Suppression of copulatory behavior by intracerebroventricular infusion of antisense oligodeoxynucleotide of granulin in neonatal male rats

Physiology & Behavior 68 (2000) 707–713

Suppression of copulatory behavior by intracerebroventricular infusion of antisense oligodeoxynucleotide of granulin in neonatal male rats Masatoshi Suzukia, Makoto Bannaia, Mie Matsumuroa, Yasufumi Furuhataa, Ryota Ikemuraa, Erina Kuranagaa, Yasufumi Kanedab, Masugi Nishiharaa,*, Michio Takahashia a

Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan b Division of Gene Therapy Science, Osaka University School of Medicine, Osaka 565-0871, Japan Received 17 May 1999; received in revised form 9 June 1999; accepted 30 November 1999

Abstract Sexual dimorphism of the rodent brain is manifested by the epigenetic action of gonadal steroids. Our previous research identified the granulin (grn) precursor gene as a sex steroid-inducible gene, which was shown to be expressed more abundantly in male than female neonates at the mediobasal hypothalamic area. Grn is a 6-kDa polypeptide promoting or inhibiting the growth of epithelial cells and hematocytes in vitro. In this study, effects on male sexual behavior of male were pursued under conditions in which grn gene expression was suppressed during the critical period. To this end, an antisense oligodeoxynucleotide (ODN) of the grn precursor gene was designed, incorporated into inactivated Sendai virus (HVJ)–liposome complexes, and infused into the third ventricle of 2-day-old male rats. Two different control treatments were used: the first consisted of a control sequence ODN that had little homology to known mRNAs; the second of vehicle (HVJ–liposome) alone. After maturation, animals treated with antisense ODN of grn displayed significantly lower scores than control males on various parameters assessing sexual behavior; i.e., mount, intromission, and ejaculation. The antisense ODN, however, did not affect body growth or serum concentrations of testosterone and luteinizing hormone. Further, there was no significant difference in the volume of the sexual dimorphic nucleus of the preoptic area between antisense ODN-treated and control animals. It was shown that inadequate expression of the grn gene in the brain of male neonatal rats during the critical period suppressed the induction of some type of male sexual behavior, suggesting the grn was involved in the process of masculinization of the rat brain. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Granulin; Sex steroid; Sexual differentiation of the brain; Antisense oligodeoxynucleotide

1. Introduction Sex-dependent differentiation of the rat brain occurs in a sex steroid-dependent manner during the perinatal period known as the critical period. The presence of androgen during the critical period induces the masculinization of the central nervous system, while the absence of androgen permits feminization [1,2]. Androgen, largely through its conversion to estrogen by aromatase in brain cells [3], has a permanent organizing effect on neuronal cells within the brain regions that control sexually dimorphic reproductive function. It has been recently proposed that sex steroids may interact in the brain with a variety of cytotropic factors, and thereby induce neuronal survival and differentiation by activating signaling cascades of these cytotrophic factors [4,5]. We previously identified the granulin (epithelin or grn) pre* Corresponding author. Tel: ⫹81-3-5841-5387; Fax: ⫹81-3-5841-8017 E-mail address: [email protected]

cursor gene as a sex steroid-inducible gene in the neonatal rat hypothalamus by means of the cDNA subtraction method [6]. The grn precursor gene encodes polypeptides that have been shown to modulate the growth of epithelial carcinoma cells in vitro [7,8]. Throughout the critical period of sexual differentiation of the brain, grn gene expression remained high in males, while that in females precipitously decreased with the passage of days. In addition, grn mRNA was detected in the arcuate (ARC) and ventromedial nuclei of the hypothalamus (VMH) of neonatal males, where histological sexual dimorphism has previously been demonstrated [9–11]. These observations suggest that the grn gene is involved in the masculinization of the neonatal rat brain. To ascertain the role of the grn gene in the sexual differentiation of the rat brain, we adapted the antisense oligodeoxynucleotide (ODN) method in the present study. This method is a useful strategy to selectively block the expression of specific genes [12,13]. Its extensive use in various cell culture systems [14,15], and more recently in the neonatal [16] and adult rat brain [17–19], has demonstrated the

