Biomedical subjects

Michael D Griswold

Publications and source records attributed to Michael D Griswold.

26 records · Page 2Linked to original sources

PubMed2002

Identification and characterization of testis- and epididymis-specific genes: cystatin SC and cystatin TE-1.

Differential display-reverse transcriptase-polymerase chain reaction was used to examine Sertoli cell gene expression. As a result, two new members of the mouse cystatin multigene family were isolated and named cystatin SC (cystatin-related gene expressed in Sertoli cells) and cystatin TE-1 (cystatin-related gene highly expressed in testis and epididymis). The full-length cDNA sequence of cystatin SC contains an open reading frame that encodes a putative signal peptide of 20 amino acids and a mature protein of 110 amino acids, whereas that of cystatin TE-1 encodes a 128 amino acid protein with a predicted signal peptide of 21 amino acids. Both of the deduced amino acid sequences contain four highly conserved cysteine residues in precise alignment with other cystatin family members. The derived cystatin SC and TE-1 amino acid sequences lack some of the specific, highly conserved motifs believed to be necessary for cysteine proteinase inhibition activity. Northern blot analysis revealed that cystatin SC mRNA was detected only in the testis, whereas the cystatin TE-1 gene was highly expressed in testis and epididymis with very low expression in ovary and prostate. In situ hybridization showed that cystatin SC mRNA was localized mainly to Sertoli cells with an obvious stage-dependent expression, and that cystatin TE-1 mRNA was predominantly expressed in Sertoli cells without apparent stage-dependent expression. Cystatin TE-1 mRNA, as displayed by in situ hybridization, was expressed only in the epithelial cells of the proximal caput region of the epididymis. The unusual amino acid sequence and highly restricted expression suggests that cystatins SC and TE-1 play a very specialized role in the testis and epididymis.

Amino Acid Sequence↗

PubMed2002

Heat shock-initiated apoptosis is accelerated and removal of damaged cells is delayed in the testis of clusterin/ApoJ knock-out mice.

The secretion and localization of clusterin in the testis has led to the hypothesis that clusterin plays a role in spermatogenesis. Furthermore, the association of clusterin with apoptosis, cellular injury, disease, and regression of nongonadal tissues has led to the hypothesis that clusterin acts to protect cells from apoptosis or may be involved in tissue remodeling. To investigate the role of clusterin in the testis, we analyzed clusterin knock-out (cluKO) mice to determine the impact of the absence of clusterin on spermatogenesis. Furthermore, we investigated the cellular response to injury caused by methoxyacetic acid (MAA) toxicity and mild heat exposure in the cluKO mice to determine the extent to which clusterin protects against apoptosis or participates in tissue remodeling. We found that cluKO mice were fertile and had essentially normal spermatogenesis with the exception of some incomplete spermiation after stage VIII. No differences in testicular morphology or the incidence of apoptosis in the testis were seen between the cluKO and clusterin wild-type (cluWT) mice after MAA treatment. In contrast, apoptosis was delayed in the cluWT mice compared with the cluKO mice after heat exposure, suggesting that clusterin does have a slight protective effect against apoptosis under some conditions. Also, a dramatic loss of germ cells after heat stress occurred earlier in the cluWT testes than in the cluKO testes. Clusterin is clearly acting in a dual role in that cells can be protected from damage and dead cells can be more easily removed after some types of cellular damage but not after others.

Acetates↗

PubMed2002

Biological activity and enrichment of spermatogonial stem cells in vitamin A-deficient and hyperthermia-exposed testes from mice based on colonization following germ cell transplantation.

Spermatogenesis is a complex process in which spermatogonial stem cells divide and subsequently differentiate into spermatozoa. This process requires spermatogonial stem cells to self-renew and provide a continual population of cells for differentiation. Studies on spermatogonial stem cells have been limited due to a lack of unique markers and an inability to detect the presence of these cells. The technique of germ cell transplantation provides a functional assay to identify spermatogonial stem cells in a cell population. We hypothesized that vitamin A-deficient (VAD) and hyperthermically treated testes would provide an enriched in vivo source of spermatogonial stem cells. The first model, hyperthermic treatment, depends on the sensitivity of maturing germ cells to high temperatures. Testes of adult mice were exposed to 43 degrees C for 15 min to eliminate the majority of differentiating germ cells. Treated donor testes were 50% of normal adult testis size and, when transplanted into recipients, resulted in a 5.3- and 19-fold (colonies and area, respectively) increase in colonization efficiency compared to controls. The second model, VAD animals, also lacked differentiating germ cells, and testes weights were 25% of control values. Colonization efficiency of germ cells from VAD testes resulted in a 2.5- and 6.2-fold (colonies and area, respectively) increase in colonization compared to controls. Hyperthermically treated mice represent an enriched source of spermatogonial stem cells. In contrast, the low extent of colonization with germ cells from VAD animals raises important questions regarding the competency of stem cells from this model.

Animals↗

PubMed2002

Identification of a murine testis complementary DNA encoding a homolog to human A-kinase anchoring protein-associated sperm protein.

