepsin L ({type:entrez-protein,attrs:{text:NP_666023,term_id:22202619}}NP_666023; NM-145918), testin does not possess protease nor protease inhibitor activity.11 Equally important, Sertoli cell testin originally identified in the rat testis is entirely different from a tumor suppressor gene called TESTIN (gene name Tes) (GenBank™ Accession Numbers: {type:entrez-nucleotide,attrs:{text:NM_015641,term_id:325197160}}NM_015641, {type:entrez-protein,attrs:{text:NP_056456,term_id:7661666}}NP_056456), which was identified and cloned around the same time rat Sertoli cell testin was identified.12-14 The tumor suppressor TESTIN14 shares no significant homology in its amino acid sequence (only ~1–3%) with rat Sertoli cell testin. Moreover, Sertoli cell testin does not contain any characteristic LIM domains or zinc finger motifs, which are found in the tumor suppressor TESTIN, illustrating that these two proteins are distinctly different and evolutionarily unrelated. A mouse genetic model is available for the tumor suppressor TESTIN, and its deletion was found to associate with hematopoietic malignancies and epithelial tumors.15 However, findings from this genetic model are not applicable to Sertoli cell testin since they are two entirely different proteins.

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells culturedin vitro

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100nM E2 (17β-oestradiol) or 100nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2pmol/cell) at similar rates during the 50h. After 25h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.

Influence of 5α-dihydrotestosterone and 17β-estradiol on human Sertoli cells metabolism

Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell-enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using (1) H-NMR spectra analysis, in the absence or presence of 100 nm 17β-estradiol (E(2) ) or 100 nm 5α-dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi-quantitative RT-PCR. Cells cultured in the absence (control) or presence of E(2) consumed the same amounts of glucose at similar rates during the 50 h. During the first 15 h of treatment with DHT, glucose consumption and glucose consumption rate were significantly higher. Nevertheless, DHT-treated cells secreted a significantly lower amount of lactate than control and E(2) -treated cells. Such a decrease was concomitant with a significant decrease in lactate dehydrogenase A mRNA levels after 50 h treatment in DHT-treated groups. Finally, alanine production was significantly increased in E(2) -treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells on those conditions. These results support the existence of a relationship between sex steroid hormones action and energy metabolism, providing the first assessment of androgens and oestrogens as metabolic modulators of human Sertoli cells.

Understanding the testes immunological sanctuary: an in vitro human Sertoli cell study

OBJECTIVE: The immunological “sanctuary” of the testis is theorized to have 2 components: a passive, physical “blood-testis” barrier formed by inter-Sertoli tight junctions and a localized active immunosuppressive “field effect”. To better understand active immunosuppression of human testis, we analyzed the presence of an immunoregulatory lectin, Galectin-1, in testis cryosections and in a primary human Sertoli cell model. DESIGN: Controlled immunohistochemical studies in human testis and in an in vitro human Sertoli cell model. Jurkat E-6 T cell proliferation assays in the model. MATERIALS AND METHODS: Human Sertoli cells were isolated from fresh cadaveric tissue. Intact tissue and derived primary Sertoli cell were analyzed by immunostaining and by FACS. Sertoli cells were cultured as monolayers, or as polarized monolayers or spheroids in semipermeable transwell chambers by proprietary methods. Immunostaining was performed with GATA4 (positive Sertoli marker), and Galectin-1. The effect of Sertoli cell conditioned media on Jurkat E-6 T cell proliferation was analyzed by metabolic dyes. The effect of direct Sertoli cell contact on CFSE-stained Jurkat T cell proliferation was assessed by FACS. RESULTS: Galectin-1 and GATA4 localized to Sertoli cells in the testis cryosections, and >90% of Sertoli cell-enriched cultures expressed GATA4. Immunoblots confirmed Galectin-1 expression and secretion by Sertoli cells. Conditioned media from Sertoli monolayers, and direct Sertoli cell contact in transwells reduced Jurkat proliferation. CONCLUSIONS: The immunosuppressive Galectin-1 localizes to human Sertoli cells in testes and in vitro. T cell proliferation was suppressed by conditioned media, and by direct Sertoli cell contact. The results suggest that active immunosuppression may exist in the human seminiferous tubule and that secreted Galectin-1 could be important for this field effect.

