Search PubMed⌕ Search

Biomedical subjects

E M Eddy

Publications and source records attributed to E M Eddy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Gene expression during mammalian meiosis.

The expression of a wide variety of genes is developmentally regulated during mammalian meiosis. Drawing mainly on studies in spermatogenesis, this review shows that some of these genes are transcribed exclusively in germ cells, while others are also transcribed in somatic cells. Some of the genes expressed exclusively in spermatogenic cells are unlike any expressed in somatic cells, while others are isologous to genes expressed in somatic cells and are in the same gene family. Some of the developmentally regulated genes also expressed in somatic cells produce spermatogenic cell-specific transcripts, while others produce transcripts that are apparently the same in somatic and germ cells. Possible answers to why so many genes have atypical patterns of expression during meiosis are that: (1) all cell types express certain genes that define their cell type and lineage, (2) spermatogenesis is a developmental process that progresses according to a genetic program directing the sequential and coordinate expression of specific genes, (3) some genes are expressed that encode proteins required for meiosis, (4) some genes are expressed that encode proteins not required until after meiosis, (5) some genes are expressed to compensate for other genes that become inactivated with X chromosome condensation, and (6) it has been suggested that regulation of gene expression becomes leaky during spermatogenesis due to changes in DNA organization, leading to production of irrelevant transcripts. However, it is largely unknown how extrinsic cues from the endocrine system and surrounding somatic cells interact with intrinsic mechanisms of germ cells to activate signal transduction processes regulating transcription during mammalian meiosis.

Animals↗
PubMed1997

Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp 70-2 knockout mice.

The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 26 and in juvenile Hsp 70-2 knockout mice. Adult Hsp 70-2 knockout males are infertile and lack spermatids and spermatozoa (Dix et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp 70-2 gene product (heat-shock protein 70-2 [HSP70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1;marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp 70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes.

Animals↗
PubMed1997

HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes.

Cyclin B-dependent CDC2 kinase activity has a key role in triggering the G2/M-phase transition during the mitotic and meiotic cell cycles. The Hsp70-2 gene is expressed only in spermatogenic cells at a significant level. In Hsp70-2 gene knock-out (Hsp70-2(-/-)) mice, primary spermatocytes fail to complete meiosis I, suggesting a link between HSP70-2 heat-shock protein and CDC2 kinase activity during this phase of spermatogenesis. Members of the HSP70 protein family are molecular chaperones that mediate protein de novo folding, translocation and multimer assembly. This study used immunoprecipitation-coupled western blot and in vitro reconstitution experiments to show that HSP70-2 interacts with CDC2 in the mouse testis, appears to be a molecular chaperone for CDC2, and is required for CDC2/cyclin B1 complex formation. Previous studies reported that most CDC2 kinase activity in the mouse testis is present in pachytene spermatocytes. Although CDC2 kinase activity for histone H1 was present in the testis of wild-type mice, it was nearly absent from the testis of Hsp70-2(-/-) mice, probably due to defective CDC2/cyclin B1 complex formation. Furthermore, addition of HSP70-2 to freshly prepared extracts of testis from Hsp70-2(-/-) mice not only restored CDC2/cyclin B1 complex formation but also reconstituted CDC2 kinase activity in vitro. It appears that one cause of failure to complete meiosis I during spermatogenesis in Hsp70-2(-/-) mice is disruption of CDC2/cyclin B1 assembly in pachytene spermatocytes, thereby preventing development of the CDC2 kinase activity required to trigger G2/M-phase transition. These studies provide novel in vivo evidence for a link between an HSP70 molecular chaperone and CDC2 kinase activity essential for the meiotic cell cycle in spermatogenesis.

Animals↗
PubMed1997

HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes.

Spermatogenic cells synthesize a unique 70-kDa heat shock protein (HSP70-2) during prophase of meiosis I, and targeted disruption of the Hsp70-2 gene has shown that this protein is required for spermatogenic cell differentiation in adult mice. HSP70-2 is associated with synaptonemal complexes formed between paired homologous chromosomes during meiotic prophase. The present study focuses on the nearly synchronous first wave of spermatogenesis in 12- to 28-day old juvenile mice to determine more precisely when HSP70-2 is required and what meiotic processes are affected by its absence. Spermatogenesis in homozygous mutant mice (Hsp70-2[-/-]) proceeded normally until day 15 when increasing numbers of pachytene spermatocytes became apoptotic and differentiation of cells beyond the pachytene stage began to falter. Synaptonemal complexes assembled in Hsp70-2(-/-) mice and spermatocytes developed through the final pachytene substage. However, synaptonemal complexes failed to desynapse and normal diplotene spermatocytes were not observed. Metaphase spermatocytes were not seen in tissue sections from testes of Hsp70-2(-/-) mice, and expression of mRNAs and antigens characteristic of late pachytene spermatocytes (e.g., cyclin A1) and development of spermatids did not occur. Thus, HSP70-2 is required for synaptonemal complex desynapsis, and its absence severely impairs the transition of spermatogenic cells through the late meiotic stages and results in apoptosis beginning with the first wave of germ cell development in juvenile mice.

