Abstract:Previous studies reported the presence of choline acetyltransferase (ChAT) mRNA and protein in the mammalian testis. We have now found that none of the ChAT mRNAs produced in the testis is capable of encoding a full‐length ChAT protein. Two ChAT cDNAs were isolated from an adult rat testis cDNA library encoding N‐terminally truncated ChAT proteins of 450 and 414 amino acids (aa), respectively, the former containing a novel N‐terminal extension of 69 residues. Rapid Amplification of cDNA Ends (RACE) analysis re… Show more
“…figure 3). For lack of space, absence of conclusive experimental evidence, or stylistic reasons, the event mechanism may not be mentioned in the abstract text (e.g., [1,2,3]). In these cases, the event may be missing but the presence of other word chunks may give enough bases to consider them positive sentences (category 3).…”
The mechanisms for generating transcript diversity have been studied experimentally, using various biochemical methods including variants of PCR, S1 nuclease assays and blot hybridizations. The conclusion about the mechanism(s) involved, can be reached after nucleotide sequencing and computational analysis. Hence, sentences describing events that generate TD (Supp figure 3) may contain event mechanisms, results of the experimental methods or statements describing observations or presumptions.
“…figure 3). For lack of space, absence of conclusive experimental evidence, or stylistic reasons, the event mechanism may not be mentioned in the abstract text (e.g., [1,2,3]). In these cases, the event may be missing but the presence of other word chunks may give enough bases to consider them positive sentences (category 3).…”
The mechanisms for generating transcript diversity have been studied experimentally, using various biochemical methods including variants of PCR, S1 nuclease assays and blot hybridizations. The conclusion about the mechanism(s) involved, can be reached after nucleotide sequencing and computational analysis. Hence, sentences describing events that generate TD (Supp figure 3) may contain event mechanisms, results of the experimental methods or statements describing observations or presumptions.
The presence of muscarinic receptors (MR) in the ovary of different species has been recognized, but the identity of these receptors as well as ovarian sources of their natural ligand, acetylcholine (ACh), have not been determined. Because luteinized human granulosa cells (GC) in culture express functional MR, we have determined whether the group of the related MR subtypes, M1R, M3R, and M5R, are present in vivo in human and rhesus monkey ovaries. To this end, ribonucleic acids (RNAs) of different human and monkey ovaries as well as RNAs from human GC and monkey oocytes were reverse transcribed and subjected to PCR amplification, followed by sequencing of the amplified complementary DNAs. Results obtained showed that M1R, M3R, and M5R messenger RNAs are present in adult human and monkey ovaries; oocytes express exclusively the M3R subtype, whereas GC express M1R and M5R. To determine the ovarian source(s) of the natural ligand of these ACh receptors, we attempted to localize the enzyme responsible for its synthesis with the help of a monoclonal antibody recognizing choline acetyltransferase for immunohistochemistry. In neither human nor monkey sections did we detect immunoreactive choline acetyltransferase-positive fibers or nerve cells, but, surprisingly, GC of antral follicles showed prominent staining. To determine whether GC can produce ACh, human cultured GC derived from preovulatory follicles were analyzed using a high pressure liquid chromatography technique. The results showed that these cells contained ACh in concentrations ranging from 4.2-11.5 pmol/10(6) cells. Samples of a rat granulosa cell line likewise contained ACh. Thus, the ovary contains multiple MR, and GC of antral follicles are able to synthesize ACh, the ligand of MR. We propose that ACh may serve as an as yet unrecognized factor involved in the complex regulation of ovarian function in the primate, e.g. regulation of cell proliferation or progesterone production.
The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that nonneuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight a and three b subunits of CHRN in testis. Expression of the a7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the a4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.
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