In vitro effect of FSH and testosterone on Sertoli cell nursing function in wall lizard Hemidactylus flaviviridis (Rüppell)

The present in vitro Sertoli cell-enriched culture study was aimed to understand the precise role of FSH and testosterone either alone or in combination in regulation of Sertoli cell nursing function such as lactate production, which in turn is utilized by the germ cells as an energy substrate. The stimulatory effect of FSH on lactate production by Sertoli cell-enriched culture was significantly enhanced when FSH and dihydrotestosterone were supplemented in the medium either simultaneously or DHT was added 12 h later to FSH (0 h FSH + 12 h DHT). On the contrary, DHT alone or added 12 h prior to FSH (0 h DHT + 12 h FSH) in the incubation medium, significantly reduced the lactate secretion by Sertoli cells as compared to control or FSH alone/FSH + DHT simultaneously/0 h FSH + 12 h DHT, respectively. This provides the conclusive evidence that androgen has additive effect on nursing function of Sertoli cell-enriched clusters only when they are in an activated state in response to gonadotropin.

Male Reproductive Toxicology

R.E. Chapin and J.J. Heindel, Introduction. C. Chubb, Male Mouse Sexual Behaviour Test. M.D. Culler, In Vitro Techniques for Assessing Pituitary Secretory Function. R.A. Hess and B.J Moore, Histological Methods for Evaluation of the Testis. W.F. Blazak, KA. Treinen, and P.E. Juniewicz, The Application of Testicular Spermatid Head Counts in the Assessment of Male Reproductive Toxicity. C.C. Linder and M.D. Griswold, Stage Synchronization in Rat Seminiferous Tubules Using Vitamin A Depletion and Repletion. M.L. Meistrich and Maria E.A.B. van Beek, Spermatogonial Stem Cells: Assessing Their Survival and Ability to Produce Differentiated Cells. W.M. Generoso and W. W. Piegorsch, Dominant Lethal Tests in Male and Female Mice. M. Parvinen, J. Toppari, and J. Laehdetie, Transillumination-Phase Contrast Microscopic Techniques for Evaluation of Male Germ Cell Toxicity and Matagenicity. G.R. Klinefelter, W.R. Kelce, and M.P. Hardy, The Isolation and Culture of Leydig Cells from Adult Rats. L.B. Biegel J.C. Cook, and M.E. Hurtt, Isolation and Primary Culture of Leydig Cells. A.H. Payne and L. Sha, Purification and Primary Culture of Leydig Cells. W.W. Ku and R.E. Chapin, The Preparation and Use of Sertoli Cell-Enriched Cultures from 18-Day-Old Rats. A. Steinberger and J-P. Clinton, Two-Compartment Cultures of Sertoli Cells: Applications in Testicular Toxicology. D.A. O'Brien, Isolation, Separation, and Short-Term Culture of Spermatogenic Cells. G.L. Rehnberg, The Collection of Interstitial Fluid and Seminiferous Tubule Fluid from the Rat Testis. G.R. Klinefelter, The Isolation and Culture of Epididymal Epithelial Cells from Adult Rats. B.J Danzo, Evaluation of Protein Synthesis by the Epididymis. G.P. Toth, E.J. Read, and M.K. Smith, Utilizing Cryo Resources CellSoft Computer-Assisted Sperm Analysis System for Rat Sperm Motility Studies. V.L. Slott and S. D, Perreault, Computer-Assisted Sperm Analysis of Rodent Epididymal Sperm Motility Using the Hamilton-Thorn Motility Analyzer. R. Filler, Evaluation of Rat Epididymal Sperm Morphology. J. Williams, Semen Analysis and Fertility Assessment in the Rabbit. S.M. Schrader, General Techniques for Assessing Male Reproductive Potential in Human Field Studies.

Mouse testin: complementary DNA cloning, genomic organization, and characterization of its proximal promoter region.

