PubMed Health⌕ Search

Biomedical subjects

M D Griswold

Publications and source records attributed to M D Griswold.

At least 55 records · Page 3Linked to original sources
PubMed1992

Testicular synchrony: evaluation and analysis of different protocols.

Using the vitamin A depletion-replacement rat model to obtain testicular synchrony, we examined the reproducibility and degree of synchronization obtained by two different protocols. In the original protocol (A), synchrony was achieved by use of retinol alone. In protocol B, retinoic acid was used during the final days of vitamin A depletion as a supplement to retinol. With protocol A, a total of 56 rats were analyzed by an adaptation of a previously published method for quantifying synchrony. Animals treated by protocol A demonstrated a reproducible degree of synchrony although variability was high among individual animals. A smaller group of animals treated with protocol B demonstrated a lower degree of synchrony. In contrast, the midpoint of synchrony (point in the cycle at which 50% of the stages are more advanced and 50% are less advanced) was a more constant value and was not different between the two treatments. The midpoints of synchrony obtained from both protocols were used to calculate a cycle duration of 300 h for our strain of Sprague-Dawley rats. Our results indicate that while the use of either protocol can reproducibly provide testicular synchrony, protocol A results in a higher degree of synchrony. The ability to synchronize testes to selected stages provides sufficient experimental material for the study of the molecular and cellular events of spermatogenesis.

Animals↗
PubMed1992

Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis.

Three murine epididymal secretory proteins have been characterized by their site of synthesis, sperm association, and tissue localization by use of polyclonal antisera and immunochemistry. Mouse epididymal protein 7 (MEP 7) was localized initially within the supranuclear regions of some principal epithelial cells in the proximal corpus while other cells remained unstained. In the mid-proximal corpus, all principal cells and stereocilia were stained, and luminal staining increased from corpus to cauda. Some clear cells in the distal corpus and cauda also showed immunoperoxidase staining. Sequential extraction of caudal spermatozoa indicated that MEP 7 was predominantly loosely associated with spermatozoa and that only a small amount of MEP 7 required detergent to extract it from spermatozoa. Examination of other rodent caudal fluids revealed a related protein in rat caudal fluid of 32 kDa, and amino acid sequence analysis of MEP 7 showed a 68% sequence similarity with rat proteins AEG and D/E. MEP 9 immunolocalized within the cytoplasm of all principal cells of the distal caput. In a transition zone between the distal caput and the corpus, some principal cells were stained while others were not. Distal to the corpus, the principal cell staining gradually decreased. In the distal caput and proximal corpus, large heavily stained droplets associated with spermatozoa were seen in the lumen. The staining intensity of these droplets also decreased from corpus to cauda. The clear cells of the distal corpus and cauda did not stain with the antibody to MEP 9. Sequential extraction of caudal spermatozoa showed that some MEP 9 was extractable under low-salt conditions, whereas extraction with 0.1% Triton X-100 was required to remove all MEP 9, indicating it was firmly associated with spermatozoa. The antibody to MEP 9 cross-reacted with a 25-kDa protein present in rat caudal fluid. MEP 10 was localized within the cytoplasm of the principal cells, the stereocilia, and the lumen of the epididymis at the junction of the distal caput and corpus. In the distal corpus, a large number of clear cells were stained, but very few of these cells stained in the cauda. MEP 10 dissociated completely from caudal spermatozoa under low-salt conditions, indicating that it was not firmly bound to spermatozoa. The antiserum to MEP 10 cross-reacted with proteins present in rat and guinea pig caudal fluid. The related rat protein migrated at approximately 20 kDa. Amino acid sequence analysis of MEP 10 revealed an 86% sequence similarity with rat proteins B and C.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Structural organization of the follicle-stimulating hormone receptor gene.