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efficacy of antisense ODN in blocking target protein synthesis. In addition, incorporation of inactivated Sendai virus (Hemagglutinating virus of Japan; HVJ) into liposomes is an effective delivery for antisense ODN into the central nervous system [20]. Here, we employed HVJ–liposome–antisense ODN complexes expecting to inhibit grn expression in the neonatal rat brain, and evaluated sexual behavior after maturation. 2. Materials and methods 2.1. Animals Wistar–Imamichi rats were housed in a temperature- (23 ⫾ 1⬚C) and light-controlled room (lights on 0500–1900 h), and laboratory chow and water were available ad lib. After mating, pregnant females were allowed to deliver pups naturally. Litter size was adjusted to eight at the day of birth. 2.2. Preparation of HVJ–liposome–ODN complexes Phosphorothioate oligonucleotides (ODNs) were obtained from Takara (Kyoto, Japan). Twenty-mer antisense ODN spanning the putative translation start codon was constructed based on the nucleotide sequence of the rat grn gene previously reported [21]. The sequence for the antisense ODN was 5⬘-CACCAGGATCCACATGGTCT-3⬘. Underlining indicates the sequence antisense to the translation start codon. The 19-mer ODN, 5⬘-CTTCGTCGGTACCGTCTTC-3⬘, which has little or no homology to mRNA sequences in the GenBank database [22], was used as a control ODN. Detailed preparation of HVJ–liposomes has been described by Kitajima et al. [23]. Briefly, liposomes consisting of 6 mg of phosphatidylcholin, 3 mg of cholesterol, and 0.75 mg of 3␤-[N-(N⬘,N-dimethylaminoethane) carbomoyl] cholesterol were dissolved in chloroform and evaporated using a rotary evaporator. The dried mixture was hydrated with 200 ␮L balanced salt solution (137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6) containing 3 ␮g ODNs by vigorous vortexing, then sonicated. The liposome–ODN was then fused with HVJ that had been inactivated by ultraviolet light. Free HVJ was removed from the HVJ–liposome–ODN complexes by sucrose density gradient centrifugation through a 30% (w/w) sucrose layer at 62,800 ⫻ g for 90 min. Final concentration of ODNs in the complexes was 6 ng/␮L. CaCl2 was added to equal 100 ␮M just before use. 2.3. Cell culture and estimation of Grn mRNA Rat pheochromocytoma cells (PC12) and human epidermoid carcinoma cells (A431) were provided by RIKEN Cell Bank (Tukuba, Japan). PC12 cells were maintained in collagen-precoated six-well culture dishes (Iwaki, Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% horse serum, 5% fetal calf serum, 50 units/ mL penicillin G, and 0.1 mg/mL streptomycin sulfate. A431

