Alteration of the menstrual cycle in baboons placed on tethering devices and moved to individual housing--a stress model for a follicular phase defect.

BACKGROUND: During an attempt to identify endocrine characteristics in the baboon that would more precisely predict ovulatory status for assisted reproductive techniques, we observed severe alterations in the menstrual cycle length upon introducing an environmental stress. This environmental stress involved moving animals from their baseline gang cage environment to individual indoor caging and placing them on a tethering apparatus. METHODS: Five adult female baboons were followed for changes in sex skin indicative of menstrual cycle timing and move from outdoor gang gages to individual indoor cages during the early follicular phase of their cycle. A tether device including a surgically implanted cannula was then installed to facilitate daily blood draws without sedation. Radioimmuonoassays were performed to monitor serum estradiol levels and lapraroscopic surveillance was used to confirm time of ovulation. RESULTS: Complete data sets were collected from four of the female baboons. In each case, a prolongation of the menstrual cycle was noted either during the cycle during which the females were moved to indoor caging or during the cycle immediately following the move. This prolongation was isolated to the follicular phase of the affected cycle. CONCLUSIONS: We conclude that otherwise normal handling procedures, including movement to new caging, and/or installation of a tether device, can impart a stress effect on reproductively cycling adult female baboons, such that folliculogenesis is delayed.

Rat synapsin 1 promoter mediated transgene expression in testicular cell types.

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.

Ultraviolet B-induced melanoma in Monodelphis domestica occurs in the absence of alterations in the structure or expression of the p53 gene.

Monodelphis domestica, a South American opossum, has been established as a mammalian model for sporadic ultraviolet radiation (UVR)-induced melanoma. Using this model system, we investigated the role of changes in the p53 gene in the development of cutaneous melanocyte-derived lesions. Cutaneous melanocytic hyperplasias, benign melanomas and metastatic primary melanomas, plus affected lymph nodes and visceral organs, were screened for mutations in the Monodelphis p53 gene by single-strand conformation polymorphism analysis and direct sequencing. With the exception of a silent point mutation found in a single benign melanocytic hyperplasia sample, no p53 mutations were detected. Furthermore, a relative quantitative reverse transcriptase-polymerase chain reaction approach was used to analyse p53 gene expression at different stages of primary melanoma progression and revealed no substantial changes in p53 mRNA levels. These results suggest that, as in humans, UVR-induced melanoma in the Monodelphis model is initiated and progresses on the basis of predominantly p53-independent molecular pathways.

Characterization of the CDKN2A and ARF genes in UV-induced melanocytic hyperplasias and melanomas of an opossum (Monodelphis domestica).

We examined the involvement of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in the pathogenesis of ultraviolet (UV) radiation-induced melanomas in an opossum (Monodelphis domestica) melanoma model in which suckling young were exposed to UVB to produce melanocytic lesions. Monodelphis CDKN2A and alternated reading frame (ARF) cDNAs were cloned and sequenced, and the expression patterns of these genes were determined by reverse transcription-polymerase chain reaction in normal tissues, 39 primary melanocytic skin lesions, and two tumor-derived cell lines, one nonmetastatic and one metastatic. Primary melanocytic lesions, including hyperplasias, benign melanomas, melanomas metastatic to lymph nodes, and melanomas metastatic to nodes and additional visceral organs, were categorized accordingly as types I-IV. Levels of CDKN2A transcripts were most abundant in type III tumor samples and the metastatic cell line but absent in the nonmetastatic cell line. ARF transcripts were expressed in all tumors and cell lines. A UV-signature mutation was detected with the wild-type allele at the CDKN2A locus in type II and III primary tumor samples and in the nonmetastatic cell line. Interestingly, in the metastatic cell line, only the mutant allele was present and expressed. These data suggest dynamic changes in the expression and/or structure of the CDKN2A and ARF genes represent one molecular defect associated with the etiology of melanoma formation and progression in the Monodelphis model system.

Changes in DNA loop domain structure during spermatogenesis and embryogenesis in the Syrian golden hamster.

