The role of neurotrophin receptors in female germ-cell survival in mouse and human

Norah Spears ) 2 3 Michael D. Molinek 2 3 Lynne L. L. Robinson 1 2 Norma Fulton 1 2 Helen Cameron 2 3 Kohji Shimoda 2 3 Evelyn E. Telfer 0 2 Richard A. Anderson 1 2 David J. Price 2 3 0 Institute of Cellular and Molecular Biology, University of Edinburgh , Darwin Building, Kings Buildings, Edinburgh , UK 1 Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The University of Edinburgh Chancellor' s Building, 49 Little France Crescent, Edinburgh EH16 4SA , UK 2 Key words: Trk , Oogonia, Oocyte, Survival, Human, Mouse, Neurotrophin 3 Biomedical Sciences, University of Edinburgh , Hugh Robson Building, George Square, Edinburgh EH8 9XD , UK The role of neurotrophin receptors in female germ-cell survival in mouse and human - During mammalian ovary formation, the production of ovarian follicles is accompanied by an enormous loss of germ cells. It is not known how this loss is regulated. We have investigated the role of the Trk tyrosine kinase receptors, primarily TrkB, in this process. The ovaries of TrkB/ and TrkC/ mice with a mixed (129Sv C57BL/6) genetic background were examined shortly after birth. Around 50% of TrkB/ mice had grossly abnormal ovaries that contained greatly reduced numbers of follicles. No defects were found in the ovaries of TrkC/ mice. Congenic TrkB/ mice were generated on 129Sv and C57BL/6 backgrounds: whereas the former had a mixed ovarian phenotype similar to that of the original colony of mice, the ovaries of all offspring of the C57BL/6 congenic line contained reduced numbers of follicles. RT-PCR showed that mRNA encoding TrkB and its two ligands, neurotrophin 4 (NT4) and brain-derived neurotrophic factor (BDNF), were present throughout the period of Female embryonic mice produce tens of thousands of germ cells as the ovary forms. Shortly before birth, germ cells (termed oogonia at this stage) stop mitosis, initiate meiosis (which is halted at the diplotene stage) and associate closely with somatic pregranulosa cells to form primordial follicles. There is now a finite supply of female germ cells (now termed oocytes), which cannot be replenished if lost. Concurrent with these processes, there is a massive wave of cell death that results in the death of 80-90% of oocytes in mice and humans (Brambell, 1927; Baker, 1963; Hirshfield, 1991). The same general pattern of oocyte loss occurs in all mammals. In mice, this wave of oocyte death is most pronounced around the time of birth, when follicle formation is at its peak; in humans, it occurs at around five months of gestation. This process is vital to the reproductive potential of all female mammals because their reproductive lifespan is determined by the supply of primordial follicles, but its regulation is not understood. Primordial follicles consist of an oocyte surrounded by flattened granulosa cells, and are considered to be at a resting stage of development. Follicles can remain at this stage follicle formation in the mouse. In situ hybridisation showed that TrkB was expressed primarily in the germ cells before and after follicle formation. Mouse neonatal and fetal ovaries and human fetal ovaries were cultured in the presence of K252a, a potent inhibitor of all Trk receptors. In mice, K252a inhibited the survival of germ cells in newly formed (primordial) follicles. This effect was rescued by the addition of basic fibroblast growth factor (bFGF) to the culture medium. Combined addition of both BDNF and NT4 blocking antibodies lowered germ-cell survival, indicating that these TrkB ligands are required in this process. The results indicate that signalling through TrkB is an important component of the mechanism that regulates the early survival of female germ cells. throughout the reproductive lifespan of a female. The first sign of further development of the primordial follicle is the rounding up of granulosa cells. When follicles contain primarily rounded granulosa cells, they are considered to have entered the growth phase and are termed primary follicles. This process first occurs shortly after birth in the mouse. The neurotrophins are a small family of closely related peptide factors. Nerve growth factor (NGF) was the first to be discovered; BDNF, NT3, NT4 and NT6 have since been identified (Snider, 1994). The neurotrophins act on both high and low affinity cell-surface receptors. Many of the effects of the neurotrophins on cell survival and neuronal growth are mediated by high affinity glycoprotein tyrosine receptor kinases, or Trk receptors. Trk receptors consist of an extracellular domain, which contains the neurotrophin-binding site, a short transmembrane segment, and an intracellular domain that encodes a tyrosine kinase. The neurotrophins bind selectively to the high affinity Trk receptors, which form homodimers and autophosphorylate to trigger the intracellular cascade (Segal and Greenberg, 1996). There are three members of the Trk receptor family: TrkA, the receptor for NGF; TrkB, the receptor for BDNF and NT4; and TrkC, the receptor for NT3. The functions of truncated forms of the TrkB and TrkC receptors, which lack the intracellular tyrosine kinase domains (Klein et al., 1990; Dechant, 2001), are unclear. In addition to the Trk receptors, all neurotrophins bind with relatively equal, low affinity to a membrane receptor known as p75, a member of the tumour necrosis receptor superfamily. The p75 receptor lacks tyrosine kinase activity, but it does appear to have signalling capabilities. It might modulate cellular responses to the neurotrophins by enhancing the sensitivity of the Trk receptors (Hantzopoulos et al., 1994), whereas in the absence of Trk receptors it can induce cell death (Friedman, 2000). The neurotrophins are implicated in a variety of developmental processes at numerous neural sites. Their bestknown roles are in the regulation of cell survival. Thus, neurons that contain one or more of the Trk receptors might require the presence of sufficient concentrations of the appropriate neurotrophin(s) for their continued survival. They might also be involved in the regulation of neuronal differentiation, growth and migration (Ghosh and Greenberg, 1995; Segal and Greenberg, 1996). All three Trk receptors are expressed around the time of follicle formation in rats and humans (Dissen et al., 1995; Anderson et al., 2002). In rats, expression of TrkB mRNA increases sharply and TrkA mRNA decreases abruptly during the period of follicle formation whereas TrkC remains constant throughout. Expression of NT4 mRNA increases concomitantly with that of its ligand TrkB. In humans, the expression pattern of NT4 mRNA changes as follicles start to form, with expression, which is predominantly in oogonia before follicle formation, switching predominantly to the somatic pregranulosa cells around the time of follicle formation (Anderson et al., 2002). Thus, the location of NT4 mRNA production moves from the germ cell to the somatic cell just as germ ce (...truncated)