Biomedical subjects

M D Griswold

Publications and source records attributed to M D Griswold.

At least 37 records · Page 2Linked to original sources

PubMed1997

Specific testicular cellular localization and hormonal regulation of the PKIalpha and PKIbeta isoforms of the inhibitor protein of the cAMP-dependent protein kinase.

We have previously demonstrated that there exist two distinct genes for the thermostable inhibitor protein of the cAMP-dependent protein kinase, PKIalpha and PKIbeta (Van Patten, S. M., Howard, P., Walsh, D. A., and Maurer, R. A. (1992) Mol. Endocrinol. 6, 2114-2122). We have also shown that in the testis, at least eight forms of PKIbeta exist, differing as a result of at least post-translational modification and alternate translational initiation (Kumar, P., Van Patten, S. M., and Walsh, D. A. (1997) J. Biol. Chem. 272, 20011-20020). We now report that in the testis, there is a unique cellular distribution of protein kinase inhibitor forms, with PKIbeta being essentially (if not exclusively) a germ cell protein and PKIalpha being expressed primarily in Sertoli cells. Furthermore, there is a progressive change in the forms of PKIbeta that are present within germ cells with development that is initiated in testis tubules and continues as the germ cells migrate through the epididymis. These conclusions are derived from studies with isolated cell populations and with the at/at germ cell-deficient mouse line, by in situ hybridization, and by following the developmental expression of these proteins in both testis and epididymis. We have also shown that follicle-stimulating hormone (FSH) can increase the expression of both PKIalpha and PKIbeta. The FSH-regulated expression of PKIalpha in the Sertoli cell likely occurs via the normal route of second messenger signal transduction. In contrast, the FSH-dependent PKIbeta expression must arise by some form of Sertoli cell-germ cell intercommunication.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Follicle-stimulating hormone (FSH) regulates the expression of FSH receptor messenger ribonucleic acid in cultured Sertoli cells and in hypophysectomized rat testis.

FSH acts on Sertoli cells via interaction with a transmembrane receptor (FSHr). Control of expression of the receptor is surely a factor in the regulation of the action of FSH. The regulation of FSHr by FSH and testosterone was studied both in culture and in vivo. Sertoli cells from 18- to 20-day-old male rats were cultured in the presence of 25 ng/ml ovine (o) FSH. At 8 h after addition of FSH, expression of FSHr mRNA decreased significantly. Addition of FSH and actinomycin D to cells did not result in a further decrease in FSHr mRNA levels, suggesting that FSH does not alter turnover of FSHr mRNA. Treatment of cells with 40 ng/ml testosterone did not have any significant effect on the expression of FSHr mRNA. Hypophysectomy of 20-day-old male rats resulted in an increase in expression of FSHr mRNA as compared to that in sham-hypophysectomized animals. This increase was measured at 24 h posthypophysectomy and was maintained at 72 h after surgery. Injection of rats with 0.2 U oFSH at 48 h posthypophysectomy resulted in a reduction in FSHr mRNA when compared to the levels in hypophysectomized rats. Treatment with 2 mg testosterone propionate had no effect on FSHr mRNA levels. The findings confirm that FSH plays an important role in regulating mRNA expression of the FSHr in Sertoli cells in culture and show for the first time that FSHr mRNA is regulated in vivo by FSH in the immature rat testis.

Animals↗

PubMed1997

Accumulation of clusterin/sulfated glycoprotein-2 in degenerating pachytene spermatocytes of adult rats treated with methoxyacetic acid.

Clusterin is a ubiquitous glycoprotein that is produced constitutively by Sertoli cells at relatively high amounts. Its association with apoptosis, damage, disease, and repair in nongonadal tissues led us to investigate whether clusterin could be part of a damage-induced response in Sertoli cells brought on by apoptosis of an adjacent cell type. Therefore, the objective of this study was to treat adult rats with methoxyacetic acid (MAA) to selectively destroy pachytene spermatocytes, examine the localization and expression of testicular clusterin, and relate this to the timing of DNA fragmentation, a hallmark of apoptosis. Clusterin protein was localized to the cytoplasm of pachytene spermatocytes at 6 h post-MAA, whereas clusterin mRNA was localized to Sertoli cells. Morphological degeneration of dying cells and DNA fragmentation were not seen until 12 h. Thus, Sertoli cell-derived clusterin had accumulated in the cytoplasm of degenerating spermatocytes early in the apoptotic process. On the basis of these results and the known binding of clusterin to hydrophobic macromolecules, we hypothesize that clusterin is produced by Sertoli cells as a mechanism to "clear" potentially harmful cellular components during the degeneration of germ cells and remodeling of their membranes that occur normally during spermatogenesis.