Testin is a secretory protein that was initially identified from rat Sertoli cell-enriched cultures and has been suggested to be a sensitive marker to monitor the integrity of Sertoli-germ cell junctions. However, the expression of the testin gene in other species and the molecular mechanisms that govern its transcription are unknown. To address these issues, we cloned and characterized the mouse testin gene. A full-length mouse testin cDNA encoding a polypeptide of 333 amino acid residues was isolated by library screening. Sequence analysis revealed that mouse testin shares 90.1%, 58.9%, 62.2%, and 64.6% identity with rat testin and cathepsin L of mouse, rat, and human, respectively, at the amino acid level. Reverse transcription-polymerase chain reaction and Southern blot analysis demonstrated that mouse testin transcripts were predominantly expressed in the gonads. The mouse testin gene spans over 21 kilobases (kb) and contains eight exons interrupted by seven introns. Primer extension analysis and 5' rapid amplification of cDNA ends identified a major transcription start site located 134 base pairs upstream from the translation initiation codon. Analysis of a 2.3-kb mouse testin 5'-flanking region revealed that it lacked TATA and CAAT boxes, and the region was not GC rich. By the use of deletion analysis, in vitro DNase I footprinting, and site-directed mutagenesis, we identified within the proximal promoter region three closely spaced putative binding sites for GATA, sex-determining factor, and steroidogenic factor 1 that are important for testin gene transcription in mouse Sertoli (MSC-1) cells. These cis-acting elements are also present in the conserved Mullerian-inhibiting substance (MIS) proximal promoters, raising a possibility that the transcriptions of testin and MIS genes are controlled by similar mechanisms.

Hormonal regulation of inhibin B secretion by immature rat sertoli cells in vitro: possible use as a bioassay for estrogen detection.

The influences of follicle-stimulating hormone (FSH), gonadal steroids, and culture time were studied in relation to inhibin B production by Sertoli cells of immature rats cultured in vitro. Sertoli cell-enriched cultures were established from 18-day-old rats and were maintained in medium supplemented with insulin, transferrin, and epidermal growth factor at 34 degrees C. A recently developed ELISA for the measurement of inhibin B was used to assess the effects of recombinant human FSH (rh FSH), testosterone (T), and estradiol (E2) on inhibin B production and accumulation in the culture media of Sertoli cell-enriched cultures and to optimize the cell culture system to serve as a bioassay for the detection and quantification of estrogens and estrogenlike substances. Prolonging the incubation time (24, 48, or 72 hours) of Sertoli cells with control medium without rh FSH, T, or E2 resulted in a time-dependent increase of inhibin B production. Incubation with rh FSH (1, 2.5, 5, or 10 U/L) caused a dose- and time-dependent increase of inhibin B production by Sertoli cells (but not by cultured Leydig cells), reaching a plateau at 5 U/L rh FSH. Addition of T in concentrations of 2.88, 5, or 50 ng/ml to medium without rh FSH and E2 significantly lowered the daily production rate of inhibin B (P < 0.05). In contrast, addition of E2 (0.01 and 0.1 ng/ml) caused a dose-responsive increase in inhibin B production after 24 and 48 hours. The relative increment of inhibin B production induced by E2 was maximal after 24 hours in the presence of 2.5 U/L rh FSH (acting synergistically) and in the absence of T. When these conditions are implemented, the Sertoli cell culture system may serve as a bioassay for estrogenic substances, and it may reflect the possibly harmful effect they may have on spermatogenesis.

Evidence for cross-talk between Sertoli and germ cells using selected cathepsins as markers.

