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M D Griswold

Publications and source records attributed to M D Griswold.

At least 19 recordsLinked to original sources
PubMed2002

Transplantation of bovine germinal cells into mouse testes.

To develop techniques for spermatogonial transplantation in bulls, it is essential to have an effective bioassay procedure to evaluate the transplantation efficiency of spermatogonial stem cell collection, purification, and culture techniques. The objective of the present study was to develop a mouse bioassay model to evaluate transplantation efficiency of fresh and cultured bovine germ cells. Bull calves of four ages (1, 2, 3, and 4 mo) were used as a source of donor testes cells. Two calves were used for each age point, one calf was experimentally made cryptorchidistic at 1 wk of age and the other left normal. A STO (mouse fibroblast) feeder cell line was used to culture bovine testes cells for 2 wk preceding transfer into recipient testes. Immunodeficient nude mice (nu/nu) in which endogenous spermatogenesis had been abolished by busulfan treatment served as recipient animals for transplantation. Donor bovine germ cells were microinjected into mouse seminiferous tubules. Mouse testes were analyzed 2 wk after transplant with the use of a bovine-specific antibody and whole-mount immunohistochemistry for the presence of bovine donor germ cells. Bovine testis cells were present in all recipient mouse testes analyzed. Fresh bovine testes cells were observed as colonies of round cells within mouse seminiferous tubules, indicating spermatogonial expansion and colonization; however, cultured bovine testes cells appeared as fibrous tissue and not as spermatogenic colonies. The average number of colonies resulting from donor cryptorchid testes was not different (P > 0.05) from noncryptorchid, 56+/-4 and 78+/-7, respectively. Fresh donor cells from calves older than 1 mo gave rise to a greater average number of colonies within recipient testes (P <0.05) (1 mo, 33+/-4; 2 mo, 70+/-8; 3 mo, 63+/-6; 4 mo, 87+/-9). Fresh bovine germ cells are capable of colonization in the busulfan-treated nude mouse testis, making it a suitable model for evaluation and development of spermatogonial transplant techniques in bulls.

Animals↗
PubMed2001

Clusterin, a binding protein with a molten globule-like region.

Clusterin is a heterodimeric glycoprotein found in many tissues of the body and is the most abundant protein secreted by cultured rat Sertoli cells. The function of clusterin is unknown, but it has been associated with cellular injury, lipid transport, apoptosis, and it may be involved in the clearance of cellular debris caused by cell injury or death. Consistent with this last idea, clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins and the hydrophobic probe 1-anilino-8-naphthalenesulfonate (ANS). Given this variety of ligands, clusterin must have specific structural features that provide the protein with its promiscuous binding activity. Using sequence analyses, we show that clusterin likely contains three long regions of natively disordered or molten globule-like structures containing putative amphipathic alpha-helices. These disordered regions were highly sensitive to trypsin digestion, indicating a flexible nature. The effects of denaturation on the fluorescence of the clusterin-ANS complex were compared between proteins with structured binding pockets and molten globular forms of proteins. Clusterin bound ANS in a manner that was very similar to that of molten globular proteins. Furthermore, we found that, when bound to ANS, at least one cleavage site within the protease-sensitive disordered regions of clusterin was protected from trypsin digestion. In addition, we show that clusterin can function as a biological detergent that can solubilize bacteriorhodopsin. We propose that natively disordered regions with amphipathic helices form a dynamic, molten globule-like binding site and provide clusterin the ability to bind to a variety of molecules.

Amino Acid Sequence↗
PubMed2001

Mechanisms involved in the homologous down-regulation of transcription of the follicle-stimulating hormone receptor gene in Sertoli cells.

The action of follicle-stimulating hormone (FSH) in spermatogenesis is regulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells. By controlling the number of receptors, the cell is able to modulate the timing and magnitude of subsequent signal transduction in response to FSH. One mechanism of control is the down-regulation of the steady state levels of the FSH receptor gene after exposure to FSH or agents that stimulate or prolong the cAMP signal transduction cascade (homologous down-regulation) in Sertoli cells. The goals of this study were to examine possible mechanisms involved in the down-regulation of mRNA levels of this gene. Analysis of transcription and processing by a PCR-based assay showed that treatment of Sertoli cells with FSH caused at least a 50% reduction of hnRNA for the FSH receptor gene. Reporter genes controlled by 5' flanking sequences of the FSH receptor gene that were transiently transfected into Sertoli cells were not down-regulated. In electrophoretic mobility shift assays (EMSA), cAMP-inducible nuclear protein complex containing c-Fos formed on the activator protein-1/cAMP responsive element-like site located at -216 to -210 in the promoter of the rat FSH receptor gene. We concluded from this study that there was no evidence for the putative role of ICER in the down-regulation of the FSH receptor promoter. In addition, the FSH-induced down-regulation of the transcription of the FSH receptor gene in Sertoli cells was prevented by the treatment of Sertoli cells with trichostatin A prior to the addition of FSH. This experiment coupled with other observations suggested that the down-regulation may be mediated by changes in chromatin structure.