Acetates↗

PubMed1996

Role of E box and initiator region in the expression of the rat follicle-stimulating hormone receptor.

The promoter for the rat follicle-stimulating hormone receptor (FSHR) gene contains a conserved consensus E box sequence and an initiator-like region (InR) sequence. Deletion analysis and transient transfections showed that a 114-base pair region (-143 to -30) that encompasses the E box and the InR was sufficient for greater than 75% of promoter function. DNase I footprint analysis showed that the E box and InR were protected by nuclear proteins from rat Sertoli cells, and the E box region was shown by electrophoretic mobility shift assays (EMSA) to be a site of Sertoli protein interactions. Mutations in the E box disrupted these interactions and reduced FSHR promoter activity. Co-transfection of the inhibitor of DNA binding (Id) with an FSHR/luciferase construct into mouse Sertoli 1 cells reduced FSHR promoter activity. Using EMSA, the upstream stimulatory factor was shown to be a component of the complexes that interacted with the E box in the FSHR promoter. Binding of proteins from rat Sertoli cells to the InR was demonstrated using EMSA. Also, an oligonucleotide that represented the sequence of the terminal deoxynucleotidyltransferase InR displaced the complexes at the FSHR InR. Mutations in the InR resulted in a significant reduction of FSHR promoter activity.

Animals↗

PubMed1996

Steady-state luteinizing hormone receptor messenger ribonucleic acid levels and endothelial cell composition in bovine normal- and short-lived corpora lutea.

The short-lived corpus luteum (CL) contributes to reproductive inefficiency during the postpartum period in beef cows. The cause for the early demise of the short-lived CL is not fully understood but is believed to involve a premature release of prostaglandin F2 alpha. The objectives of this study were to evaluate norgestomet-hCG-induced normal-lived CL and hCG-induced short-lived CL in postpartum cows with respect to serum progesterone (P4) and 13,14-dihydro-15-keto, prostaglandin F2 alpha (PGFM) concentrations and luteal LH receptor (LH-R) concentrations, LH-R mRNA levels, and vascularity. Although serum P4 profiles from the time of hCG administration (Day 0) until luteectomy (Day 6, 7, or 8) were similar between CL life span groups, PGFM concentrations were elevated (p < 0.05) on Day 8 in cows expected to have short-lived CL compared to normal-lived CL. The LH-R concentrations were similar between normal- and short-lived CL on all days measured. Irrespective of luteal life span and day of luteectomy, all CL possessed a 4.4-kb LH-R transcript. Actin-normalized LH-R mRNA levels were similar between normal- and short-lived CL on Days 6 and 7; however, Day 8 short-lived CL contained less (p < 0.05) LH-R mRNA than Day 8 normal-lived CL. Although the area of luteal tissue occupied by capillaries in normal- and short-lived CL was similar on Days 6 and 7, the area occupied by capillaries in short-lived CL was lower (p < 0.05) than that for normal-lived CL on Day 8. Collectively, these results indicate that there is a decrease in steady-state LH-R mRNA and a reduction in luteal vascularity in CL expected to be short-lived. These changes occur concomitantly with a rise in serum PGFM, but prior to a decline in serum P4.

Animals↗

PubMed1995

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

Animals↗

PubMed1995

The molecular biology of the FSH receptor.

The actions of follicle stimulating hormone (FSH) mediated through its receptor are necessary for the proper functioning of mammalian gonads. The FSH receptor is localized on granulosa cells of the ovary and Sertoli cells of the testis. The expression of the FSH receptor (FSHR) in Sertoli cells varies in vivo as a function of the stage of the cycle of the seminiferous epithelium and in culture as a result of the addition of exogenous hormones. The gene for the FSH receptor is large and has been shown to be related in structure to the genes for luteinizing hormone (LH) receptor and thyroid stimulating hormone (TSH) receptor. The promoter region of the gene for FSHR does not contain a TATA box and has multiple transcriptional start sites. Less than 280 bp of the promoter are sufficient in transient transfection assays to direct expression of the chloramphenicol acetyl transferase gene (CAT) in a number of different cell types including non-gonadal cells. However, the promoter does direct the expression of a marker gene only into testis and ovary of transgenic mice.