To examine whether proteases are possibly involved in cellular migration and/or spermiation when developing germ cells translocate across the seminiferous epithelium during spermatogenesis, in situ hybridization was used to localize messenger RNA (mRNA) transcripts of cathepsin L, D, and S in the epithelium at different stages of the spermatogenic cycle in the rat. Cathepsin L mRNA was found to localize almost exclusively near the basal lamina of the epithelium. At stages VI and VII of the cycle before spermiation, the signal of cathepsin L mRNA was so intense that it formed a complete dark precipitate near the basal lamina encircling the entire tubule. At stage VIII, the expression of cathepsin L was completely abolished, and no staining of cathepsin L mRNA was seen in the epithelium. The mRNA of cathepsin D and S was found near the basal lamina, a finding consistent with their localization in Sertoli cells and possibly primary spermatocytes in almost all stages, but peaked at stages VII-IX and VII-VIII of the cycle, respectively, at the time before and during spermiation. These results illustrate the possible involvement of these proteases in facilitating germ cell movement and spermiation. To examine whether germ cells express any of these cathepsin genes, spermatocytes with a purity of greater than 95% were isolated from 15-day-old rat testes by Percoll gradient centrifugation for reverse transcriptase-polymerase chain reaction. It was found that primary spermatocytes expressed multiple cathepsin genes, including cathepsin B, C, D, H, L, and S. Furthermore, the expression of cathepsin L by germ cells isolated from 15-day-old rats (largely spermatocytes and spermatogonia) can be stimulated by Sertoli cell-enriched culture medium in a dose-dependent manner, but not by germ cell-conditioned medium. These results reveal that germ cell function can be regulated by Sertoli cells. Moreover, these results suggest that germ cells may play an active role in the overall testicular protease expression. Also, we present evidence suggesting there is cross-talk between Sertoli and germ cells, since the expression of cathepsin L in each cell type is regulated by one another via either soluble factors or cell-cell contact.

Rat testicular extracellular superoxide dismutase: its purification, cellular distribution, and regulation.

Using multiple HPLC steps, we have identified and purified a 68-kDa polypeptide (as estimated by gel permeation HPLC) to apparent homogeneity, from primary Sertoli cell-enriched culture medium, that consisted of two monomers of 35 (alpha chain) and 33 kDa (ss chain) on SDS-polyacrylamide gel running under reducing conditions. Partial N-terminal amino acid sequence analysis of these two monomers revealed sequences of NH2-DXGESGVDLADRL (SODEX-alpha) and NH2-XXDTGESGVDLADXL (SODEX-ss), which are identical to rat extracellular superoxide dismutase (SODEX) with the exceptions that SODEX-alpha and SODEX-ss are missing, respectively, four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids from their N-termini, compared to rat SODEX, suggesting that the cleavage sites of the SODEX gene in the testis are different from that of other organs. Studies by sequential use of reverse transcription and polymerase chain reaction (PCR) using two SODEX primers have demonstrated the expression of SODEX in the heart, brain, lung, kidney, epididymis, testis, Sertoli, and germ cells, with low expression in the liver and ovary and no expression in the uterus, spleen, or thymus. Nucleotide sequence analysis of this 447-base pair PCR product from Sertoli cells revealed that its sequence is equivalent to the sequence of previously published rat SODEX. During testicular maturation, the SODEX steady-state mRNA level increased significantly from 20 to 60 days of age and then declined at 90 days of age. Such an increase in the testicular SODEX expression during maturation is not likely a result of an up-regulation by germ cells, since germ cells isolated from either 20- or 60-day-old rats when cocultured with Sertoli cells failed to elicit an increase in SODEX expression in the cocultures. Using primary Sertoli cell cultures in vitro, it was found that Sertoli cell SODEX expression was stimulated by interleukin-1alpha but not by either interferon-gamma or basic fibroblast growth factor. These results illustrate that Sertoli cells as well as germ cells synthesize and/or secrete a testicular variant of SODEX that may provide essential clues to understanding superoxide radical-mediated damage in the gonad.

Creatine metabolism in the seminiferous epithelium of rats. II. Effect of modulators of cellular biochemical function on creatine secretion by cultured Sertoli cells.