Animals↗
PubMed1996

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Animals↗
PubMed1996

Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis.

Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer- extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5'-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp7-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene beta- galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp7O-2 and Hsp7O-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp7O-2 and Hsp7O-2/lacZ was specific to the meiotic phase of spermatogenesis.

Animals↗
PubMed1996

Boar proacrosin expressed in spermatids of transgenic mice does not reach the acrosome and disrupts spermatogenesis.

Transgenic mice that express boar proacrosin were produced to examine mechanisms for targeting hydrolytic enzymes to the acrosome. A 2.3 kb transgene was constructed by ligating the cDNA for boar preproacrosin with the mouse protamine 2 promoter region. Six founder mice that incorporated the transgene were identified by polymerase chain reaction and Southern blot analysis. Northern blots indicated that the two male founders (Ac.2 and Ac.5) and male progeny from three female founders (Ac.3, Ac.4, Ac.6) expressed the transgene mRNA in testis, but not in somatic tissues. In these transgenic animals boar proacrosin was detected by immunohistochemistry in condensing spermatids, but was not localized in the acrosome. This acrosomal targeting defect of the transgene product may result from its delayed expression during the later steps of haploid differentiation. Furthermore, both male founders and all Ac.4 and Ac.6 males were infertile, as determined by multiple matings for at least 2 months. Ac.3 males were either infertile or rarely transmitted the transgene to their offspring. The infertile males mated, produced copulatory plugs, and had seminal vesicle weights and testosterone levels within the normal range. However, they produced significantly fewer spermatozoa and had lower testis weights than controls. Although the mitotic and meiotic phases of spermatogenesis appeared normal by histological criteria, condensing spermatids were missing from most tubules, and multinucleated cells were present in the lumen of seminiferous tubules and in the epididymis. We hypothesize that boar proacrosin which fails to reach the acrosome is activated in these transgenic mice, and that its proteolytic activity disrupts spermatogenesis during spermatid formation.

Acrosin↗
PubMed1996

Testis-specific expression of mRNAs for a unique human type 1 hexokinase lacking the porin-binding domain.

Several enzymes in the glycolytic pathway are reported to have spermatogenic cell-specific isozymes. We reported recently the cloning of cDNAs representing three unique type 1 hexokinase mRNAs (mHk1-sa, mHk1-sb, and mHk1-sc) present only in mouse spermatogenic cells and the patterns of expression of these mRNAs (Mori et al., 1993: Biol Reprod 49:191-203). The mRNAs contain a spermatogenic cell-specific sequence, but lack the sequence for the porin-binding domain that somatic cell hexokinases use to bind to a pore-forming protein in the outer mitochondrial membrane. We now report the cloning of cDNAs representing three unique human type 1 hexokinase mRNAs (hHK1-ta, hHK1-tb, and hHK1-tc) expressed in testis, but not detected by Northern analysis in other human tissues. These mRNAs also contain a testis-specific sequence not present in somatic cell type 1 hexokinase, but lack the sequence for the porin-binding domain. The hHK1-tb and hHK1-tc mRNAs each contain an additional unique sequence. The testis-specific sequence of the human mRNAs is similar to the spermatogenic cell-specific sequence of the mouse mRNAs. Furthermore, Northern analysis of RNA from mouse, hamster, guinea pig, rabbit, ram, human, and rat demonstrated expression of type 1 hexokinase mRNAs lacking the porin-binding domain in the testes of these mammals. These results suggest that hexokinase may have unique structural or functional features in spermatogenic cells and support a model proposed by others for hexokinase gene evolution in mammals.

Amino Acid Sequence↗
PubMed1996

HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes.

Mouse spermatogenic cells are known to express HSP70-2, a member of the HSP70 family of heat-shock proteins. The purpose of the present study was to characterize further the expression and localization of HSP70-2 in meiotic cells of mice and hamsters. After separating mouse spermatogenic cells into cytoplasmic and nuclear fractions, proteins were separated by two-dimensional gel electrophoresis and detected with HSP-specific antibodies. Of several HSP70 proteins identified in the cytoplasm, only HSC70 and HSP70-2 were also detected in the nucleus. Immunocytological analyses of spermatocyte prophase cells revealed that HSP70-2 was associated with the synaptonemal complex. Surface-spread synaptonemal complexes at pachytene and diplotene stages labeled distinctly with the antiserum to HSP70-2. Synaptonemal complexes from fetal mouse oocytes failed to show any evidence of HSP70-2. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses of gene expression confirmed this sex specificity; Hsp70-2 mRNA was detected in mouse testes, but not ovaries. These findings are suggestive of a previously unsuspected sexual dimorphism in structure and/or function of the synaptonemal complex.