Female↗

PubMed1995

Interactions between germ cells and Sertoli cells in the testis.

The "nurse cell" function of Sertoli cells in spermatogenesis was originally tacitly assumed on the basis of the anatomical relationships between cells in the testis. In mammals, from very early in prenatal development to the onset of meiosis and to the ultimate production of spermatozoa, the relationship between the germinal cells and the Sertoli cells is important and apparently obligatory. The experimental evidence supports the notion that the primary endocrine regulation of spermatogenesis via FSH and testosterone is manifested through actions on the Sertoli cells. Although diverse strategies are used by Sertoli cells to support germ cell development, one of the most important roles of Sertoli cells is the regulation of the intratubular and intercellular environment adluminal to the tight junctional complexes. The meiotic and post-meiotic germ cells are sequestered by Sertoli-Sertoli junctional complexes in an adluminal compartment that is isolated from the serum or lymph. As a result of this sequestering activity, the secretion products of the Sertoli cells and the meiotic germ cells determine the composition of this local environment that can influence meiosis as well as spermatid and spermatocyte development. Evidence is accumulating that paracrine and autocrine factors from Sertoli and germ cells are important in the functioning of both cell types. While it is important to know what Sertoli cells and germ cells make, it is equally important to know when they make it. Distinct but well-defined groups of germ cells interact with Sertoli cells in a cyclic pattern. These recurring groups of germ cells define the cycle of the seminiferous epithelium during which sperm are produced in an asynchronous fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Isolation and culture of Sertoli cells from the testes of adult Siberian hamsters: analysis of proteins synthesized and secreted by Sertoli cells cultured from hamsters raised in a long or a short photoperiod.

In the present study, we describe two procedures for isolation and culture of Sertoli cells from the testes of adult Siberian hamsters. In procedure I, collagenase and pancreatin were used for differential enzymatic digestion of the tissue at 32 degrees C, and effects of several attachment factors were examined. Sertoli cells isolated from adult hamsters were not affected by addition of Na selenite, epidermal growth factor, insulin, and transferrin, with or without 5% fetal bovine serum, to Dulbecco's modified Eagle's medium + Ham's F-12. Significant increase in the yield and plating efficiency of Sertoli cells was achieved by developing a novel procedure (procedure II) for Sertoli cell isolation. In this procedure, testicular tissue was exposed to only one enzyme (collagenase I) for two consecutive digestions at 37 degrees C, and the total period of enzymatic exposure was reduced relative to that of procedure I. Another objective of this study was to compare the functions of Sertoli cells isolated from spermatogenetically active and inactive adult testes. Isolated Sertoli cells from 60 +/- 5-day-old hamsters raised in long day conditions (LD, 16L:8D) or in short day conditions (SD, 6L:18D) were stimulated with FSH and testosterone in the presence of 35S-methionine for 24 h on Day 4 of culture. The medium was concentrated, and equal amounts of radioactive proteins from LD and SD hamster Sertoli cell cultures were analyzed by two-dimensional PAGE. Compared to Sertoli cells derived from SD hamsters, Sertoli cells from LD animals produced greater amounts of two secretory proteins and a smaller amount of one.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Activity and form of sulfated glycoprotein 2 (clusterin) from cultured Sertoli cells, testis, and epididymis of the rat.

Sulfated glycoprotein 2 (SGP 2) is a 73-kDa highly glycosylated disulfide-linked heterodimer. It is a major secreted protein of Sertoli cells, is found in high abundance within the seminiferous tubule fluid (STF) and epididymal fluid (EPF), and can be found on the surface of spermatozoa. Due to its high abundance and location it is believed to play a major role in the development of spermatozoa; however, its specific function(s) within the reproductive tract is not known. Purified and renatured SGP 2 were found to have the ability to inhibit complement activity with a mean concentration of 66 mg/ml for 50% inhibition. Extraction of epididymal sperm with various reagents showed that a major fraction of the SGP 2 in EPF was free or was loosely associated with the spermatozoa whereas a smaller fraction was more tightly associated and disruption of the lipid bilayer was required for its complete removal. Ultracentrifugation techniques and gel permeation chromatography revealed that SGP 2 in plasma, STF, and EPF formed complexes with other proteins and/or lipids but was not specifically associated with the apolipoprotein-like particles containing apolipoprotein A1 (apo A1).