cells were cultured in six-well culture dishes in DMEM containing 10% fetal calf serum, 50 units/mL penicillin G, and 0.1 mg/mL streptomycin sulfate. Cells were harvested in a 5% CO2 humidified atomosphere. HVJ–liposomes (20 ␮l) with 120 ng of each ODN was added to 2 ⫻ 105 cells per 1 mL culture medium. After incubation for 48 h, total RNA was extracted using the TRIzol reagent (GIBSON BRL., Life technologies Inc., Rockville, MD). The same preparations of each treatment of the RNA extraction were used for assessing the viability of the cells with Dojindo Cell Counting Kit (Dojindo Co., Kumamoto, Japan). 2.4. Intracerebroventricular (i.c.v.) infusion of ODNs Two-day-old male pups were divided into an antisense ODN infusion group (AS) and two control infusion groups. HVJ–liposome (1.5 ␮l) solution containing 9 ng of ODN was infused into the third ventricle of cold-anesthetized pups by the aid of a modified rat stereotaxic apparatus [24]. The volume of infusion (1.5 ␮L) was determined preliminarily using 1% brilliant blue solution to localize the diffusing area, mainly in the mediobasal hypothalamus. Control groups were given either HVJ–liposome–control ODN (CO) or HVJ–liposomes only (vehicles; VE). For analysis of grn expression in the hypothalamic tissues treated with antisense ODN, rats were sacrificed by decapitation at 8 h after the infusion. The brain was immediately removed, and the entire hypothalamus was cut, bordered anteriorly by the optic chiasma—laterally by the hypothalamic fissures, and posteriorly by the mammillary body—and was dissected out. Its depth from the basal surface of the hypothalamus was 2 mm. After dissection, the hypothalamic tissue was frozen in liquid nitrogen and the total RNA was extracted using the TRIzol reagent. 2.5. Determination of grn gene expression of cultured cells and hypothalamic tissues Grn gene expression was determined by RT-PCR, as reported previously [25]. Briefly, 350 ng of total RNA was incubated at 37⬚C with Moloney murine leukemia virus reverse transcriptase for the synthesis of the first strand of cDNA. After incubation, grn cDNA was amplified by PCR with AmpliTaq DNA polymerase using the appropriate number of cycles, by which the linear relationship between the graded doses of template, and the amount of RT-PCR products was obtained. Primers were synthesized on the basis of the sequences of rat grn precursor cDNA [21]. The upstream primer was 5⬘-AGTTCGAATGTCCTGACT-3⬘, and the downstream primer was 5⬘-ATGTGGTCTTCACAACAC-3⬘. The amplification profile involved denaturation for 0.5 min at 94⬚C, primer annealing for 1 min at 55⬚C, and primer extention for 1 min at 72⬚C. The PCR was run for 37 cycles. As an internal control, rat ␤-actin (RTPCR Amplimer Set; CLONETECH, CA) was used. The predicted size of the PCR product was 561 bp for grn and

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289 bp for ␤-actin. PCR products were electrophoresed on a 1.2% agarose gel in 1 ⫻ TAE buffer, and the bands were detected by ethidium bromide staining. After washing with distilled water, the agarose gel was photographed and scanned by FAS-III (TOYOBO, Tokyo, Japan), and the relative intensity of the ethidium bromide fluorescence of each band was densitometrically analyzed with NIH Image software.

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0.1% cresyl violet before coverslipping. Pictures of the sexual dimorpohic nucleus of the preoptic area (SDN-POA) were digitized, and the area of the nucleus was measured using the NIH Image software. Nuclear volume was calculated as total cross-sectional area times section thickness according to the procedure of Block and Gorski [27].

2.6. Copulatory behavioral tests The AS-, CO-, and VE-treated pups were kept with their original dam, and weaned around 28 days of age. They were weighed at every 7 days until 8 weeks of age. The following analyses were performed when the subjects were 8–15 weeks of age. Copulatory behavior tests were carried out as reported previously [26]. In each behavioral test, an experimental male was placed in a plastic cage (40 ⫻ 22 ⫻ 18 cm). After a 4-h adaptation period, a highly estrous ovariectomized female, primed with a subcutaneous estradiol silastic tubing (70% cholesterol/30% 17␤-estradiol, Sigma, MO) for 5 days and injected with 1 mg progesterone 6 h before the test, was introduced into the cage. Male sexual behavior was observed for 30 min after the introduction of the female. The female was replaced by another similarly prepared female every 10 min to eliminate the influence of partner affinity. The following standard measures of male copulatory behavior were recorded: (1) latency to first mount, intromission, and ejaculation following the introduction of the female; (2) frequency of mount, intromission, and ejaculation during the 30 min; and (3) interval between first ejaculation and subsequent mount (postejaculation interval, PEI). Regarding the frequency of mount and intromission, when the male ejaculated during the 30 min of the experimental period, the number of mounts and intromissions from the point of female introduction to the first ejaculation was converted into the number per 30 min. 2.7. Hormone assays Blood samples were obtained from the tail vein under light ether anesthesia at 8 weeks of age. Serum concentration of LH was determined by double antibody radioimmunoassay with materials supplied by Amersham (Rat luteinizing hormone [125I] assay system; Amersham Life Science Ltd., UK). Serum testosterone concentration was measured by DELFIA testosterone assay kit (Pharmacia, Sweden). 2.8. Morphometric measurements of brain nuclei Upon completion of the experiment, animals were overdosed with Nembutal and intracardially perfused with 0.1 M phosphate buffer, followed by 4% paraformaldehyde. Fixed brains were sectioned on a freezing microtome, and 30 ␮mthick sections were taken from the entire preoptic area. Sections were mounted in serial order onto MAS-precoated slides (Matsunami Inc., Osaka, Japan) and stained with