We have characterized a series of rat genomic clones that code for the FSH receptor (FSHR) gene and approximately 14.8 kilobases of DNA up-stream of the transcriptional start sites. Southern blot analysis indicated that there was only a single gene for the FSHR. Primer extension and S1 nuclease experiments revealed the presence of two major transcriptional start sites at positions -80 and -98 relative to the translational start site. Transient expression studies of a fusion gene containing 830 basepairs of DNA 5' to the translational start site linked to the reporter gene chloramphenicol acyltransferase have shown that this portion of the gene is capable of acting as a transcriptional promoter in rat Sertoli cells. The FSHR gene contained 10 exons and nine introns. The first nine exons encoded the extensive amino-terminal domain of the receptor, while the last exon encoded the transmembrane-spanning and cytoplasmic domains. A repeated motif similar to that observed in the leucine-rich glycoprotein family was delineated within exons 2-9. Comparison of the FSHR gene to the LH receptor gene revealed a number of striking similarities which clearly indicate that these receptors evolved through gene duplication. The ancestral gene for these receptors presumably arose from a series of tandem duplications of the leucine-rich motif, which when combined with the common ancestral gene of the G-protein-coupled receptor family led to the current gene structure of the glycoprotein hormone receptors.

Amino Acid Sequence↗
PubMed1991

Variations in the level of transferrin and SGP-2 mRNAs in Sertoli cells of vitamin A-deficient rats.

Stage-specific levels and long-term effects of vitamin A deficiency on transferrin and sulfated glycoprotein-2 (SGP-2) mRNAs were analyzed in normal rats and in rats fed a vitamin A-deficient diet for 49 days (49 day VAD) and 77 days (77 day VAD). Histological sections of testes from these animals were hybridized in situ with single stranded 3H-labeled RNA probes complementary to the transferrin and SGP-2 (clusterin) mRNAs prepared from specific cDNAs subcloned in the SP65 vector. In all cases the probes were visualized and quantified by radioautography. Vitamin A deficiency (49 day VAD rats) differentially affected the levels of these mRNAs by increasing significantly the level of SGP-2 transcripts and decreasing the level of transferrin mRNA. However, in both cases the stage-specific pattern characteristic of the normal testes remained cyclic indicating that the specific interactions between germ cells and Sertoli cells may play an important role in modulating the cyclic activities of Sertoli cells. The data also indicated that dividing spermatocytes, secondary spermatocytes and steps 13-14 spermatids were associated with Sertoli cells expressing high levels of transferrin mRNA, while steps 7, 8 and particularly 19 spermatids were associated with Sertoli cells expressing high levels of SGP-2 mRNA. In contrast to the shorter term, a longer term of vitamin A deficiency (77 day VAD rats) produced a significant decrease in the levels of both transferrin and SGP-2 mRNAs.

Animals↗
PubMed1991

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat.

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.

Animals↗
PubMed1991

Expression of receptors during the cycle of the seminiferous epithelium.

The data presented in this manuscript are all based on some inferences drawn from past experimental observations. The first is that the synchronized testis model is representative of the normal testis. Support for this premise comes from the studies on SGP-2, SGP-1 and transferrin where results using in situ hybridization and Northern blots are similar for normal and for synchronized testes. The second inference is that normalizing all of the data to the relative levels of SGP-1 mRNA adjusts for the possible differential loading of mRNA samples. The logic of this practice is based on the observation obtained using in situ hybridization that SGP-1 mRNA levels did not change across the cycle. The third assumption was that total mRNA levels do not change greatly across the cycle. Wholesale changes in testicular mRNA such as the doubling of all of the mRNA transcripts per testis would not be accounted for by these studies. We feel that this is an unlikely complication because of the strong correlation between much of the data and the known biology. In addition, there is a strong correlation between our data on the FSHR mRNA and the binding data for FSH obtained by another laboratory and different techniques. The available data in the literature reveals that most of the Sertoli cell products which change in relative concentrations during the cycle of the seminiferous epithelium appear to have a maximum in either stages VII or IX or in stages XIII to III. Thus, the Sertoli cells in the cycle could be described has having two different functional modes. In mode A maximal levels of mRNA for a specific Sertoli cell product are roughly found in stages VII-IX and in mode B the maximal levels are found in stages XIII to III (Fig. 5). The distribution of the receptor mRNA and ABP mRNA can also be described in terms of these two modes of Sertoli cell function. Transferrin receptors, retinoic acid receptors, and androgen-binding protein appear to fall into mode A while FSH and androgen receptors fall into mode B (Table 1). Products which have antithetical functions such as FSH and inhibin or cystatin and cathepsin L are found in different modes. We propose that most of the actions of the Sertoli cell during the cycle can be specified by the dual modes described above rather than by an infinite number of operational modes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

A transferrinlike (hemiferrin) mRNA is expressed in the germ cells of rat testis.