Animals↗

PubMed1994

Relationship of a mouse Sertoli cell line (MSC-1) to normal Sertoli cells.

Recently, a mouse Sertoli cell line (MSC-1) was established from transgenic mice carrying a fusion gene composed of human Müllerian inhibitory substance transcriptional regulatory sequences linked to the SV40 T-antigen gene. This cell line contained a morphologically heterogeneous population of cells that expressed characteristic Sertoli cell mRNAs such as transferrin, the beta-subunit for inhibin, and sulfated glycoprotein-2 (SGP-2). In this study, we compared various characteristics of MSC-1 cells to primary cultures of Sertoli cells from immature rats or adult mice. Our observations indicated that: 1) These cells were ultrastructurally similar to mouse Sertoli cells in culture; 2) MSC-1 cells expressed mRNA for androgen-binding protein (ABP) and SGP-1, but not the receptor for FSH; 3) The expression of SGP-2 mRNA and secretion of SGP-2 increased approximately 2-fold when cells were cultured at 41 degrees C, the nonpermissive temperature for the SV40 virus, while SGP-1 and transferrin mRNA levels and secretion were unaffected; 4) Proliferation of these cells was serum-dose dependent and temperature dependent and could be inhibited by incubating MSC-1 cells at 41 degrees C; 5) Proliferation was also significantly reduced after cells were incubated in the presence of dibutyryl cAMP (dbcAMP) for 6 days; 6) Fifteen subcultures produced from single MSC-1 cells displayed similar levels of mRNA expression for transferrin or SGP-1. Together these data indicate that the MSC-1 cell line is composed of a single cell type displaying numerous characteristics of Sertoli cells. This cell line may provide a unique source of tissue for studying various aspects of Sertoli cell behavior.

Androgen-Binding Protein↗

PubMed1994

Accessibility of rat and human follitropin receptor primary sequence (R265-S296) in situ.

The primary sequence of FSH receptor (FSHR) is homologous to LH and TSH receptors (LHR and TSHR). This family of receptors belong to the G-protein-coupled class of membrane-bound receptors. A very large extracellular domain suggests that interaction of ligand with receptor is likely to be complex. Secondary structure analysis of the FSHR R265-S296 primary sequence, which has little homology to LHR, predicted a helix-turn-helix motif. An objective of these studies was to test directly the hypothesis that FSHR R265-S296 is accessible in FSHR and plays a role in hormone binding. Rat FSHR (rFSHR) was expressed in insect cells and used as a source of receptor for binding studies. Recombinant receptor had a Kd in the picomolar range with about 200,000 receptors/cell and appeared as two forms (180 and 75 kilodaltons) by Western blot analysis. Functional coupling of the rat FSHR to adenylate cyclase in insect cells was demonstrated. Antipeptide antibodies against FSHR R265-S296 inhibited binding of radiolabeled hFSH to insect cell rat FSHR. In contrast, neither nonimmune rabbit serum nor antipeptide antibodies against FSHR G150-L183 inhibited the binding of radiolabeled hFSH to rat FSHR in insect cells. Similar results were obtained with recombinant human FSHR in Y1 cells, measuring progesterone production as an end point. Confocal microscopy using antihuman FSHR R265-S296 demonstrated that recombinant human FSHR on Chinese hamster ovary cells existed as discrete patches on the surface. In summary, the data offer compelling evidence that portions of the peptide sequence FSHR R265-S296 are accessible to the antipeptide antibodies and may be involved in hormone binding.

Adenylyl Cyclases↗

PubMed1993

Expression of the FSH receptor in the testis.

