Current Neurobiology 2010; 1 (2): 103-108

In transgenic Mini rat, in which the expression of intrinsic growth hormone (GH) gene is suppressed by the antisense GH transgene are characterized by definite somatic growth retardation at puberty. Compared to age-matched wild-type Wister (non-Mini) rat, Mini rat showed low blood GH levels and GH cell depletion in the anterior pituitary (AP). The AP and periventricular hypothalamic nucleus (PeVN) were processed for immunohistochemical analysis with antisera to Luteinizing hormone (LH) and somatostatin (SOM), respectively. Although LH-immunoreactive (IR) cells showed the decrease in number in Mini rat from 4 to 8 weeks of age, there was the significant increase in non-Mini rat. Interestingly, SOM-IR cells in the PeVN showed the marked increase in number in Mini rat in contrast to the decrease in non-Mini rat during the same periods. The testis weights were not significantly different between Mini and non-Mini rat at 4 weeks of age, whereas at 6 and 8 weeks of age the value of Mini rat was significantly smaller than non-Mini rat. It was concluded that the onset and development of LH-testicular axis need intrinsic GH regulation, which is involved the SOM cell interaction from PeVN, and the SOM plays a crucial role in the development of pituitary-testicular maturation during the puberty. Introduction Accepted August 07 2010 The transgenic Mini rat is carrying an antisense RNA transgene which directly suppresses the transcription of intrinsic rat growth hormone (GH) gene. This antisense gene is mainly expressed in the anterior pituitary (AP) [1, 2] and indirectly suppresses the AP formation and GH cell number. Hence, Mini rat has mainly affected the development of the AP and the number of total GH cells during the puberty [3]. In addition, the changes of GH in the transgenic animal are reported to affect hypothalamic-pituitary-gonadal (HPG) axis which dues to the reduction of GH-releasing hormone (RH) and luteinizing hormone (LH) RH neurons in the arcuate nucleus and eventually the increase of somatostatin (SOM) neurons in the periventricular hypothalamic nucleus (PeVN) [4,5]. With respect to the SOM release pattern in sexual dimorphism was reported in which testicular development and production of sex steroids may have strongly affected hypothalamic SOM-immunoreactive (IR) cells in the PeVN [6]. Male rat and mice have higher SOM mRNA levels in the PeVN nucleus and higher SOM-IR in the median eminence, compared to female [7, 8, and 9]. That sexually dimorphic is associated with the onset of GH secretion pattern at the puberty. In this point, 40–70% of SOM neurons with androgen receptor (AR) in the PeVN decided the sex differences of the GH secretion in the male rat [10]. Such sex differences on HPG axis may depend on the exposure of sex steroids during the puberty. However, the exact role of the HP axis by the hypothalamic regulation of both LH and GH production remains uncertain. Therefore, the purpose of this study was to investigate the effect of GH action on the alteration of gonadal development with respect to SOM-IR cells and LH-IR cells by applying immunohistochemical and morphometric methods of the hypothalamus and the AP. Materials and Methods Transgenic rat (ARGHGEN-1Nts, Mini rat) was donated by the Nippon Institute for Biological Science (NIBS) of Japan. Wild rat (Wistar rat, non-Mini rat) was used as a control. All methods were conducted according to the standards of humane animal care. Animals were housed in stable temperature and humidity conditions with lights on between 6 a.m. and 8 p.m. and food and water available ad libitum. At 4, 6, and 8 weeks of age, male Mini rat (n = 5) and non-Mini rat (n = 5) were deeply anesthetized with sodium pentobarbital (40 mg/kg i.p.), weighed, and immediately decapitated. The AP, hypothalamus, and testis were fixed in 10% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h. These specimens were then dehydrated in a graded series of ethanol and xylene and embedded in paraffin. The tissues were cut into sequential 6 m-thick sections. These tissue sections were dewaxed in xylene and rehydrated in a graded series of ethanol solutions. Sequential sections of the AP and hypothalamus were processed for immunohistochemistry to investigate LHand SOM-IR expression. For LH and SOM immunohistochemistry, sections were incubated in 10% normal goat serum in 0.02 M phosphate-buffered saline (PBS) with 0.2% Triton X-100 for 30 min at room temperature. Tissues were treated with 0.3% H2O2 dissolved in 0.02 M PBS to inactivate endogenous peroxidase activity. AP sections were incubated with anti-rabbit LH antibody (1:3000 dilutions, NIDDK) for 1 h at 35°C. Hypothalamus sections were incubated with anti-rabbit SOM antibody (1:2000, DAKO) overnight at 4°C. All treated sections were processed with peroxidase-labeled protein A (Organon Teknika, USA). Subsequently, all sections were treated with 0.006% diaminobenzidine and 0.003% hydrogen peroxide in 0.02 M PBS for 5–10 min at room temperature. For identification of the stages of spermatogenesis and acrosomal formation on the testis, glycosylated substrates on the testis were stained by periodic acid-Schiff (PAS) reaction. A morphometric study for LH-IR cell was carried out according to our previous reports [3] Statistical analysis was performed using Sigma stat for Windows. The data of testis weight, GSI and LH-IR cell numbers were analyzed by twoway analysis of variance (ANOVA) and post hoc tests with Tukey-Kramer's test. Analyses were considered to be statistically significant at P < 0.001.