Expression of Nerve Growth Factor and its Receptors trka and p75 and Inhibin α-subunit in the Ovarian Interstitial Cells of Lactating Golden Hamsters

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1 Journal of Reproduction and Development, Vol. 54, No. 5, 2008, Research Note Expression of Nerve Growth Factor and its Receptors trka and p75 and Inhibin α-subunit in the Ovarian Interstitial Cells of Lactating Golden Hamsters Qiang WENG 1,2), Zhanquan SHI 2,3), Maiko KAWAGUCHI 4), Gen WATANABE 2,3) and Kazuyoshi TAYA 2,3) 1) College of Biological Science and Technology, Beijing Forestry University, Beijing , China, 2) Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo , 3) Department of Basic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu and 4) Department of Toxicology and Pharmacology, Faculty of Pharmacology, Musashino University, Tokyo , Japan Abstract. Characteristic daily increases in the plasma levels of luteinizing hormone (LH) are present every afternoon during lactation in golden hamsters. The objective of this study was to investigate the effect of the diurnal rhythm of increases in LH on expression of nerve growth factor (NGF), its receptors trka and p75 and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters. Both lactating and non-lactating groups of postpartum golden hamsters were used in this study. The expression of NGF, its receptors trka and p75 and inhibin α-subunit were determined by immunohistochemistry. Positive staining of NGF, trka and p75 was found in the interstitial cells of the lactating group, and no immunoreactivity for NGF, trka or p75 was observed in the ovarian interstitial cells of the nonlactating group. In addition, immunostaining of inhibin α-subunit was also observed in the interstitial cells of the lactating group but not in those of the non-lactating group. Immunostaining of the inhibin/activin β A- and β B-subunits was observed in the granulosa cells of antral follicles, but not in the interstitial cells of the lactating and non-lactating animals. These results suggest that the diurnal rhythm increases in LH can induce expression of NGF, trka, p75 and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters and that NGF, its receptors trka and p75 and inhibin α-subunit may have the capacity for autocrine or paracrine modulation of interstitial cell differentiation in golden hamsters. Key words: Golden hamster (Mesocricetus auratus), Inhibin α subunit, Interstitial cell, Nerve growth factor (NGF), p75, trka (J. Reprod. Dev. 54: , 2008) he nerve growth factor (NGF), a 26-kDa polypeptide [1], belongs to a family of related proteins required for the survival, maintenance and development of discrete neuronal populations in the central and peripheral nervous systems [2, 3]. The effect of NGF has been shown to be mediated through specific membrane receptors high-affinity tyrosine kinase A (trka), and it is responsible for the biological activities of NGF [4, 5]. Furthermore, the effect of NGF is also mediated via low affinity p75 receptor and it is also other neurotropins receptor [6]. It is now well known that NGF and its receptors are expressed in the mammalian ovary. More and more evidence indicates that NGF and its receptors play a critical role in development of the mammalian ovary, oogenesis and folliculogenesis [7 13]. Inhibin/activin subunits are present in numerous mammalian tissues of both endocrine and nonendocrine organs. The dimeric proteins formed from the subunits are members of the transforming growth factor (TGF) β superfamily of growth and differentiation factors and have been isolated and characterized as gonadal peptides. Inhibins and activins are structurally related dimeric gonadal Accepted for publication: June 13, 2008 Published online in J-STAGE: July 16, 2008 Correspondence: Q. Weng ( weng3@msn.com) proteins with the ability to regulate follicle stimulating hormone (FSH) secretion from pituitary glands [14]. They are also dimeric glycoproteins formed by two of three different subunits (α, β A and β B). Inhibins consist of β-subunits dimerized with a common α- subunit (α:β A and α:β B; inhibin A and inhibin B, respectively). Activins are dimers of β-subunits (β A: β A, β A:β B and β B:β B; activin A, activin AB and activin B, respectively). It is now clear that inhibin/activin subunits can play local autocrine and paracrine roles in steroidogenesis and folliculogenesis within developing ovaries [15 19]. There are many unique and interesting features in the reproductive physiology of golden hamsters. For example, follicular atresia and luteolysis occur rapidly, and the ovaries are transformed into an almost solid mass of interstitial tissues after parturition [20]; therefore, there are no antral follicles and functional corpora lutea in the ovaries of lactating golden hamsters. Characteristic daily increases in the plasma levels of luteinizing hormone (LH) and FSH are observed every afternoon during lactation, and the LH elevation is thought to be responsible for interstitial hyperplasia during lactation in golden hamsters [21, 22]. In our previous studies, NGF and its two receptors trka and p75 were present in ovaries and uteri, demonstrating that they have important autocrine and paracrine regulatory roles in ovarian function and in uterine growth