FSH has multiple and changing roles in the regulation of spermatogenesis. The first function of FSH is to increase the number of Sertoli cells by stimulation of their mitotic activity. During the prepubertal phase of development, FSH is important for the maturation of the Sertoli cells. Hormonal stimulation of tight junction formation and specific protein secretion are essential. In the adult rat, some of the functions carried out by FSH in prepubertal animals are assumed by testosterone. However, there is evidence that even in the adult rat, FSH is important for quantitatively normal spermatogenesis. The gene for the FSH receptor is large (greater than 85 kb) and complex (10 introns) and is structurally similar to the genes for the LH and TSH receptor. The promoter region of the FSHR gene has been identified and is active in the expression of transgenes in transfected Sertoli cells. We have shown that the FSH receptor mRNA is present in the testes of the adult rat and that the levels of this mRNA are changing during the cycle of the seminiferous epithelium. The presence of relatively high levels of FSHR mRNA in stages XIV-II of the cycle and the relatively low levels in stages VII-VIII suggest that the FSH receptor is carefully regulated in adult rats and presumably has an important function in spermatogenesis. The levels of FSHR mRNA in cultured Sertoli cells are immediately reduced in the presence of FSH or phorbol esters, but the levels soon return to normal.

Animals↗

PubMed1993

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Animals↗

PubMed1993

Localization and molecular heterogeneity of sulfated glycoprotein-2 (clusterin) among ventral prostate, seminal vesicle, testis, and epididymis of rats.

In the rat reproductive tract, sulfated glycoprotein 2 (SGP-2) is present in the ventral prostate, seminal vesicle, testis, and epididymis. In the ventral prostate, SGP-2 is associated with the process of programmed cell death, while in the testis and epididymis a role for SGP-2 in sperm maturation has been proposed. Available information suggests that there are both inter- and intra-organ variations in SGP-2 localization, molecular forms, and response to androgen ablation. In the present study, localization of SGP-2 within the ventral prostate, seminal vesicle, and epididymis was compared by immunohistochemistry. In the ventral prostate of intact rats, immunoreactive SGP-2 was confined to a discrete population of epithelial cells lining the proximal ducts. Epithelial cells in other regions of the ventral prostate did not stain for SGP-2. A similar staining pattern was observed for the seminal vesicle; a small population of SGP-2-expressing epithelial cells was found in epithelium that did not stain for SGP-2. The epididymis also demonstrated a non-uniform staining pattern. The caput displayed strong immunoperoxidase reaction over the apical membrane and stereocilia of all principal cells. Principal cells also showed variable degrees of cytoplasmic staining ranging from weak to strongly positive. The corpus and cauda showed a similar staining pattern. After castration, all epithelial cells in the ventral prostate and seminal vesicle became intensely positive for SGP-2 staining. In the caput and cauda epididymis there was an increase in the number of principal cells demonstrating strong intracellular staining for SGP-2. These results suggest that as observed previously in the regressing ventral prostate, increased intracellular SGP-2 staining may also be associated with the regressing epididymis and seminal vesicle. Differences in molecular forms of SGP-2 were investigated by two-dimensional Western and lectin blots. Molecular forms of SGP-2 differed between testis and epididymis but were similar between ventral prostate and seminal vesicle. Prostate and seminal vesicle forms of SGP-2 differed from those of both testis and epididymis. Analysis of terminal carbohydrate present on the various SGP-2 molecular forms also confirmed the existence of heterogeneity. These results demonstrate the presence of multiple molecular forms of SGP-2 in various organs of the male reproductive tract in rats and suggest a possible variation in functional activity and/or half-life of SGP-2 in these organs.

Animals↗

PubMed1993

Expression of prohibitin in rat seminiferous epithelium.

Subtractive hybridization was used to isolate cDNAs highly expressed in stages IX-XI of the cycle of the seminiferous epithelium in the rat. One of the cloned cDNAs was sequenced and shown to be homologous to a previously described cDNA encoding rat prohibitin. Northern blot analyses showed that 1.9- and 1.2-kb transcripts were present in Sertoli cells whereas 1.5-, 1.2-, and 0.7-kb transcripts were expressed in germ cells. Western blot analyses with anti-peptide antibody to prohibitin revealed only a single 30-kDa protein in testis. Immunocytochemistry demonstrated that prohibitin protein was expressed constitutively in adult Leydig cells and Sertoli cells at all stages. Immunoreactivity of prohibition was very low in preleptotene spermatocytes, very high in leptotene spermatocytes, and very low in zygotene spermatocytes. In pachytene spermatocytes, immunoreactivity was very high in stages VII-XI and was minimal during stages XII and XIV. No protein was detected in spermatogonia and spermatocytes undergoing mitotic and meiotic divisions, respectively. These studies show that the prohibitin gene is expressed differentially in testis. The expression pattern of the prohibitin gene in rat testis appears to correlate with a proposed antiproliferative role of prohibitin.