In the testis, germ cells which are separated from the serum by the blood-testis barrier rely primarily on the Sertoli cell to obtain nutrients. For example, transferrin synthesized by the Sertoli cell is important in delivering iron from the serum to the developing germ cells. Because of its role in the testis, Sertoli cell transferrin protein and mRNA have been extensively studied. By using RNA blot analysis of rat testicular tissue, we detected a transcript of 2.6 kb which is attributed to transferrin. In addition, we detected a novel mRNA of 0.9 kb which had sequence similarity to the 3' end of transferrin. This 0.9-kb mRNA was present in germ cells, but not Sertoli cells, liver, or brain. The primary source of this mRNA in the testis was round spermatids. Sequence analysis of a cDNA clone showed that this mRNA encoded a protein with sequence similarity to the carboxy terminus of transferrin. Polysome analysis indicated that this transcript was translated and may therefore have importance in the iron metabolism of germ cells. The evolutionary implications between the transferrinlike mRNA germ cells and the gene duplication event which resulted in the diferric binding of transferrin are discussed.

Amino Acid Sequence↗
PubMed1991

Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.

The rat prostate is a complex ductal system with branches and subbranches extending from one end to another. Owing to the relative distance of various regions from the urethra, the entire length of the ductal system can be divided into three segments, i.e. the proximal, intermediate, and distal segments. The present study was carried out to examine the pattern of localization of sulfated glycoprotein-2 (SGP-2), a marker protein associated with programmed cell death, in various regions of the prostatic ductal system under normal conditions and during castration-induced regression. SGP-2 has been considered an androgen-repressed gene product in the rat prostate and has previously been known as castration-induced protein or TRPM-2. In the normal rat prostate, immunoreactive SGP-2 was localized in epithelial cells lining the proximal segment in which signs of programmed cell death were apparent. Cells lining the distal and intermediate segments were, however, devoid of SGP-2. This observed regional variation in SGP-2 localization did not support an earlier hypothesis which stated that SGP-2 was constitutively expressed by all prostatic epithelial cells in the normal rat prostate. After castration in adult rats, there was a shift in the location of cells containing SGP-2 from the proximal segment toward the distal segment. Therefore, there is a regional variation in the distribution of SGP-2 in the rat prostate both before and after castration in the host. These findings are likely to be associated with a regional variation in cellular responsiveness to androgen stimulation and androgen depletion in the prostatic ductal system. Results also support the view that SGP-2 localization is associated with an early manifestation of programmed cell death in the rat prostate.

Androgens↗
PubMed1991

Expression of follicle-stimulating hormone receptor mRNA in rat testes and Sertoli cells.

In order to better understand the role of FSH in reproduction, we have studied the expression of its receptor in the male rat. A DNA probe for the rat FSH receptor (FSHR) was generated by the polymerase chain reaction, and a partial genomic clone was isolated. Northern blot analysis revealed two transcripts for the FSHR, a 2.6-kilobase (kb) transcript, which is the predominant mRNA, and a 4.5-kb transcript. The two transcripts were specific to the testis and ovary, and in the testis, the expression of the mRNA appeared to be primarily in the Sertoli cells. Northern blot analysis showed the presence of FSHR mRNA in testes from rats ranging in age from 10-60 days as well as in Sertoli cells cultured from 20-day-old and adult animals. FSHR mRNA levels in testes synchronized to specific stages of the seminiferous epithelium were shown by Northern blot analysis to be highest in stages XIII-II and to decrease to a minimum at stages VII-VIII. The levels of FSHR mRNA underwent more than a 3-fold change during the cycle of the seminiferous epithelium.

Aging↗
PubMed1990

Structural analysis of sulphated glycoprotein 2 from amino acid sequence. Relationship to clusterin and serum protein 40,40.