2 398 WENG et al. and proliferation during the estrous cycle [13, 23 25]. Shi et al. [23] found that the LH surge can stimulate expression of NGF, trka and p75 in the interstitial cells of cyclic golden hamsters. In our previous studies, we reported similar observations for inhibin α- subunit in the ovarian interstitial cells of cyclic golden hamsters, and this indicates that the LH surge may be an important factor for inducing production of inhibin α-subunit in the interstitial cells of the cyclic golden hamster [26, 27]. To extend our understanding of the mechanisms that regulate ovarian interstitial cells differentiation in golden hamsters, the present study investigated the immunolocalization of NGF, its receptors trka and p75 and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters. The objective of this study was elucidate the effect of daily increases in LH on expression of NGF, its receptors trka and p75 and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters. Materials and Methods Animals Adult female golden hamsters (Mesocricetus auratus) with at least two consecutive estrous cycles were used in the present study. Mating was carried out in the evening of proestrus by putting female golden hamsters into a cage holding fertile male golden hamsters. The golden hamsters delivered pups after gestation for 15 days, and the day of parturition was designated as day 0 of lactation. Golden hamsters nursing 8 pups were used in this experiment. All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Tokyo University of Agriculture and Technology. Treatment After delivery, the animals were divided into two groups, a lactating group in which pups were nursed until day 10 of lactation and a non-lactating group in which all pups were removed soon after delivery. Five animals were used in each group. Sampling The ovaries from two groups were removed at 1100 h on day 10 of lactation and were fixed in 4% paraformaldehyde and embedded in paraffin wax. Serial sections (4 μm) were mounted on slides coated with poly-l-lysine (Sigma-Aldrich Chemical, St. Louis, MO, USA.) for immunohistochemistry. Immunohistochemistry The procedures for immunohistochemistry have been described previously [23]. Ovarian sections were incubated with 10% normal goat serum to reduce background staining caused by the second antibody. NGF and its receptors trka and p75 were detected using polyclonal antibodies against NGF (M-20), trka (763) and p75 (H- 92) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibodies against each inhibin subunit were anti-[tyr 30 ]inhibin-αchain (1-30)-NH 2 conjugated to rabbit serum albumin, anticyclic inhibin β A (81-113)-NH 2 and anticyclic inhibin β B (80-112)-NH 2. The inhibin α-subunit peptide was kindly provided by Dr. Ling N (Neurocrine Biosciences, San Diego, CA, USA), and the anticyclic inhibin β A (no D) and anti-cyclic inhibin β B (no D) were kindly provided by Dr. Vale W (Salk Institute for Biological Studies, La Jolla, CA, USA). The sections were incubated with primary antibody to NGF, trka, p75, inhibin α-subunit (1:2000), inhibin β A (1:2,000) and inhibin β B (1:2,000) for 12 h at room temperature. The sections were then incubated with a second antibody, goat anti-rabbit IgG conjugated with biotin and peroxidase with avidin, using a rabbit ExtrAvidin TM staining kit (Sigma) and then visualized with 30 mg 3,3-diaminobenzidine (Wako, Osaka, Japan) solution in 150 ml of 0.05 M Tris-HCl buffer (ph 7.6) plus 30 μl H 2O 2. Finally, the reacted sections were counterstained with haematoxylin solution (Wako, Tokyo, Japan). The control sections were treated with normal rabbit serum (Sigma) instead of the primary antisera. Results Effect of pups removal on ovarian follicular development of postpartum golden hamsters In the non-lactating group, the normal estrous cycle was restored after removal of the pups. Vaginal discharge was observed 3 and 7 days after delivery, whereas no vaginal discharge was observed until day 10 of lactation in the lactating animals. The animals in the non-lactating group 10 days after delivery exhibited a proestrous vaginal cytology pattern. Immunohistochemical detection of NGF, its receptors trka and p75 and inhibin α-subunit Immunohistochemical detection of NGF, trka, p75 and inhibin α-subunit are shown in Fig. 1. Immunostaining for NGF, trka and p75 was present in the ovarian interstitial cells of the lactating group (Fig. 1a, b and c), whereas no positive was observed for NGF, trka and p75 in the ovarian interstitial cells of the non-lactating group (Fig. 1d, e and f). Immunoreactivity for inhibin α- subunit was also observed in the interstitial cells of the lactating groups but not in the non-lactating group (Fig. 1g and l). No immunohistochemical signals for inhibin β A and β B were found in the interstitial cells of both the lactating and non-lactating groups (Fig. 1h, i, j and k). Immunoreactivity for the inhibin α, β A and β B-subunits was clearly observed in the granulose cells of the antral follicles of the lactating and non-lactating animals (Fig. 1g, h, i, j, k and l). No immunostaining was detected in control sections in which normal rabbit serum was substituted for the primary antibody (data not shown). Discussion This is the first immunohistochemical description of the localization of NGF, trka, p75 and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters. The principal findings are that the diurnal rhythm of increases in LH may stimulate the expression of NGF, trka, p75 and inhibin α-subunit in ovarian interstitial cells, which suggests that NGF, its receptors and inhibin α-subunit may have the capacity for autocrine or paracrine modulation of interstitial cell differentiation in lactating golden hamsters. Previous studies in the golden hamster have shown that there are