Animals↗
PubMed2001

Intrinsically disordered protein.

Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Models, Molecular↗
PubMed2001

Site-specific methylation of the promoter alters deoxyribonucleic acid-protein interactions and prevents follicle-stimulating hormone receptor gene transcription.

In the male gonad, the FSH receptor (FSHR) gene is expressed only in Sertoli cells. To date, the mechanism(s) responsible for Sertoli cell-specific expression of the FSHR gene are unknown. In this study, DNA methylation at specific sites in the promoter are shown to lead to changes in the DNA-protein interactions at those sites and, subsequently, to transcriptional repression of the gene. The extent of methylation of cytosine residues within the core promoter region of genomic DNA isolated from cells/tissues that expressed, or did not express, the FSHR gene was analyzed by the sodium bisulfite conversion technique. All seven cytosine residues in CpG dinucleotides within the core promoter region were found to be unmethylated in primary cultured rat Sertoli cells that were actively expressing FSHR mRNA. In contrast, in tissues not expressing FSHR the same region of the gene was methylated at each of the CpG dinucleotides examined. In addition, DNA-protein interactions in three primary regulatory regions of the promoter were examined by electrophoretic mobility shift assays (EMSA) with synthetic oligonucleotides containing selectively methylated cytosine residues. Methylation of a CpG sequence within a consensus E box element (CACGTG, -124/-119) decreased the binding affinity of USF1/2 transcription factors for this element. Methylation of the CpG sequence in the Inr region (CCGG, -85/-82) allowed the formation of an additional DNA-protein complex. Methylation at both cytosine residues in the E2F element ((m)CG(m)CG) generated a new methylcytosine-specific DNA-protein complex. The core FSHR promoter region of a mouse Sertoli cell line (MSC-1) that does not express FSHR was shown to be methylated at four CpG dinucleotides. The demethylation of these four sites by treatment of the MSC-1 cells with 5-aza-2'-deoxycytidine (5-azaCdR) activated the transcription of the FSHR gene. Taken together, these results suggest that cytosine methylation is a major factor in the repression of the expression of the FSHR gene.

Animals↗
PubMed2001

Distribution of type A spermatogonia in the mouse is not random.

The distribution of type A spermatogonia was studied using drawings of cross-sectioned tubules at various stages of the spermatogenic cycle of perfusion-fixed, epoxy-embedded mouse testis. Spermatogonia were classified as either positioned opposite the interstitium or opposite the region where two tubules make contact or in a defined, intermediate region at which the two tubules diverged. At stage V, the population of type A spermatogonia, comprised of A(s) through A(al) cells, is randomly positioned around the periphery of the seminiferous tubule. The A(s) through A(al) population becomes nonrandomly distributed beginning at stage VI, being located primarily in regions where the tubule opposes the interstitium, and remains nonrandom through stage III of the next cycle. The A(1) spermatogonia of stage VII, derived from most A(pr) and A(al) spermatogonia, and the A(2) spermatogonia of stage IX, derived from the A(1) spermatogonia, are also nonrandomly positioned opposing the interstitium. However, the A(3) population of stage XI becomes randomly distributed around the tubule. To our knowledge, these are the first data to show that the more primitive spermatogonial types (A(s) to A(al)) move to specific sites within the seminiferous tubule. Division of the regularly spaced, more primitive spermatogonia (A(s) to A(al)) leads to the spread of their progeny (A(1) to A(4)) laterally along the base of the seminiferous tubule. The lateral spread from more or less evenly spaced foci ensures that spermatogenesis is conducted uniformly around the entire tubule. The data also suggest that the position of a seminiferous tubule in the mouse is stabilized in relationship to other seminiferous tubules.

Animals↗
PubMed2001

Identification of a novel retrovirus expressed in rat Sertoli cells and granulosa cells.

Differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to identify a novel retrovirus, designated SC1, that is expressed at high levels in rat granulosa cells and prepubertal Sertoli cells. The initial DDRT-PCR screen was performed using RNA from cultured prepubertal rat Sertoli cell, liver, and brain samples. SC1 was detected in the prepubertal rat Sertoli cell samples but not in those from liver and brain. SC1 cDNA was 6 kilobases in length and contained regions encoding for the gag, pol, and env retroviral proteins. Northern blot analysis failed to detect expression of the SC1 gene in total RNA isolated from adult brain, heart, spleen, lung, liver, skeletal muscle, kidney, prostate, and epididymis. Similarly, Northern blot analysis of testes from rats at various ages of development showed that high-level expression of the SC1 gene was limited to prepubertal testis samples. In situ hybridization analysis localized the SC1 mRNA to the seminiferous tubules of prepubertal testes and at a much lower level in Sertoli cells of adult testes. Northern blot analysis of total RNA isolated from Sertoli cells from 20-, 27-, and 35-day-old rat Sertoli cells and type A spermatogonia, pachytene spermatocytes, and round spermatids showed expression of the SC1 gene to be restricted to 20- and 27-day-old Sertoli cells, with no expression detected in germ cells. Furthermore, Northern blot analysis also showed expression of the SC1 gene in rat ovaries, and the level of expression was affected during eCG/hCG-induced ovulation. Expression of SC1 mRNA was localized by in situ hybridization of eCG-treated ovaries to the granulosa cell layer in developing follicles. Southern blot analysis showed SC1 to be endogenous in the rat and absent in mouse and human cell genomes. Transient transfection assays using the SC1 promoter region showed high promoter activity in MSC-1 and cultured prepubertal rat Sertoli cells, and no activity in 3T3 or MCF-7 cell lines.

Animals↗
PubMed2001

Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation.

The spermatogonial stem cell transplantation technique was employed to determine if murine germ cells require functional androgen receptors to complete qualitatively normal spermatogenesis. Testicular cells from testicular feminized mice were injected into the seminiferous tubules of azoospermic mice expressing functional androgen receptors. Recipient testes were analyzed between 110 and 200 days following transplantation. Multiple colonies of complete and qualitatively normal donor-derived spermatogenesis were seen within the seminiferous tubules of each recipient testis, demonstrating that murine germ cells do not require functional androgen receptors to complete spermatogenesis.

Animals↗
PubMed2000

E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis.

Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.

Animals↗
PubMed2000

Spermatogonial transplantation - an update for the millennium.

Spermatogonial transplantation as developed in the laboratory of Ralph Brinster has been a technological breakthrough in the study of Sertoli-germ cell interactions. For the first time, germ cells can be transferred from one animal to another and from one species to another. The transfer technology combined with developments in freezing germ cells, long-term culture of germ cells, and enrichment of stem cell populations portend even more significant breakthroughs in the new millennium. The ultimate application of germ cell transfer would allow the in vitro genetic manipulation of cultured stem cells that could then be transplanted into recipient syngeneic or xenogeneic recipients and give rise to functional male gametes. Clearly, this achievement would have applications in basic science, human medicine, and domestic and wild animal reproduction. While progress in this direction has been significant and swift, significant barriers such as immunological response and mechanisms for introducing genetic material into the stem cells remain to be examined. This report is a chronological review of the technological advances made and conceptual insights gained since the first report of successful transplantation in 1994.

Animals↗
PubMed2000

Follicle-stimulating hormone inhibits all-trans-retinoic acid-induced retinoic acid receptor alpha nuclear localization and transcriptional activation in mouse Sertoli cell lines.

The regulation of retinoic acid receptor alpha (RARalpha) signal transduction has not been well characterized. In this study, we determined whether all-trans-retinoic acid (tRA) and follicle-stimulating hormone (FSH) modulate RARalpha receptor subcellular localization, leading to changes in its transcriptional activity and protein expression in mouse Sertoli cell lines. We found that tRA induced the nuclear localization of RARalpha within 30 min and that longer term exposure increased the receptor transcriptional activity and RARalpha protein expression. Conversely, FSH suppressed the tRA-induced nuclear localization, transcriptional transactivation, and protein expression of RARalpha. Treatment with two different protein kinase A-selective antagonists reversed the inhibitory actions of FSH on tRA-dependent RARalpha nuclear localization and transcriptional activity. These results are consistent with the involvement of protein kinase A in mediating the inhibitory effects of FSH. For the first time, we demonstrate a unique signaling convergence between the RARalpha and the FSH-mediated signaling pathways, which may have significant implications in the testis because both are critical regulators of testis physiology.

Animals↗
PubMed2000

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat.

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.

Amino Acid Sequence↗
PubMed2000

Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis.