The DNA in eukaryotic cells is organized into loop domains that are 25 to 100 kilobases long and attached at their bases to the nuclear matrix. This organization plays major roles in DNA replication and transcription. We examined changes in DNA loop structure of the 5S rDNA gene cluster in the Syrian golden hamster as a function of cellular differentiation by direct visualization with fluorescent in situ hybridization. The 5S rDNA cluster is large enough to encompass more than one loop domain but small enough that individual loop domains can still be resolved. We found that the sizes of the 5S rDNA loops are much smaller, and that the numbers of loops per locus are larger, in all pluripotent cell types than they are in adult somatic tissue. Within the pluripotent spermatogenic cell lineage, the loop domain organization was cell specific. The loop size decreased during the early stages of spermatogenesis but did not change during spermiogenesis, suggesting that DNA loop structure is independent of the chromatin condensation that occurs when protamines replace histones. In early embryonic cells, the loop structure remained small, but in differentiated somatic cells, it became much larger. We suggest that these changes in the 5S rDNA loop domain structure may be related to the maintenance or loss of developmental potential.

Primate spermatogonial stem cells colonize mouse testes.

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.

Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity.

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

An abundance of X-linked genes expressed in spermatogonia.

Spermatogonia are the self-renewing, mitotic germ cells of the testis from which sperm arise by means of the differentiation pathway known as spermatogenesis. By contrast with hematopoietic and other mammalian stem-cell populations, which have been subjects of intense molecular genetic investigation, spermatogonia have remained largely unexplored at the molecular level. Here we describe a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissues. We identified 25 genes (19 of which are novel) that are expressed in only male germ cells. Of the 25 genes, 3 are Y-linked and 10 are X-linked. If these genes had been distributed randomly in the genome, one would have expected zero to two of the genes to be X-linked. Our findings indicate that the X chromosome has a predominant role in pre-meiotic stages of mammalian spermatogenesis. We hypothesize that the X chromosome acquired this prominent role in male germ-cell development as it evolved from an ordinary, unspecialized autosome.

The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development.

Differences in DNA methylation distinguish the maternal and paternal alleles of many imprinted genes. Allele-specific methylation that is inherited from the gametes and maintained throughout development has been proposed as a candidate imprinting mark. To determine how methylation is established in the germline, we have analyzed the allelic methylation patterns of the maternally expressed, paternally methylated H19 gene during gametogenesis in the mouse embryo. We show here that both parental alleles are devoid of methylation in male and female mid-gestation embryonic germ cells, suggesting that methylation imprints are erased in the germ cells prior to this time. In addition, we demonstrate that the subsequent hypermethylation of the paternal and maternal alleles in the male germline occurs at different times. Although the paternal allele becomes hypermethylated during fetal stages, methylation of the maternal allele begins during perinatal stages and continues postnatally through the onset of meiosis. The differential acquisition of methylation on the parental H19 alleles during gametogenesis implies that the two unmethylated alleles can still be distinguished from each other. Thus, in the absence of DNA methylation, other epigenetic mechanism(s) appear to maintain parental identity at the H19 locus during male germ cell development.

Human spermatogenesis as a model to examine gene potentiation.

The first tier of control over the expression of genic domains utilizes chromatin structure. Before the onset of transcription, the chromatin domain that encompasses the gene(s) must assume an open conformation. This renders large segments of the genome available to the tissue-specific and ubiquitous trans-factors necessary for proper expression of the genes present. This process has been termed potentiation. It is a necessary obligate, but alone it is not sufficient for gene expression. Spermatogenesis, the development of a viable fertile male gamete, provides a unique model to begin to address the underlying mechanism(s) governing differentiation and tissue-specific gene expression. Male gametogenesis is typified by the activation of numerous genes whose products have novel functions, as well as testis-specific forms of constitutively expressed somatic genes. We have shown that mouse spermatogenesis represents a selective potentiative process (Kramer et al., 1998: Development 125:4749-4655), but little is known about its human counterpart. To fill this void we have examined the potentiative state of several spermatid-expressed genes during the latter stages of human spermatogenesis. We have shown that spermatidexpressed genes are potentiated by the pachytene stage of differentiation. Furthermore, we establish that a chromatin domain functions as a discrete structural unit during differentiation. Interestingly, some of these open structures are maintained in the mature spermatozoon.

Concerted regulation and molecular evolution of the duplicated SNRPB'/B and SNRPN loci.