Fig. 1. Effect of antisense oligodeoxynucleotide (ODN) treatment to granulin (grn) mRNA on grn gene expression in PC12 cells (A) and A431 cells (B). Gene expression of grn was normalized using expression of ␤-actin and shown as a percentage of the VE values. Each symbol and vertical bar represent the mean ⫾ SEM (n ⫽ 3 for each group). AS, antisense ODN; CO, control ODN; VE, vehicle (HVJ-liposome). *p ⬍ 0.05 versus CO; † p ⬍ 0.05 versus VE.

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effect of antisense ODN on grn mRNA levels was determined using cultured cells. After PC12 and A431 cells were cultured with antisense or control ODN for 48 h, total RNA was extracted, and grn gene expression was determined by RT-PCR. This is the first observation that grn mRNA was expressed in PC12 cells, as well as in A431 cells that were known to express the grn gene [28]. Antisense ODN decrease grn gene expression in PC12 by 92%, F(2, 6) ⫽ 48.01, p ⫽ 0.0002 (Fig. 1A) and in A431 cells by 47%, F(2, 6) ⫽ 82.51, p ⬍ 0.0001 (Fig. 1B). Although control ODN also significantly decreased grn mRNA levels in A431 cells, the effect was much smaller than that of antisense ODN. ODN treatments did not affect the cell viability in PC12 and A431 cells (data not shown). 3.2. Grn gene expression in the neonatal hypothalamus

Fig. 2. Effect of antisense oligodeoxynucleotide (ODN) treatment to granulin (grn) mRNA on grn gene expression in the neonatal hypothalamus. Gene expression of grn was normalized using expression of ␤-actin and shown as a percentage of the VE values. Each symbol and vertical bar represent the mean ⫾ SEM (n ⫽ 4 for each group). AS, antisense ODN; CO, control ODN; VE, vehicle (HVJ-liposome). *p ⬍ 0.05 versus CO; † p ⬍ 0.05 versus VE.

2.9. Statistics All the data except that regarding the number of animals showing copulatory behavior were analyzed by one-way functional ANOVA. When a significant effect was observed, Fisher’s PLSD analysis was used as a post hoc test. Data regarding the number of animals showing copulatory behavior were analyzed by Fisher’s exact probability test. Differences were considered significant when p ⬍ 0.05. 3. Results 3.1. Grn gene expression in PC12 and A431 cells As a preliminary experiment to investigate whether antisense ODN could inhibit grn gene expression in vitro, the

Antisense ODN complementary to grn mRNA was infused into the third ventricle of male rats at 2 days of age. After 8 h of the injection, total RNA of the hypothalamic tissue was extracted, and grn gene expression was determined by RT-PCR. An approximately 40% decrease in grn expression in the hypothalamus was observed in antisense ODN-treated animals, F(2, 9) ⫽ 30.82, p ⬍ 0.0001 (Fig. 2). 3.3. Copulatory behavior Antisense ODN of grn mRNA was infused into the third ventricle of male rats at 2 days of age. Until 8 weeks of age, there was no difference in growth rate among experimental groups (data not shown). Copulatory behaviors of sexually inexperienced males were tested using estrogen-primed progesterone-injected ovariectomized females for an experimental period of 30 min. The numbers of rats showing copulatory behaviors, especially that of ejaculation and postejaculation mount, were decreased by antisense ODN treatment, though not significantly (Table 1). Frequencies of mount, F(2, 38) ⫽ 4.634, p ⬍ 0.05, intromission, F(2, 38) ⫽ 4.208, p ⬍ 0.05, and ejaculation, F(2, 38) ⫽ 3.651, p ⬍ 0.05, in the antisense ODN-treated group were significantly lower than those in the vehicle (HVJ–liposome)-treated group (Fig. 3). The frequency of mount in the antisense ODN-treated group was also significantly lower than that in the control-ODN group. The antisense ODN treatment tended to prolong the latencies of mount, intromission, and ejaculation, though differ-