The Sertoli cells have been identified as the primary locus for creatine synthesis within the seminiferous epithelium. The purpose of the studies reported here was to examine the effect of modulators of Sertoli cell function on creatine secretion by primary cultures of these cells. Sertoli cell-enriched cultures, maintained in a defined medium, secreted creatine into the incubation medium in a manner that was linear with time over at least 6 h, but which had reached a plateau within 24 h. Secretion was stimulated by physiological and toxicological modulators of Sertoli cell function. Incubation of Sertoli cell-enriched cultures in the presence of FSH (> or = 40 mU ml-1), dibutyryl cyclic AMP (> or = 0.1 mmol l-1), mono-(2-ethylhexyl) phthalate (> or = 1 mumol l-1) or cadmium (> or = 3 mumol l-1) increased the secretion of creatine into the incubation medium by at least 85% over 24 h. Creatine secretion by Sertoli cell-enriched cultures, incubated over 4 h in a balanced salt solution, was independent of exogenous L-glutamine. However, the stimulation of secretion induced by 1 mmol dibutyryl cyclic AMP l-1 was dependent on the presence of 4 mmol L-glutamine l-1 in the incubation medium, which suggests that an increase in creatine secretion occurs as a consequence of stimulated glutamine oxidation.

Creatine metabolism in the seminiferous epithelium of rats. I. Creatine synthesis by isolated and cultured cells.

The testis synthesizes creatine from both arginine and glycine precursors, but when rat testicular tissue is separated into seminiferous tubules and interstitial cells, creatine synthesis occurs only in the tubular fraction. The purpose of the work presented here was to define the locus of creatine synthesis within the seminiferous tubules, by using cell separation and culture techniques to examine synthesis in the Sertoli cells and germ cells. The total creatine content, in the cellular compartment and incubation medium, of Sertoli-germ cell co-cultures and of Sertoli cell-enriched cultures, largely free of germ cells, increased by similar amounts over a 24 h incubation period. Sertoli cell-enriched cultures incorporated radioactivity from L-[guanidino-14C]arginine and [1-14C]glycine into both creatine and its biosynthetic precursor, guanidinoacetic acid. Isolated germ cells did not incorporate radioactivity from L-[guanidino-14C]arginine into either creatine or guanidinoacetic acid when incubated at a similar density and protein concentration under similar conditions. It is concluded that the synthesis of creatine observed in isolated rat seminiferous tubules occurs within the Sertoli cells and not the germ cells.

PURIFICATION AND CELLULAR LOCALIZATION OF β 2-MICROGLOBULIN IN THE TESTIS

Using multiple high performance liquid chromatography (HPLC) steps, a protein of 12 kDa was purified to apparent homogeneity from rat Sertoli cell-enriched culture medium (SCCM). Partial N-terminal amino acid sequence analysis revealed a sequence of NH 2-IQKTPQIQVYS which is identical to β 2-microglobulin (β 2MG) previously identified in the brain. Studies by sequential reverse transcription and polymerase chain reaction (RT-PCR) indicated that β 2MG mRNA was expressed in Sertoli but not in germ cells suggesting that Sertoli cells are the source of this protein in the seminiferous epithelium behind the blood-testis barrier. The steady-state β 2MG mRNA level in Sertoli cells cultured in vitro was not affected by either follicle stimulating hormone (FSH), testosterone, estradiol, dexamethasone or several cytokines such as interleukin-1β (IL-1β), interleukin 6 (IL-6), and transforming growth factor β (TGF-β), with the exception of interferon-γ (INFγ) which induced a dose-dependent stimulation of β 2MG mRNA. The possible physiological significance of this protein in the male reproductive tract is discussed.

Purification, cDNA cloning, and developmental changes in the steady-state mRNA level of rat testicular tissue inhibitor of metalloproteases-2 (TIMP-2).