In mice, the juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis followed by failure of type A spermatogonial stem cells to repopulate the testis, rendering male animals sterile. It is not clear whether the defect in jsd resides in a failure of the somatic component to support spermatogenesis or in a failure that is intrinsic to the mutant's germ cells. To determine if the jsd intratesticular environment is capable of supporting spermatogenesis, germ cell transplantation experiments were performed in which C57BL/6 ROSA germ cells were transplanted into jsd recipients. To determine if jsd spermatogonia are able to develop in a permissive seminiferous environment, jsd germ cells were transplanted into W/W(v) and busulfan-treated C57BL/6 animals. The data demonstrate that up to 7 mo after transplantation of normal germ cells, jsd seminiferous tubules are capable of supporting spermatogenesis. In contrast, when jsd germ cells were transplanted into busulfan-treated C57BL/6 testis, or into testis of W/W(v) mice, no jsd-derived spermatogenesis was observed. The data support the hypothesis that the jsd phenotype is due to a defect in the germ cells themselves, and not in the intratubular environment.

Animals↗
PubMed2000

Clusterin gene in rat sertoli cells is regulated by a core-enhancer element.

Clusterin is a ubiquitous glycoprotein that is promiscuously expressed at a low basal level but can be highly induced by a variety of stress conditions. In contrast, in some secretory cells associated with tissue-fluid interfaces such as the Sertoli cells in the testis, clusterin demonstrates high constitutive expression. In this study, we address the mechanisms that regulate the constitutive expression of the clusterin gene by using primary cultures of immature rat Sertoli cells. We have identified a region of the rat clusterin gene promoter that activated transcription only in Sertoli cells and that mapped between positions -426 and -311. Sequence analysis of this region revealed a high concentration of potential regulatory elements. Using gel-shift assays combined with hydroxyl radical footprinting, we identified the elements recognized by the Sertoli cell nuclear factors. Comparison of the interactions with this region of the nuclear factors from different cell types demonstrated that recognition of the core-enhancer element is specific for the Sertoli cells, and in vitro, the core region was recognized by the transcription factor CBF. Transient transfections showed that a core enhancer is responsible for more than a half of the total promoter activity and is an essential element for the cell-specific activity of the Sertoli-specific region. In addition to the core enhancer, tandem Sp1 sites are also required for maximal activity of this region.

Animals↗
PubMed2000

Advances in spermatogonial stem cell transplantation.

Spermatogonial stem cell transplantation was first reported by Ralph Brinster's laboratory in 1994. It has proven to be a technological breakthrough in the study of both stem cells and Sertoli cell-germ cell interactions. This technique can be used to transfer testicular stem cells successfully from one animal to another of the same species (referred to as syngeneic transplants) and sometimes to an animal of a different species (xenogeneic transplants). This transfer technique, combined with developments in cryopreservation, long-term culture, and the enrichment of stem cell populations makes more significant breakthroughs likely in the near future. Ultimately, the application of spermatogonial stem cell transfer will allow transplantation of cultured stem cells manipulated genetically in vitro to give rise to functional male gametes with an altered genotype. This achievement will have applications in basic science, human medicine, and domestic and wild animal reproduction. Although progress toward this goal has been swift, potentially significant barriers, such as the stable incorporation of genetic material into stem cells and immunological responses to the introduced germ cells, remain to be overcome. This article is a review of the scientific advances made since the initial report of successful transplantation in 1994.

Animals↗
PubMed1999

Clusterin in the male reproductive system: localization and possible function.

Clusterin is a glycoprotein that was initially isolated from the male reproductive system. Subsequently, clusterin has been found to be widely distributed in a variety of tissues in mammals. One characteristic of the expression of clusterin is that it is induced as a result of cellular injury, death, or pathology. Despite the efforts of many laboratories working in diverse biological systems, the function of clusterin remains unknown. Recent studies have revealed a 'heat-shock element' in the promoter of the gene that may account for the inducible nature of the clusterin gene. Overall, the evidence suggests that function of clusterin is to protect surviving cells after damage. This protection may result from a detergent-like action of the protein.

Animals↗
PubMed1998

The central role of Sertoli cells in spermatogenesis.

Sertoli cells are the somatic cells of the testis that are essential for testis formation and spermatogenesis. Sertoli cells facilitate the progression of germ cells to spermatozoa via direct contact and by controlling the environment milieu within the seminiferous tubules. The regulation of spermatogenesis by FSH and testosterone occurs by the action of these hormones on the Sertoli cells. While the action of testosterone is necessary for spermatogenesis, the action of FSH minimally serves to promote spermatogenic output by increasing the number of Sertoli cells.

Animals↗