Table 1 Number of rats showing copulatory behaviors Number of rats showing

Antisense Control Vehicle

Total number of rats

Mount

Intromission

Ejaculation

Ejaculation and subsequent mount

18 12 11

14 (77.8%) 12 (100%) 11 (100%)

12 (67.0%) 11 (92.0%) 11 (100%)

10 (56.0%) 11 (92.0%) 10 (91.0%)

7 (38.9%) 9 (75.0%) 8 (72.7%)

Values in the parentheses are percent of the total.

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Fig. 3. Effect on male sexual behavior of neonatal treatment of grn mRNA with antisense oligodeoxynucleotide (ODN). Each column and vertical bar represent the mean ⫾ SEM (n ⫽ 18 for AS, 12 for CO and 11 for VE). Regarding the frequency of mount and intromission, when the male ejaculated during the 30 min of the experimental period, the number of mounts and intromissions from the point of female introduction to the first ejaculation was converted into the number per 30 min. AS, antisense ODN; CO, control ODN; VE, vehicle (HVJ–liposome). *p ⬍ 0.05 versus CO; † p ⬍ 0.05 versus VE.

ences were not significant (Table 2). PEI was not statistically different among the three experimental groups. 3.4. Serum hormone levels and SDN-POA volume As shown in Fig. 4, the serum concentrations of LH and testosterone in mature males were not affected by antisense ODN treatment at the neonatal stage. The estimated volume of SDN-POA are shown in Fig. 5, respectively. SDN-POA volume did not differ among the experimental groups. As expected, the volume of SDN-POA was significantly smaller in females than in males, F(3, 15) ⫽ 4.612, p ⬍ 0.05. 4. Discussion Intracerebroventricular infusion of a grn antisense ODN to the brain of the neonate male rat significantly decreased the frequency of mount, intromission, and ejaculation in adulthood, strongly suggesting that grn is involved in the process of sexual differentiation of the portion of the brain controlling copulatory behaviors. Although several previous investigations have described the presence of grn peptides or mRNA in various tissues including those of the brain [21], the physiological roles of grn in vivo remained to be elucidated. This is the first report indicating that grn may play certain roles in vivo in the sexual differentiation of the rat brain.

Fig. 4. Effect of neonatal treatment with antisense oligodeoxynucleotide (ODN) of grn mRNA on serum LH and testosterone (T) concentrations. Each column and vertical bar represent the mean ⫾ SEM (n ⫽ 10 for each group). AS, antisense ODN; CO, control ODN; VE, vehicle (HVJ–liposome).

As a preliminary assessment to investigate whether antisense ODN could inhibit grn gene expression, the effect of antisense ODN on grn mRNA levels was determined using PC12 and A431 cells in vitro. It was found for the first time that grn mRNA was expressed in PC12 cells as well as in A431 cells that were already known to express the grn gene

Table 2 Latency of copulatory behaviors

Antisense Control Vehicle

Total number of rats

Mount latency (s)

Intromission latency (s)

Ejaculation latency (s)

PEI (s)

18 12 11

587 ⫾ 142 (14) 396 ⫾ 115 (12) 324 ⫾ 114 (11)

796 ⫾ 174 (12) 568 ⫾ 107 (11) 418 ⫾ 136 (11)

1190 ⫾ 124 (10) 898 ⫾ 118 (11) 835 ⫾ 163 (10)

395 ⫾ 77 (7) 347 ⫾ 19 (9) 415 ⫾ 70 (8)

Values in the parentheses are number of rats exhibiting each specified behavior. PEI; postejaculation interval.

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Fig. 5. Effect of neonatal treatment with antisense oligodeoxynucleotide (ODN) of grn mRNA on the volume of sexual dimorphic nucleus of the preoptic area (SDN-POA). Each column and vertical bar represent the mean ⫾ SEM (n ⫽ 5 for each group). AS, antisense ODN; CO, control ODN; VE, vehicle (HVJ–liposome); NF, normal female. *p ⬍ 0.05 versus all other groups.