The human small nuclear ribonucleoprotein SNRPB ' /B gene is alternatively spliced to produce the SmB or SmB' spliceosomal core proteins. An ancestral duplication gave rise to the closely related SNRPN paralog whose protein product, SmN, replaces SmB'/B in brain. However, the precise evolutionary and functional relationship between these loci has not been clear. Genomic, cDNA and protein analyses presented here in chicken, two marsupials (South American opossum and tammar wallaby), and hedgehog, suggest that the vertebrate ancestral locus produced the SmB' isoform. Interestingly, three eutherians exhibit radically distinct splice choice expression profiles, producing either exclusively SmB in mouse, both SmB and SmB' in human, or exclusively SmB' in hedgehog. The human SNRPB ' /B locus is biallelically unmethylated, unlike the imprinted SNRPN locus which is unmethyl-ated only on the expressed paternal allele. Western analysis demonstrates that a compensatory feedback loop dramatically upregulates SmB'/B levels in response to the loss of SmN in Prader-Willi syndrome brain tissue, potentially reducing the phenotypic severity of this syndrome. These findings imply that these two genes encoding small nuclear ribonucleoprotein components are subject to dosage compensation. Therefore, a more global regulatory network may govern the maintenance of stoichiometric levels of spliceosomal components and may constrain their evolution.

Asynchronous replication of imprinted genes is established in the gametes and maintained during development.

Genomic imprinting is characterized by allele-specific expression of multiple genes within large chromosomal domains that undergo DNA replication asynchronously during S phase. Here we show, using both fluorescence in situ hybridization analysis and S-phase fractionation techniques, that differential replication timing is associated with imprinted genes in a variety of cell types, and is already present in the pre-implantation embryo soon after fertilization. This pattern is erased before meiosis in the germ line, and parent-specific replication timing is then reset in late gametogenesis in both the male and female. Thus, asynchronous replication timing is established in the gametes and maintained throughout development, indicating that it may function as a primary epigenetic marker for distinguishing between the parental alleles.

Construction and preliminary characterization of a series of mouse and rat testis cDNA libraries.

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18-day-old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6-, 8-, 17-, and 18-20-day-old mice, and peritubular cells from 18-20 day old mice, all recovered from outbred white Swiss (CD-1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 Bl6/J, C3 HEB, BDF-1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20-day-old Sprague-Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85-98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni-Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 x 10(5) to 2.9 x 10(8) plaque-forming units, and insert sizes averaged 1.0-1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided.

Reprogramming the male gamete genome: a window to successful gene therapy.

Hematopoiesis and spermatogenesis both initiate from a stem cell capable of renewal and differentiation. Each pathway reflects the expression of unique combinations of facultative, i.e. tissue-specific and constitutive, i.e. housekeeping, genes in each cell type. In spermatogenesis, as in hematopoiesis, commitment is mediated by the mechanism of potentiation whereby specific chromatin domains are selectively opened along each chromosome. Within each open chromatin domain, a unique battery of gene(s) is availed to tissue-specific and ubiquitous transacting factors that are necessary to initiate transcription. In the absence of an open domain, trans-factor access is denied, and the initiation of transcription cannot proceed. Cell-fate is thus ultimately defined by the unique series of open-potentiated cell-specific chromatin domains. Defining the mechanism that opens chromatin domains is fundamental in understanding how differentiation from stem cells is controlled and whether cell-fate can be modified. A recent examination of the mammalian spermatogenic pathway [Kramer, J.A., McCarrey, J.M, Djakiew, D., Krawetz, S.A., 1998. Differentiation: the selective potentiation of chromatin domains. Development 125, 4749-4755] supports the view that cell fate is mediated by global changes in chromatin conformation. This stride underscores the possibility of moderating differentiation through chromatin conformation. It is likely that gene therapeutics capable of selectively potentiating individual genic domains in populations of differentiating and/or replicating cells that modify cellular phenotype will be developed in the next millennium.

Multiple elements influence transcriptional regulation from the human testis-specific PGK2 promoter in transgenic mice.

The PGK2 gene is expressed in a strictly tissue-specific manner in meiotic spermatocytes and postmeiotic spermatids during spermatogenesis in eutherian mammals. Previous results indicate that this is regulated at the transcriptional level by core promoter sequences that bind ubiquitous transcription factors and by sequences in a 40-base pair (bp) upstream enhancer region (E1/E4) that bind tissue-specific transcription factors. Transgenic mice carrying different PGK2 promoter sequences linked to the chloramphenicol acetyltransferase (CAT) reporter gene, one containing only the 40-bp E1/E4 enhancer sequence plus the core promoter and two containing 515 bp of PGK2 promoter but with either the E1/E4 enhancer region or the Sp1-binding site in the core promoter disrupted by in vitro mutagenesis, all showed levels of expression reduced to less than half that of the wild-type 515 PGK2/CAT transgene. These results indicate that multiple factor-binding regions normally regulate initiation of transcription from the PGK2 promoter. The single disruption of any one of these binding activities reduced, but did not abolish, transgene expression. This is consistent with an "enhanceosome"-like function in this promoter involving multiple bound activator proteins that interact in a combinatorial manner to synergistically promote testis-specific transcription.

