scholarly journals Diurnal Rhythmicity of the Clock Genes Per1 and Per2 in the Rat Ovary

Endocrinology ◽  
2006 ◽  
Vol 147 (8) ◽  
pp. 3769-3776 ◽  
Author(s):  
Jan Fahrenkrug ◽  
Birgitte Georg ◽  
Jens Hannibal ◽  
Peter Hindersson ◽  
Søren Gräs
Keyword(s):  
In Situ Hybridization ◽  
Circadian Clock ◽  
Estrous Cycle ◽  
Clock Genes ◽  
Continuous Darkness ◽  
Peripheral Tissues ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Rat Ovary

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12-h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0–2 and peaked at Zeitgeber time 12–14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis.

Expression of transforming growth factor alpha during development

1988 ◽  
Vol 66 (8) ◽  
pp. 1113-1121 ◽  
Author(s):  
V. K. M. Han ◽  
A. J. D'Ercole ◽  
D. C. Lee
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
Egf Receptors ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Factor Alpha

Transforming growth factors (TGFs) are polypeptides that are produced by transformed and tumour cells, and that can confer phenotypic properties associated with transformation on normal cells in culture. One of these growth-regulating molecules, transforming growth factor alpha (TGF-α), is a 50 amino acid polypeptide that is related to epidermal growth factor (EGF) and binds to the EGF receptor. Previous studies have shown that TGF-α is expressed during rodent embryogenesis between 7 and 14 days gestation. To investigate the cellular sites of TGF-α mRNA expression during development, we have performed Northern analyses and in situ hybridization histochemistry on the conceptus and maternal tissues at various gestational ages. Contrary to previous reports, both Northern analyses and in situ hybridization histochemistry indicate that TGF-α mRNA is predominantly expressed in the maternal decidua and not in the embryo. Decidual expression is induced following implantation, peaks at day 8, and declines through day 15 when the decidua is being resorbed. In situ hybridization revealed that expression of TGF-α mRNA is highest in the region of decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and embryo. In addition, we could not detect TGF-α mRNA expression in other maternal tissues, indicating that the induction of TGF-α transcripts in the decidua is tissue specific, and not a pleiotropic response to changes in hormonal milieu that occur during pregnancy. The developmentally regulated expression of TGF-α mRNA in the decidua, together with the presence of EGF receptors in this tissue, suggests that this peptide may stimulate mitosis and angiogenesis locally by an autocrine mechanism. Because EGF receptors are also present in the embryo and placenta, TGF-α may act on these tissues by a paracrine or endocrine mechanism.

scholarly journals Expression and localization of the mRNA for DNA (cytosine-5)- methyltransferase in mouse seminiferous tubules.

1994 ◽  
Vol 42 (9) ◽  
pp. 1271-1276 ◽  
Author(s):  
M Numata ◽  
T Ono ◽  
S Iseki
Keyword(s):  
In Situ Hybridization ◽  
Significant Role ◽  
Meiotic Prophase ◽  
Oligonucleotide Probe ◽  
Mouse Testis ◽  
Seminiferous Tubules ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry

DNA (cytosine-5)-methyltransferase (DNA MTase) is the only enzyme known to be involved in the methylation of mammalian DNA. Although the expression of DNA MTase gene is abundant in the testis, little is known about the role of this enzyme during spermatogenesis. We examined the distribution of DNA MTase mRNA in mouse testis by in situ hybridization histochemistry with an oligonucleotide probe. The mRNA signal was observed in the seminiferous tubules and was localized predominantly in spermatogonia and spermatocytes, particularly during the earlier steps of meiotic prophase I, with maximal intensity in the early pachytene cells. These results suggest some significant role for DNA MTase in spermatogenesis.

Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain

1992 ◽  
Vol 68 (3) ◽  
pp. 756-766 ◽  
Author(s):  
T. M. Perney ◽  
J. Marshall ◽  
K. A. Martin ◽  
S. Hockfield ◽  
L. K. Kaczmarek
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
K Channel ◽  
Rnase Protection ◽  
Hybridization Histochemistry ◽  
Adult Rat ◽  
In Situ Hybridization Histochemistry ◽  
Adult Rat Brain ◽  
Developing Rat

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Rapid and efficient generation of PCR-derived riboprobe templates for in situ hybridization histochemistry.

1993 ◽  
Vol 41 (5) ◽  
pp. 773-776 ◽  
Author(s):  
J H Sitzmann ◽  
P K LeMotte
Keyword(s):  
In Situ Hybridization ◽  
Coding Region ◽  
Biopsy Material ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Gene Coding ◽  
Suprabasal Cell ◽  
Cell Layers ◽  
Negative Controls

In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3' end of the human cytokeratin 1 (K1) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes were then applied in ISH to human skin sections made from routinely fixed and paraffin-embedded clinical biopsy material. Consistent with published results, we observed strong expression of K1 mRNA in the suprabasal cell layers of the epidermis but only weak to undetectable signals in the basal and cornified cell layers and in the dermis. With this experimental procedure we see no decrease in probe efficiency or quality compared to conventional methods. The use of PCR-derived riboprobe templates for ISH makes it possible to detect expression of any desired gene of known sequence rapidly and efficiently.

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