Using multiple high-performance liquid chromatography (HPLC) steps and high-performance electrophoresis chromatography (HPEC) in conjunction with an [125I]collagen film assay to identify inhibitors of metalloproteases, we have purified a 22-kDa polypeptide to apparent homogeneity from primary Sertoli cell-enriched culture medium. Partial N-terminal amino acid sequence analysis revealed that this protein is similar to the human tissue inhibitor of metalloproteases-2 (TIMP-2). To determine the similarity of rat testicular TIMP-2 to the human homolog, a full-length cDNA coding for rat testicular TIMP-2 was isolated from a rat Sertoli cell cDNA expression library and sequenced. Analysis of the nucleotide sequence and the deduced amino acid sequence of the rat testicular TIMP-2 cDNA revealed an 84 and 98% homology with the human TIMP-2 nucleotide and amino acid sequences, respectively. A survey of its mRNA transcripts in different tissues by northern blots revealed the presence of two mRNA species of 3.7 and 1.3 kb in the testis and brain but not in the kidney, spleen, epididymis, and liver in adult male rats. Studies using polymerase chain reaction (PCR) and Southern blot to detect the TIMP-2 mRNA using total RNA isolated from germ cells, Sertoli cells, and Leydig cells have shown that only Sertoli and Leydig cells expressed TIMP-2 mRNA. These results indicate that Sertoli cells are the major source of TIMP-2 in the testis behind the blood-testis barrier (seminiferous tubule barrier). During testicular development from 3 to 60 days of age, the testicular steady-state TIMP-2 mRNA level increased steadily with an advancement of age. Such an increase in the steady-state testicular TIMP-2 mRNA level apparently is not the result of an up-regulation by germ cells because germ cells cocultured with Sertoli cells failed to elicit an increase in the Sertoli cell steady-state TIMP-2 mRNA level. The results of this study suggest that TIMP-2 secreted by Sertoli cells may play a role in tissue restructuring and germ cell migration during spermatogenesis.

The immunohistochemical localization of alpha 2-macroglobulin in rat testes is consistent with its role in germ cell movement and spermiation.

alpha 2-Macroglobulin (alpha 2-MG) is a nonspecific protease inhibitor and binding protein for peptide hormones that was recently isolated from Sertoli cell-enriched culture medium and shown to be the same protein as alpha 2-MG in serum. The present study was conducted to determine the localization of alpha 2-MG in the seminiferous epithelium in order to gain insight into its possible site(s) of action. Immunostainable alpha 2-MG was present in the lumen of the tubules consistent with its proposed role as a protease inhibitor needed to inactivate the protease released from defective spermatozoa in the male reproductive tract. Immunoreactive alpha 2-MG was also localized adjacent to the heads of elongated spermatids, the most mobile cells in the seminiferous epithelium; immunostainable alpha 2-MG was not observed adjacent to round spermatids and spermatocytes, which are relatively less mobile. The intensity of the staining around the elongated spermatids was dependent on the stage of the spermatogenic cycle. Stainable alpha 2-MG was present adjacent to the spermatids in stage XI soon after the elongation process began. Immunoreactive product was in stages XI-XIV but only faintly visible. The most intense staining reaction for alpha 2-MG was in stages I-VI; it was reduced in stage VII; and virtually no alpha 2-MG was detectable in stages VIII-X at and just after spermiation. The postnatal changes of alpha 2-MG in the testis was also examined. During the first 2 weeks after birth, alpha 2-MG was not detected in the seminiferous epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of Gonadotropin Surge-Inhibiting Factor from Sertoli Cell-Enriched Culture Medium

Gonadotropin surge-inhibiting factor (GnSIF) is a novel biological factor in follicular fluid that inhibits the pre-ovulatory LH and FSH surges. Recent studies suggest that the biological activity of GnSIF can be attributed to inhibin. Preliminary studies showed that the Sertoli cell-conditioned medium contains GnSIF-like biological activity. Using 32 liters of primary Sertoli cell-enriched culture medium and an in vitro pituitary bioassay, GnSIF was isolated. GnSIF is a single monomeric polypeptide (Mr of 37,000) with a partial N-terminal amino acid sequence of NH 2-SDXXPQL which is distinct from any existing protein sequences. The identity of the inhibin that was separated from GnSIF was confirmed by direct protein sequencing. The purified GnSIF inhibited GnRH-stimulated LH- and basal FSH-release from pituitary cells cultured in vitro dose-dependently with a half maximal effective dose of about 1.4 ng/ml and 3.5 ng/ml, respectively. The half maximal effective dose of inhibin to suppress the pituitary GnRH-stimulated LH- and basal FSH-release was 2.5 ng/ml and 0.8 ng/ml, respectively. This study demonstrates that GnSIF is a unique protein that shares some biological activities with inhibin.

Developmental regulation of the thyroid hormone receptor alpha 1 mRNA expression in the rat testis.