Sulphated glycoprotein 2 (SGP-2) is the major secreted protein product of rat Sertoli cells; likewise, clusterin is a major constituent of ram rete testis fluid. Isolation and sequencing of the intact subunits and peptides derived from clusterin show that it is the ram homologue of rat SGP-2. Human serum protein 40,40 (SP-40,40), a component of the SC5b-9 complex of complement, has recently been reported to be the human homologue of rat SGP-2. Analysis of the amino acid sequences of rat SGP-2 and human SP-40,40 show that both of these proteins have a significant relationship to the heavy chain of myosin. The regions of highest sequence similarity correspond to the major amphipathic domains in SGP-2/SP-40,40 and the long alpha-helical-tail domain of myosin, which forms a rod-like structure. SGP-2 has anomalous sedimentation behaviour which indicates that it probably exists in an extended conformation. A putative dinucleotide-binding structure has been identified in the longest stretch of identity between SGP-2 and SP-40,40. Elucidation of these features of SGP-2 and SP-40,40 may help to direct future studies into the role of these proteins in the reproductive and complement systems.

Amino Acid Sequence↗
PubMed1990

Synthesis of transferrin and transferrin mRNA in bovine Sertoli cells in culture and in vivo: sequence of partial cDNA clone for bovine transferrin.

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.

Amino Acid Sequence↗
PubMed1990

Seminal transferrin and spermatogenic capability in the bull.

The objective of this study was to determine the relationship between seminal transferrin and sperm output in ejaculates from mature dairy bulls. Caudal sperm reserves in mature Holstein bulls (n = 15) were depleted by 8 successive ejaculations during a 50-70-min period (depletion phase). Bulls were then ejaculated 6 times per week for a period of 4 weeks (6X phase). Weekly sperm output (WSO) and weekly transferrin output (WTfO) were the sums of sperm and transferrin levels in 6 ejaculates taken in 1 week of the study. Mean WSO ranged from 20.7 billion to 39.6 billion and mean WTfO ranged from 334 micrograms to 1872 micrograms among the bulls. Regression analysis of sperm and transferrin levels in ejaculates collected during the depletion phase indicated that approximately 40% of seminal transferrin was not related to sperm output and probably was from accessory fluids. A relationship between total seminal transferrin and total sperm in ejaculate was observed (p less than 0.01, r = 0.54). This relationship was stronger when the transferrin was corrected for accessory fluid contribution (p less than 0.01, r = 0.65). The relationship between WSO and WTfO corrected for accessory fluid transferrin contribution (cWTfO) was significant (p less than 0.01, r = 0.64). The relationship between WSO and cWTfO can be interpreted to reflect the relationship between actual testicular sperm production and transferrin from testicular or epididymal origin.

Animals↗
PubMed1990

Ethylene dimethanesulfonate inhibits the function of Sertoli cells in culture at sublethal doses.

The dose-dependent effects of ethylene dimethanesulfonate (EDS) on transferrin production and viability of Sertoli cells in culture have been investigated. Sertoli cells, isolated from the testes of 20-day-old rats, exhibited decreased transferrin production in response to EDS at a dose of 0.3 mM. The suppression of transferrin synthesis by EDS reached a maximum (15% of control) with a dose of 1.1 mM, without significant cell mortality. An EDS dose of 2.7 mM proved lethal to Sertoli cells, reducing the number of viable cells per well by 85%. Sertoli cells preincubated with a sublethal dose of EDS (0.3-0.5 mM) appeared to recover upon removal of EDS from the culture medium, as evidenced by an increase in transferrin mRNA levels. Sertoli cells given 2.7 mM EDS did not recover. These data demonstrate a direct cytotoxic effect of EDS on Sertoli cell function at relatively low doses and a lethal effect of the alkylating agent at high doses. These results also indicate that EDS may affect Sertoli cells in vivo and, consequently, directly affect the function of the seminiferous epithelium.

Animals↗
PubMed1990

The regulation of retinoic acid receptor mRNA levels during spermatogenesis.

