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Canadian HealthCare News
Mammalian Oogenesis and Folliculogenesis: MATERIALS AND METHODS(2)
Conditions for PCR were as follows: 1X (3 min at 94°C); 30X (94°C for 45 sec, 55°C for 1 min, 72°C for 1 min); 1X (72°C for 5 min). PCR products were ligated into the pGEM-T Easy vector (Promega Corp., Australia) and cloned into XL-1 Blue Escherichia coli (Stratagene, La Jolla, CA). Cloned cDNAs were sequenced (Biomolecular Resource Facility, JCSMR, ANU), and nucleotide and amino acid sequence analyses were performed using Lasergene v6 software (DNASTAR Inc., Madison, WI). Sequences were deposited in GenBank, and accession numbers for rabbit Kitl DQ356265, rabbit Kit (cytoplasmic domain) DQ356267, and rabbit Kit (extracellular domain) DQ356266 were assigned respectively.
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Mammalian Oogenesis and Folliculogenesis: MATERIALS AND METHODS(1)
Materials
Unless otherwise stated, reagents were obtained from Sigma Chemical Co. (Australia).
Animals
All animal experimental procedures were approved by the CSIRO, Division of Sustainable Ecosystems, Gungahlin, Animal Experimentation Ethics Committee in accordance with National Health and Medical Research Council/CSIRO guidelines.
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Mammalian Oogenesis and Folliculogenesis: INTRODUCTION(4)
Given the limited accessibility of human ovarian tissue and the lack of information available for the activity of KITL in species other than the mouse, we identified the rabbit as an alternative animal model for the study of the roles of KITL and KIT during the early stages of folliculogenesis. The initial objective of this research was to use in situ hybridization and immunohistochemistry to provide a detailed analysis of Kitl and Kit mRNA and protein expression within the ovaries of rabbits during the assembly of primordial follicles and the first wave of folliculogenesis, which occurs between Weeks 2 and 12 of postnatal development in this species.
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Mammalian Oogenesis and Folliculogenesis: INTRODUCTION(3)
Although a role for KITL and KIT in these processes is well established in rodents, there is a paucity of information regarding the functional significance of KITL and KIT during primordial follicle activation in other species. The limited information available for the human indicates that, similar to mice, the oocytes of primordial follicles in human fetal ovaries express KIT, and expression initially intensifies in growing preantral follicles, only to decrease again with antrum formation.
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Mammalian Oogenesis and Folliculogenesis: INTRODUCTION(2)
The expression of KITL and KIT is well characterized in the developing mouse ovary. It has been reported that with the exception of oocytes undergoing the first stages of meiosis, in which Kit mRNA expression is downregulated, oocytes at all stages of development express Kit mRNA and protein. Similarly, the granulosa cells of follicles at all stages of development express Kitl mRNA and protein, although expression is very low in the squamous granulosa cells of primordial follicles. Given that KITL is derived from granulosa cells and KIT is expressed by oocytes and theca cells, this receptor-ligand pair is an excellent example of how paracrine interactions between oocytes and their companion somatic cells control many aspects of follicle formation and development.
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Mammalian Oogenesis and Folliculogenesis: INTRODUCTION(1)
Distinct Expression Patterns of Different Subunit Isoforms: DISCUSSION(9)
In summary, the expression of various isoforms of the same V-ATPase subunit in narrow and clear cells strongly indicates that these cells are fully equipped for active proton secretion and that they possess potentially important backup mechanisms that may compensate for the lack of one or more V-ATPase subunit isoforms. This underlies the important role that the V-ATPase plays in acidifying the lumen of the epididymis, a process that is important for sperm maturation and storage.
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Distinct Expression Patterns of Different Subunit Isoforms: DISCUSSION(8)
Some isoforms could serve as a possible alternative mechanism for the active role played by the predominant one in the V-ATPase holoenzyme. Mutations of some subunits of the V-ATPase, including Bl and a4, are linked to renal distal tubular acidosis and deafness. Our finding that a4 and Bl are highly expressed in narrow and clear cells of the epididymis raised the possibility that fertility might be altered in patients harboring these mutations. Long-term clinical follow-up of these young patients will be required to address this question.
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Distinct Expression Patterns of Different Subunit Isoforms: DISCUSSION(7)
Alternatively, various subunits of the V-ATPase, including subunits a, d, A, and C, have been shown to control the activity of the V-ATPase by modulating the coupling of proton transport to ATP hydrolysis. The presence of various isoforms of subunits a, d, and C in clear cells may, therefore, reflect different levels of V-ATPase activity in the membrane domains and intracellular compartments in which they are expressed.
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Distinct Expression Patterns of Different Subunit Isoforms: DISCUSSION(6)
A similar function has been previously suggested for the 56-kDa subunit (B1), which was detected in nonacidifying endosomes isolated from kidney collecting duct principal cells. Alternatively, some unidentified V0 domain subunit isoforms may be expressed in principal cells and may serve to anchor dl in their apical membrane.
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