[28]. Antisense ODN treatment decreased grn mRNA levels in both cell types. In addition, the intracerebroventriclular injection of antisense ODN significantly reduced grn gen expression in the hypothalamus 8 h after injection. Thus, it was confirmed that antisense ODN treatments decreased grn gene expression both in vitro and vivo. Several previous reports indicated that antisense ODN could decrease the corresponding mRNA content in cells with a resultant decrease in the synthesis of the protein [29,30]. The antisense ODN used in this study was shown to be effective in decreasing grn mRNA, the decrease leading to a decrease in grn peptide synthesis. In addition, our previous study demonstrated that antisense ODN injected into the brain remained at least 7 days after injection [18,20]. Thus, antisense ODN injected 2 days after birth was predicted to inhibit grn expression throughout the critical period during which sexual differentiation of the rat brain occurs. The incidence of male sexual behavior highly depends on serum testosterone concentration [31]. However, serum testosterone concentrations in antisense ODN-treated males did not significantly differ from those in the control males. This further supports our notion that the decrease in copulatory behaviors in antisense ODN-treated males is not due to hormonal influences, but, rather, to organizational changes in the portion of the brain controlling copulatory behavior. Further, serum LH concentrations of antisense ODN-treated males were normal, and the secretion of LH is regulated by GnRH neurons in the hypothalamus. It is, therefore, probable that grn expression during the critical period is not cru-

cial to the development of the hypothalamus–pituitary– gonadal axis. Grn antisense ODN was less effective in lengthening the latency to mount, intromission, and ejaculation than in decreasing the frequency of these paramiters. On the contrary, subcutaneous injection of gonadotropin-releasing hormone (GnRH) was reported to reduce the latency of each parameter, while the frequency remained unchanged [32], suggesting that latency and frequency of copulatory behaviors are controlled separately. From this aspect, grn may primarily differentiate neuronal structures involved in frequency rather than the latency component of copulatory behavior. Although the regions of the brain wherein grn modifies neuronal structures are currently unknown, strong expression of grn was detected in the VMH and ARC in the hypothalamus [6]. It was reported that animals with VMH lesions produced significantly higher levels of male sexual behavior than sham-operated animals, which indicates that the VMH appears to exert inhibitory control over male copulatory behavior in rats [33,34]. In addition, the VMH is regarded as one of the higher centers inducing the lordosis reflex in females [31]. Taken together, higher expression of the grn gene in the male VMH may exert deteriorating effects on the primary neural structure of the VMH. It has been shown that, as a result of exposure to sex steroid at the neonatal stage, the volume of the SDN-POA of male rats is two- to fivefold larger in adulthood than that of females [35,36]. It is also reported that intracerebral injection of antisense ODN to ER-␣ mRNA on Day 3 of life permanently modified the volume of the SDN-POA [16]. In our study, the volume of the SDN-POA was not influenced by neonatal treatment of grn mRNA with antisense ODN. grn does not appear to mediate the steroid action involved in the construction of the SDN-POA; this conclusion is supported by our previous observation that the grn gene was not expressed in this nucleus [6]. In contrast to its significant inhibitory effect on copulatory behaviors, the antisense ODN treatment did not affect daily food intake, wheel-running activity (our unpublished observations), and growth rate. Several previous reports have demonstrated that sexual differences in these parameters were produced by the androgen milieu during the critical period [37,38]. For example, the food intake [38] and body weight [37] of female rats were increased in adulthood in response to a single injection of testosterone propionate at the perinatal period. The present observation, that antisense ODN to grn suppressed male copulatory behaviors and did not affect other physiological parameters, suggests that the sexual differentiation processes in the brain that control these parameter do so with a high degree of specificity (that is, these paramiters appear to be independently regulated), and, further, that grn is specifically involved in the differentiation of the neuronal structure that controls male copulatory behaviors. In summary, the present study demonstrated that the administration of grn antisense ODN conjugated with HVJ–liposome

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