Retinoic acid receptor-alpha mRNAs were found in both Sertoli and germ cells of the testis. A 2.7-kilobase (kb) mRNA was expressed solely in Sertoli cells, whereas a 3.4-kb mRNA was distributed in both Sertoli and germ cells. In addition, we report two new, but minor, germ cell-specific mRNAs detected primarily in the pachytene spermatocytes. By contrast, only one transcript for retinoic acid receptor-beta was found in the testis, exclusively in Sertoli cells. These results suggest that each mRNA may have specific functions in mediating the effects of retinoids during spermatogenesis. The expression of retinoic acid receptor-alpha mRNAs was regulated during the spermatogenic cycle, showing a 7-fold increase in the level of 3.4-kb mRNA at stages VIII-IX. Since stage VIII is where the development of germ cells is arrested at the prophase of meiosis in the vitamin A-deficient testis, this result suggests that alpha mRNA transcription may be necessary before more advanced germ cells than preleptotene spermatocytes would be observed in the testis. The most striking finding was that the treatment of vitamin A-deficient rats with retinol led to a rapid increase in the retinoic acid receptor-alpha mRNA levels. The level of mRNAs was increased 3-fold at its peak, but diminished by 12 h. This precise regulation of receptor by retinol suggests that its synthesis is required before it can be used to modulate the transcription of retinoid-inducible genes. In contrast, the regulation of retinoic acid receptor-beta mRNA was different from the alpha mRNAs, in that its level remained unchanged for 48 h after the injection of retinol.

Animals↗
PubMed1990

Germ cell regulation of Sertoli cell transferrin mRNA levels.

Sertoli cells cultured in the presence of germ cells responded by increasing the level of transferrin mRNA 3-fold as determined by solution hybridization and Northern blot analysis. In contrast, the steady state levels of other mRNAs, including sulfated glycoprotein 1 (SGP-1), sulfated glycoprotein 2 (SGP-2), transferrin receptor, regulatory subunit of cAMP dependent protein kinase, and ferritin light chain, were not influenced by coculture with germ cells. The transferrin mRNA stimulatory activity was found in conditioned medium from germ cells but was not associated with germ cell membrane components. The activity was abolished by treatment of the medium with trypsin. Partial characterization and isolation of the protein(s) from conditioned medium indicated that it has an apparent mol wt between 10 and 30 K. Studies using inhibitors of protein and nucleic acid synthesis indicated that the stimulation of transferrin mRNA by germ cell conditioned medium required both transcription and translation. Sertoli cell enriched (germ cell depleted) testes were obtained from male offspring of pregnant females irradiated at the 19th day of gestation. Testicular transferrin mRNA levels from irradiated rats decreased in comparison to levels in the normal rat, whereas SGP-2 mRNA levels were unchanged. These studies demonstrate that germ cell secretions may interact with Sertoli cells to specifically increase the level of transferrin mRNA and that this interaction may be a mechanism by which germ cells regulate the flow of iron across the seminiferous epithelium.

Animals↗
PubMed1990

Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture.

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.

Amino Acid Sequence↗
PubMed1990

Pleiotypic actions of the seminiferous growth factor on two testicular cell lines: comparisons with acidic and basic fibroblast growth factors.

Bovine seminiferous growth factor (SGF) stimulated different pleiotypic responses in TM3 Leydig and TM4 Sertoli cells from those obtained with bovine acidic and basic fibroblast growth factors (aFGF, bFGF). First, the three growth factors had distinct mitogenic effects. SGF increased TM3 and TM4 cell numbers, whereas aFGF was mitogenic only for TM3 cells, and bFGF was inactive on both cell lines. Second, only SGF and bFGF stimulated TM4 cells to produce 3- and 2-fold, respectively, greater levels of extracellular, sulfated glycoprotein-1. By Northern analyses, SGF increased the steady-state levels of sulfated glycoprotein-1 mRNA approximately 1.34-fold relative to those of actin mRNA during a 48-hr period. Third, the cell lines secreted different [35S]methionine-labeled proteins. With TM3 cells, some proteins were secreted specifically, whereas other were up- or down-regulated differentially in response to SGF, aFGF, or bFGF. Likewise, with TM4 cells, the three growth factors induced qualitative and quantitative changes in the secretion of specific proteins. On immunoblots, SGF did not bind antibodies specific for an internal domain of aFGF or FGF-5, nor those directed against N-terminal, internal, and C-terminal regions of bFGF. These data suggest SGF, aFGF, and bFGF acted on TM3 Leydig and TM4 Sertoli cells through different receptors and/or diverging pathways of signal transduction to induce different pleiotypic effects.

Animals↗