Animals↗
PubMed1996

Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility.

The reproductive system of male mice homozygous for a mutation in the estrogen receptor (ER) gene (ER knock-out; ERKO) appears normal at the anatomical level. However, these males are infertile, indicating an essential role for ER-mediated processes in the regulation of male reproduction. Adult ERKO male mice have significantly fewer epididymal sperm than heterozygous or wild-type males. Although spermatogenesis is occurring in some seminiferous tubules of 3- to 5-month-old ERKO males, other tubules either have a dilated lumen and a disorganized seminiferous epithelium with few spermatogenic cells or lack a lumen and contain mainly Sertoli cells. There are no obvious differences in seminiferous tubules at 10 days of age between wild-type and ERKO mice, but the lumen in ERKO males is dilated in all seminiferous tubules by 20 days. However, spermatogenesis progresses and similar numbers of sperm are present in the cauda epididymis of ERKO and wild-type males until 10 weeks of age. Disruption of spermatogenesis and degeneration of the seminiferous tubules become apparent after 10 weeks in the caudal pole of the testis and progresses in a wave to the cranial pole by 6 months. However, the seminal vesicles, coagulating glands, prostate, and epididymis do not appear to be altered morphologically in ERKO mice. Serum testosterone levels are somewhat elevated, but LH and FSH levels are not significantly different from those in wild-type males. Sperm from 8- to 16-week-old mice have reduced motility and are ineffective at fertilizing eggs in vitro. In addition, ERKO males housed overnight with hormone-primed wild-type females produce significantly fewer copulatory plugs than do heterozygous or wild-type males. These results suggest that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.

Animals↗
PubMed1996

Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes.

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal tissue of humans and animals, its presence and tissue distribution is thought to be essential for survival. Using the techniques of homologous recombination, we have disrupted the ER gene and have produced a line of transgenic mice possessing the altered ER gene (ERKO). The mouse ER gene was disrupted by inserting a 1.8 kb PGK-Neomycin sequence into exon 2, approximately 280 bp downstream of the transcription start codon. The correct targeting of the disruption was demonstrated by Southern blot analysis and PCR. Western blot analysis of uterine preparations from ERKO females showed no detectable ER protein. Heterozygotes had one half the level of ER protein compared to wild-type animals. Estrogen insensitivity was confirmed using estrogen agonists, estradiol, hydroxy tamoxifen, diethylstilbestrol treatment for 3 days which resulted in a 3-4-fold increase in uterine wet weight and vaginal cornification in wild-type females, while ERKO mice were totally unresponsive. These data were further supported by the failure of estrogen or EGF treatment to induce DNA synthesis in uterine tissue of similarly treated mice. Lactoferrin, an estrogen-responsive gene in the uterus, was also assayed by Northern blot. Wild-type mice treated with a single estradiol injection showed a 350-fold induction in lactoferrin mRNA. while ERKO females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are infertile and demonstrate hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. Males are also infertile, with atrophy of the testes and seminiferous tubule dysmorphogenesis. Although the reproductive capabilities have been altered with a dramatic effect on the gonads, prenatal development of the reproductive tracts of both sexes appear to be independent of an ER-mediated response. Analysis of the mammary glands of the ERKO females at 4 months of age showed a primitive ductal rudiment rather than the fully developed ductal tree seen in wild-type siblings. Also absent were the terminal end buds seen during normal ductal morphogenesis. Both sexes show a decrease in skeletal bone density, supporting a direct role for ER action in bone. A single patient is described who is homozygous for a point mutation in the human ER gene at codon 157. The mutation produces a truncation of the ER protein and results in estrogen insensitivity syndrome. Most significant of the clinical findings are effects on skeletal bone density and retarded bone age. Findings from the patient and mice suggest that the absence of functional ER is not lethal. Mutation in the ER gene is present in the human population. Further characterization of the mice and identification of additional patients will be required to more fully understand the consequences of ER gene mutations.

Amino Acid Sequence↗
PubMed1995

Genomic organization of a mouse glyceraldehyde 3-phosphate dehydrogenase gene (Gapd-s) expressed in post-meiotic spermatogenic cells.