Spermatogenesis as a model system for developmental analysis of regulatory mechanisms associated with tissue-specific gene expression.

Spermatogenesis is a complex system leading to the formation of male gametes. Development of the spermatogenic cell lineage occurs throughout most of the pre- and post-natal lifetime of male mammals, and involves progression through a well-characterized series of stages and cell types. This progression is based on programmed gene expression. The mechanisms by which this tissue-, cell-type, and developmental-stage specific gene expression is regulated form the focus of an active area of investigation. In this review, I will summarize our efforts to elucidate these mechanisms through molecular analyses of the uniquely accessible mouse spermatogenic cell lineage.

Differentiation: the selective potentiation of chromatin domains.

Potentiation is requisite for the expression of our genome. It is the mechanism of opening chromatin domains to render genes accessible to tissue-specific and ubiquitous transacting-factors that enables transcription. The results presented in this study demonstrate that modulation of stage- and cell-type-specific gene expression during mammalian spermatogenesis involves selective potentiation of testis-expressed genes that reverses their repressive state when present in the spermatogonial stem cell. This directly contrasts hematopoiesis, which acts to selectively restrict lineage potential during differentiation from its permissive stem cell. These results are key to understanding how differentiative pathways are controlled and cellular phenotypes determined. A window to their modulation is presented.

9th International Congress on Isozymes, Genes, and Gene Families: an overview.

The 9th International Congress on Genes, Gene Families, and Isozymes marked a historic transition in the series formerly known as the International Congress on Isozymes. The name of the congress was changed to reflect the broadened scope of this field and the new directions in which it is moving. To recognize and promote this transition, a number of new features were incorporated into this congress. Accordingly, the broad-based program featured preeminent biologists from 29 different countries. A total of 350 people attended the congress. A special new feature of this congress was the Student/Fellow Program, which was designed to enhance participation by advanced undergraduate and graduate students and postdoctoral fellows. This congress celebrated the progress that has occurred during the past 40 years beginning with studies of isozymes and leading into studies of specific genes and gene families. As we move into the next millennium, it is clear that our field is strongly positioned and will continue to be the focus of exciting and important new research.

Mutation frequency declines during spermatogenesis in young mice but increases in old mice.

Five percent of live-born human offspring will have a genetic disorder. Of these, 20% are because of germ-line de novo mutations. Several genetic diseases, such as neurofibromatosis and Duchenne muscular dystrophy, are associated with a high percentage of de novo germ-line mutations. Until recently, a direct analysis of spontaneous mutation frequencies in mammalian germ cells has been prevented by technical limitations. We have measured spontaneous mutation frequencies in a lacI transgene by using enriched populations of specific spermatogenic cell types. Similar to previously published results, we observed a lower mutation frequency for seminiferous tubule cell preparations, which contain all stages of spermatogenesis, relative to somatic tissues. We made the unexpected observation of a decline in mutation frequency during spermatogenesis, such that the mutation frequencies of type B spermatogonia and all subsequent stages of spermatogenesis are lower than the frequency for primitive type A spermatogonia. In addition, spermatogenic cells from old mice have significantly increased mutation frequencies compared with spermatogenic cells from young or middle-aged mice. Finally, the mutation frequency was observed to increase during spermiogenesis in postreplicative cell types when spermatogenic cells were obtained from old mice.

A gene-specific promoter in transgenic mice directs testis-specific demethylation prior to transcriptional activation In vivo.

Transcription of the autosomal phosphoglycerate kinase gene, Pgk-2, is initiated at the onset of meiosis during spermatogenesis in mammals. However, in the mouse, the 5' portion of the endogenous Pgk-2 coding sequence undergoes a specific demethylation event that precedes transcriptional activation by 10-12 days. Here we show that transgenes consisting of the Pgk-2 core promoter ligated to the CAT reporter gene undergo a similar tissue-, stage-, and cell type-specific demethylation in the 5' portion of the CAT coding sequence, whereas transgenes consisting of the CAT reporter sequence alone, or of the CAT sequence ligated to the CpG island-containing transferrin gene promoter, demonstrate different patterns of demethylation. These results indicate that specific promoter sequences can influence the pattern of tissue-specific demethylation within different genes and that a signal for spermatogenic cell-specific demethylation resides within the core promoter of the mammalian Pgk-2 gene.