3 INHIBIN AND NERVE GROWTH FACTOR IN LACTATING HAMSTER OVARIES 399 Fig. 1. Immunohistochemical staining of NGF, trka, p75 and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters. NGF, trka and p75 were present in the interstitial cells of the lactating group (a, b, c), and no positive staining for NGF, trka or p75 was observed in the non-lactating group (d, e, f). Immunoreactivity for inhibin α- subunit was also observed in the interstitial cells of the lactating group (g) but not in the non-lactating group (l). Inhibin β A and inhibin β B were not found in the interstitial cells of both the lactating and non-lactating groups (h, i, j, k). Int, gr and th indicate interstitial cells, granulosa cells and theca cells, respectively. The scale bars represent 50 μm. LH/hCG binding sites in the interstitial tissues during the estrous cycle [28] and that characteristic daily increases in the plasma levels of LH are observed every afternoon during lactation [21, 22, 29]. Our previous studies showed that there is stronger positive staining of NGF, trka and p75 in the interstitial cells of the golden hamster ovary on day 1 of the estrous cycle (day 1=day of ovulation) and very weak immunostaining after LHRH-AS treatment. Together with the recovered stronger positive staining following human chorionic gonadotropin (hcg) injection after LHRH-AS treatment, which suggested that LH surge may be an important factor for inducing the expression of NGF, trka, p75 in interstitial cells of the cyclic golden hamster [23]. Previous studies have indicated that NGF acts via trka receptors in the mammalian ovary to affect cellular functions [30]. Dissen et al. [31] reported that in juvenile rats treated with equine chorionic gonadotropin, significantly elevated trka levels of mrna were found in the ovary after the first preovulatory peak of the LH surge. NGF mrna contents in the ovary also reached peak levels coinciding with the trka mrna expression was LH dependent, as verified by culture of thecal and interstitial cells treated with hcg [31]. Activation of ovarian trka appears to contribute to the cascade of events leading to ovulatory rupture, as pharmacological blockade of trka signaling or immunoneutralization of NGF activity reduces the incidence of ovulation in response to gonadotropins [31]. Similarly, the functional relationship between gonadotropins and NGF in studies of the sheep ovary consisting of in vitro experiments based on culture of follicular cells for 12 h confirm that medium-large follicles can produce NGF. The production of NGF is strictly dependent on gonadotrophin stimulation [32]. Taken together, this suggest that the present results are compatible with the broader concept that NGF and its receptors are expressed in ovarian tissues including in interstitial cells during lactation and that their expressions are LHdependent. This suggests that NGF and its receptors may play an important role in ovarian interstitial cell differentiation and growth. NGF and its trka receptor contribute to the regulation of other functions of ovarian follicles via paracrine and autocrine interactions that can take place separately within the granulosa cell and thecal-interstitial cell compartments or are involved in communication between these two cell compartments of the developing follicle [33]. Among the potential candidates for factors, growth factors of the TGF β superfamily should be considered because they are produced in ovarian mesenchymal cells, and they regulate ovarian cell growth and differentiation [34, 35]. In addition, thecal/interstitial cells by facilitating FSH-dependent events, such as induction of aromatase activity [36], steroidogenesis [37], and formation of LH receptors [37], and at least the expression of one of them (TGFβ 1) is under direct NGF transcriptional regulatory control [38]. Our previous studies have indicated that the LH surge can stimulate expression of inhibin α-subunit in the interstitial cells of cyclic golden hamsters [26, 27]. The present results provide new evidence of inhibin α-subunit staining in the ovarian interstitial cells of lactating golden hamsters. These results concerning the

4 400 WENG et al. involvement of the LH surge or administration of hcg in the regulation of interstitial α-subunit expression are also consistent with a study in the rat [39], and show that inhibin α-subunit may have the ability for autocrine or paracrine modulation of interstitial cell differentiation and growth in golden hamsters [26]. Similar to the ovarian tissues of rabbits [40], golden hamsters have large numbers of ovarian interstitial cells [41]. Though the factors regulating the differentiation of the ovarian interstitial cells of golden hamsters are unknown, previous reports have indicated that TGFβ1 and TGFβ2 [42], steriodogenic enzymes [43, 44], epidermal growth factor receptor [45] and estrogen receptors [46] are expressed in the ovarian interstitial cells of golden hamsters. These findings imply that these expression activities correspond to active interstitial cell differentiation [47]. TGFβ can amplify progesterone production and LH receptor induction [48], and TGFβ is essential to maintaining the critical level of LH receptor expression in the rat ovary for induction of ovulation [49]. Thus, the present results suggest that inhibin α-subunit may play a regulatory role in LH receptor expression and synthesis of steriods in the ovarian interstitial cell of lactating golden hamsters. In summary, the present study provides new evidence that increasing LH levels can induce expression of NGF, its receptors and inhibin α-subunit in the ovarian interstitial cells of lactating golden hamsters and that NGF, its receptors and inhibin α-subunit regulate interstitial cells differentiation in lactating golden hamsters via paracrine and autocrine interactions. 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