The Gapd-s gene encodes an isoform of the glyceraldehyde 3-phosphate dehydrogenase enzyme expressed only in post-meiotic spermatogenic cells. Two clones containing the Gapd-s gene were isolated from a mouse genomic library. Sequencing and restriction enzyme analysis demonstrated that this single-copy gene contains 11 exons and spans 9596 base pairs. The locations of Gapd-s exons and introns are conserved when compared to the corresponding portions of the chicken and human somatic Gapd genes. The promoter region contains no TATA box, although there is a potential SP1 recognition site within exon 1. Like other TATA-less genes, primer extension analysis reveals some heterogeneity in the site of transcription initiation with Gapd-s transcripts initiating from three discrete sites. Northern analysis demonstrated that a 1.5-kb Gapd-s mRNA is expressed in the testis in at least three mammalian orders, indicating that the Gapd-s gene appeared early in mammalian evolution. Using GAPD-deficient bacteria, mouse GAPD-S was shown to be capable of functioning as a glycolytic enzyme. Since GAPD has been proposed to be a key enzyme regulating glycolysis in spermatogenic cells, GAPD-S may represent a potential target for toxicological or contraceptive agents affecting fertility by interfering with glycolysis.

Animals↗
PubMed1995

Members of the 70 kDa heat shock protein family specifically recognize sulfoglycolipids: role in gamete recognition and mycoplasma-related infertility.

We have previously shown that several mycoplasma species associated with infertility bind specifically to sulfated glycolipids isolated from the mammalian reproductive tract. We now show that a germ cell-specific sulfoglycolipid binding protein (SLIP 1), which is a potent inhibitor of sperm/egg binding in vitro, is immunologically related to the heat shock protein(Hsp) 70 family of stress proteins and that Hsps are surface antigens in male germ cells. Our present data demonstrate that several mycoplasma and mammalian Hsps share this glycolipid binding specificity in vitro, and suggest that surface Hsps can function as adhesins which mediate sulfoglycolipid recognition in infectious disease and normal reproductive physiology.

Animals↗
PubMed1995

Identification of a unique mu-class glutathione S-transferase in mouse spermatogenic cells.

The fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. Two peptide sequences obtained from a tryptic digest of mouse fibrous sheath proteins exhibited high homology with mu-class glutathione S-transferases (GSTs). Using a DNA probe amplified from degenerate polymerase chain reaction (PCR) primers predicted from these two peptide sequences, a approximately 1.1 kb cDNA clone for fibrous sheath component 2 (Fsc2) was isolated which had 84% nucleic acid and 89% amino acid sequence identity with a previously reported mu-class human GST gene (hGSTM3; Campbell et al., 1990: J Biol Chem 265:4188-9193). Sequences corresponding to those of the two fibrous sheath peptides were present in the protein encoded by the Fsc2 cDNA. Northern analysis with the full length Fsc2 cDNA detected a approximately 1.1 kb mRNA in 12 of 15 somatic tissues examined, as well as in testis and isolated spermatogenic cells. However, 5'(nt--96 to 12) or 3'(nt 637 to 808) Fsc2 probes, containing mostly noncoding sequences, detected a approximately 1.1 kb mRNA abundant in testis and isolated spermatogenic cells, but absent or present at low levels in somatic tissues. Northern analysis with RNA from testes of mice of different postnatal ages and purified spermatogenic cell populations indicated that this transcript is first present during the meiotic phase of germ cell development. These results suggest that a previously unreported mu-class GST gene (mGSTM5.) is expressed at a specific time during the development of spermatogenic cells in the mouse. Immunoblot analysis indicated that a mu-class GST protein is associated with the fibrous sheath, suggesting that it becomes an integral part of the mouse sperm cytoskeleton.

Amino Acid Sequence↗
PubMed1995

Developmental regulation of genomic imprinting during gametogenesis.

Successful mammalian development requires both the male and female genomes. This is due in part to genomic imprinting, which results in offspring inheriting only one functional copy of a gene from either the mother or the father. Evidence suggests that this specialization of the parental genomes is established during gametogenesis when the imprint pattern inherited from the parent is switched to reflect the sex of the progeny. We used reverse transcription-PCR to analyze the allele-specific expression of Igf-2, Igf-2r, and H19 in the testes and ovaries of mice derived from an interspecies cross between Mus musculus and Mus spretus. Because of genomic imprinting, Igf-2 is expressed only from the paternal allele and Igf-2r and H19 only from the maternal allele, in most tissues. Although allele-specific expression was maintained in the neonatal testis and ovary, relaxation of imprinting was detected by 7 days after birth in the male and continued during testis development. In the female, relaxation of the Igf-2 and Igf-2r parental imprints was observed in the adult ovary and oocyte. These results (1) indicate that imprinted expression is relaxed during gametogenesis, presumably as a consequence or prerequisite of the imprinting mechanism, and (2) predict a subsequent imprinting event after which the allele-specific expression of Igf-2, Igf-2r, and H19 reflects the parent of origin.

Alleles↗