The multiplicity of thyroid hormone (TH) effects appears to be mediated by two TH receptors (THRs) encoded by two genes, alpha and beta, and, perhaps, by their various isoforms. The expression of THR beta is correlated with the presence of high affinity binding sites for TH, and all the mutations which cause the syndrome of generalized thyroid hormone resistance occur in THR beta. The function of THR alpha has not been clearly defined as yet. Another enigma in TH action is the effect on the testis. It has been shown that the testis of the adult rat does not respond to TH as measured by an increase in oxygen consumption. Furthermore, it has not been possible to demonstrate the presence of a nuclear high affinity binding site for TH in adult testis. To resolve these problems were measured the levels of THR alpha, its nonhormone binding variant, and THR beta mRNA in the testis at various stages of development. We discovered that the beta-message is absent at all times, whereas the alpha-message is expressed only from fetal through prepubertal stages and is absent in adult testis. THR alpha, but not the beta-mRNA, was detected in immature Sertoli cells in culture, and neither was found in adult Sertoli cell-enriched cultures. Furthermore, THR alpha and its variant mRNA was found, using in situ hybridization, in the seminiferous cords and seminiferous tubules of fetal and prepubertal testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of follistatin messenger ribonucleic acid in Sertoli cell-enriched cultures: regulation by epidermal growth factor and protein kinase C-dependent pathway but not by follicle-stimulating hormone and protein kinase A-dependent pathway

In this study the localization and regulation of steady-state follistatin messenger ribonucleic acid (mRNA) levels in testicular cell cultures were examined with a solution-hybridization assay using a specific 32P-labelled cytosolic RNA antisense probe for follistatin and a 35S-labelled cytosolic RNA antisense probe for cyclophilin as internal standard. Testes from immature rats were dispersed with collagenase and fractionated in Sertoli and Leydig cell-enriched cultures. Follistatin mRNA was mainly localized to the Sertoli cell-enriched fraction and the expression of follistatin mRNA could be stimulated in vitro with fetal calf serum, epidermal growth factor or phorbol-12-myristate-13-acetate (an activator of protein kinase C), whereas follicle-stimulating hormone and forskolin (an activator of protein kinase A) had no effect. Neither prostaglandin E2, the synthetic glucocorticoid RU 28362 or all-trans-retinoic acid, which all regulate follistatin mRNA levels in non-testicular cell types, nor extracellular adenosine triphosphate (a purinergic receptor agonist) or testosterone had any obvious influence on follistatin mRNA levels in Sertoli cell-enriched cultures. From this study it is concluded that Sertoli cells are likely to be the source of follistatin expression in the rat testis, that follistatin mRNA levels in Sertoli cell-enriched cultures are subjected to regulation by epidermal growth factor and the protein kinase C-dependent pathway but are not regulated by extracellular adenosine triphosphate, follicle-stimulating hormone, all-trans-retinoic acid, prostaglandin E2, forskolin, testosterone or the glucocorticoid RU 28362 and that the regulation of follistatin mRNA is sex- and tissue-specific.

Testicular and germ cell toxicity: In vitro approaches

Research on testicular toxicology has been advanced significantly by the introduction of in vitro testing systems. In vivo systems, however, are still essential parts of the risk assessment process, and they are unlikely to be eliminated by in vitro model systems. While in vivo systems are needed to study the integrated male reproductive system, in vitro systems are uniquely suited to investigate specific mechanisms of action in the testis. In vitro systems substantially improve the interpretation and use of in vivo systems. In vitro models can be used alone or in combination with each other to test hypotheses about testicular toxicity. Numerous systems are described in the literature, including Sertoli-germ cell cocultures, Sertoli cell-enriched cultures, germ cell-enriched cultures, Leyding cell cultures, and Leydig-Sertoli cell cocultures. These systems have been used to test relative toxicologic activity of selected chemicals in a class, to investigate the cellular response to certain toxicants, to study the metabolic capability of cells, and to describe the interaction of adjacent cell types.

Regulation of Sertoli Cell α2-Macroglobulin and Clusterin (SGP-2) Secretion by Peritubular Myoid Cells1

alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)