Aging↗

PubMed1993

Sertoli cell and germ cell cystatin C: stage-dependent expression of two distinct messenger ribonucleic acid transcripts in rat testes.

Sertoli cells were shown to synthesize and secrete cystatin C, a potent inhibitor of cysteine proteases. The evidence for this observation was obtained from protein sequencing. Western analysis using antiserum specific to cystatin C, and immunoprecipitation of 35S protein secreted by cultured cells. The Western analysis with an antiserum to human cystatin C showed that cultured Sertoli cells secrete three previously reported immunoreactive forms of cystatin C: a predominant pair of proteins at 13-14 kDa and a less abundant 20-kDa protein. Immunohistochemical localization of cystatin C in sections of rat testes showed intense staining in Sertoli cells; no immunoreactivity was observed in spermatogonia or spermatocytes. A cDNA fragment for rat cystatin C was obtained by use of the polymerase chain reaction and was used as a probe in Northern analyses to examine the steady-state levels of cystatin C mRNA in intact testes and in Sertoli and spermatogenic cells. Sertoli cells contained a 700-nucleotide cystatin C transcript, and a mixed population of spermatids and spermatocytes contained a 550-nucleotide transcript. Analysis of RNA from purified spermatogenic cells revealed that round and condensing spermatids contained the 550-nucleotide transcript, while pachytene spermatocytes contained a smaller 500-nucleotide transcript. The 700-nucleotide transcript was present in testes isolated from rats of 5-79 days of age, the 500-nucleotide transcript was detected initially in testes from 24-day-old rats, and the 550-nucleotide transcript was detected initially at 35 days of age. Both the 500- and 550-nucleotide transcripts increased in abundance until 50 days of age. RNA from stage-synchronized testes showed that steady-state levels of both the 550- and 700-nucleotide transcripts were lowest in stages VI-VII of the cycle. These data suggest that the role of cystatin C in the testis may be to inhibit the proteolytic activity of the cysteine protease cathepsin L in all stages except stages VI-VII.

Amino Acid Sequence↗

PubMed1993

Basic fibroblast growth factor is a testicular germ cell product which may regulate Sertoli cell function.

Previously, a proteinacious factor secreted by a mixture of rat testicular spermatocytes and round spermatids was shown to stimulate transferrin mRNA and protein levels in Sertoli cells. To identify the germ cell-secreted proteins which affect Sertoli cell functions, concentrated germ cell-conditioned medium was fractionated by reverse-phase HPLC. The fraction which eluted at 35% acetonitrile increased transferrin secretion in Sertoli cell cultures 2.4-fold above the basal level. Both the active fraction and a protein extract from cultured germ cells were positive for basic fibroblast growth factor (bFGF) as determined by Western blot analysis and immunoprecipitation. The apparent molecular sizes of the immunoreactive proteins were 30, 27, and 24 kilodaltons (kDa). By immunohistochemistry, bFGF was shown to be present in pachytene spermatocytes and Leydig cells. The bFGF receptor was also examined by immunohistochemistry and found to be present in Leydig cells, round and elongated spermatids, and Sertoli cells. The presence of receptors was more pronounced in stages I-VIII. Western blot analysis confirmed that the receptors were expressed in isolated round spermatids, elongated spermatids, and Sertoli cells. Two major receptor species with apparent molecular sizes of 120 and 145 kDa were detected in the rat testis. Germ cells contained both of these receptors, but Sertoli cells possessed only the 120-kDa receptor. From these experiments, it is evident that bFGF is a germ cell product which may regulate Sertoli cell function. The expression of bFGF and its receptor may be an important component of germ cell-Sertoli cell and/or germ cell-germ cell communication during spermatogenesis.

Animals↗