Using differential display reverse transcriptase-polymerase chain reaction (RT-PCR) we have cloned a cDNA that encodes a putative peptide with homology to a recently reported A-kinase anchoring protein-associated protein (ASP) in human sperm. The mouse cDNA was 864 bases in length and encoded for a putative protein of 230 amino acids that had 90% amino acid similarity with the human ASP. The N terminal amino acid sequence had 65% similarity to the rat, mouse, and human protein kinase A regulatory type II sequences. Expression of the gene encoding this ASP was specific to testicular germ cells. Northern blot analysis of testis RNA from 5-, 15-, 25-, and 40-day-old mice showed expression of the ASP gene, but similar analyses of busulfan-treated germ cell-deficient mice failed to detect its expression. In addition, Northern blot analysis did not detect expression of the ASP mRNA in cultured Sertoli cells or cultured interstitial cells. Northern blot and RT-PCR analyses did not detect the ASP mRNA in mouse spleen, brain, liver, lung, heart, kidney, skeletal muscle, ovary, or Sertoli cells. In situ hybridization analysis localized the ASP mRNA to the germ cell compartment of the seminiferous tubules in the testis.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Oligonucleotide microarray analysis of gene expression in follicle-stimulating hormone-treated rat Sertoli cells.

Spermatogenesis requires the presence of functional somatic Sertoli cells in the seminiferous tubules of the testis. Sertoli cells provide support and factors necessary for the successful progression of germ cells into spermatozoa. Sertoli cells are regulated to a large degree by the glycoprotein hormone FSH, which is required for the testis to acquire full size and spermatogenic capacity. Signaling events initiated by the binding of FSH to its receptor lead to an alteration of Sertoli cell gene expression. To characterize the changes in gene expression in FSH-treated Sertoli cells, we used the mRNA from these cells to screen Affymetrix U34A rat GeneChip oligonucleotide microarrays. Sertoli cells from 20-d-old rats were cultured in the presence of 25 ng/ml ovine FSH. At 0, 2, 4, 8, and 24 h after the addition of FSH, total RNA was purified and used to prepare biotinylated target, which was hybridized to the U34A rat microarray containing approximately 9000 rat genes. Analysis identified 100-300 transcripts at each time point that were up-regulated or down-regulated by 2-fold or greater. Genes previously reported to be FSH or cAMP regulated in rat Sertoli cells were identified, in addition to numerous genes not reported to be expressed or FSH regulated in Sertoli cells. The expression patterns of five of these genes, encoding nerve growth factor inducible gene B, PRL-1, PC3 nerve growth factor-inducible antiproliferative putative secreted protein, diacylglycerol acyltransferase, and an expressed sequence tag, in FSH- and N,O'-dibutyryl cAMP-treated rat Sertoli cells were confirmed and characterized by Northern blot analysis. Thus, we have begun to define the transcriptome induced and repressed by FSH in rat Sertoli cells, and we have generated datasets of genes available for further analysis in regard to spermatogenesis and Sertoli cell signaling.

Acyltransferases↗

PubMed2002

The expression of the follicle-stimulating hormone receptor in spermatogenesis.

Results from experiments using mouse models suggest that the role of follicle-stimulating hormone (FSH) in spermatogenesis is the regulation of Sertoli cell proliferation and, ultimately, the size and spermatogenic capacity of the testis. The regulation of the expression of the FSH receptor (FSHR) gene is very cell specific and plays an initial role in the ultimate response of the Sertoli cells to FSH. The extreme cell specificity and the importance of the FSH response to spermatogenesis have led to an extensive characterization of the promoter of the FSHR gene. Several widely expressed transcription factors - including USF 1 and 2, GATA-1, and SF-1 and potential elements such as an E2F site and an Inr region - have been shown to contribute to the maximal transcription of the transfected FSHR gene. However, these experiments have failed to provide clues as to the cell-specific expression of the FSHR gene. In both cell transfections and in transgenic mice, the promoter can direct expression of transgenes promiscuously. The rodent FSHR promoter contains conserved CpG dinucleotides that were shown to be methylated in nonexpressing cells and tissue but unmethylated in Sertoli cells. The methylated CpG sites could interfere with the binding of general transcription factors and/or lead to a repressive chromatin structure in the nonexpressing cells. While yet-undiscovered cell-specific factors may play a role in the expression of the FSHR gene, repression and activation of local chromatin structure are likely to be involved.

Animals↗

PubMedDate not supplied

Expression of testicular germ cell genes identified by differential display analysis.

Using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we identified transcripts encoding for the RNA helicase mDEAH9, Ran binding protein 5 (RanBP5), and 3 novel complementary DNAs designated GC3, GC12, and GC14 in developing testicular germ cells. Sources of RNA for the initial DDRT-PCR screen were purified mouse type A spermatogonia, adult mouse wild-type testis, and W/W(v) mutant mouse testis. We identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12, and GC14 in testis and type A spermatogonia samples from wild-type mice, but not in samples from the W/W(v) mouse testis. These same transcripts were absent in Northern blots of testis RNA from mice treated with busulfan 30 days prior, but were present in testis RNA from wild-type mice at 5, 15, 25, and 40 days of age. The mDEAH9 gene was expressed in many tissues, whereas RanBP5 and GC12 genes were expressed predominantly in the testis with much lower expression in other tissues. The expression of GC3 and GC14 were limited to the testis as evidenced by Northern blot and RT-PCR analyses. The mDEAH9 transcript was not detected in cultured interstitial cells but was found at low levels in cultured immature Sertoli cells, whereas the RanBP5, GC3, GC12, and GC14 transcripts were not detected in either cultured testicular interstitial cells or cultured Sertoli cells. RT-PCR analyses of isolated spermatogonia, pachytene spermatocytes, and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12, and GC14 genes were expressed in all 3 cellular populations. In situ hybridization analyses of testis samples from 40-day-old mice localized expression of mDEAH9, RanBP5, GC3, GC12, and GC14 to the seminiferous tubules. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium, with the highest expression in stages III through VII. Expression of GC14 was greatest in the meiotic germ cell populations.

Animals↗

PubMedDate not supplied

Long-term culture and transplantation of murine testicular germ cells.

The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.

Animals↗