Role of oestrogens in male erectile function 2

Chapter MONITORING FOOD INTAKE AND BODY WEIGHT CHANGES IN OESTROGEN TREATED ANIMAL MODELS 54 3. MONITORING FOOD INTAKE AND BODY WEIGHT CHANGES IN OESTROGEN TREATED ANIMAL MODELS 3.1. Study Groups All the animals used in this investigation were supplied and maintained by the Laboratory Animal Centre and the Animal Holding Unit of the National University of Singapore, Singapore. With due compliance of the International guiding principles for animal research, the experimental protocol was approved by the ethics committee at this centre. Cage-side examinations were conducted once daily and the animals were individually examined for abnormal behaviour or physical changes once weekly throughout the study. 3.1.1. Rat Model Taking into consideration the possibility of species difference in response to administered steroids (Biegel et al., 1998), which includes oestradiol, the time-tested and conventional rat strain, Sprague-Dawley (SD) male rats were used in this investigation. Rat is also an established whole animal / in vivo model for studies estimating sexual behaviour (Chapter 4) and cavernous pressure response to erectile nerve stimulation (Chapter 5). The animals on arrival were thoroughly assessed for general physical fitness and sixty healthy and sexually mature adult male rats were randomly divided into six experimental groups (involving two control and four treatment groups, n=10 each). The animals were housed in pairs under ambient temperature (approximately 22°C) and reversed dark light cycle (08.00am−20.00pm with the individual dark and light phases of 12 hours). All the experimental animals had ready access to food and water. The standard rat diet (Glen Forrest Stock Feeders, Western Australia) was provided as 11mm diameter pellets and the balanced formula included wheat, millmix, lupins, soya meal, fish meal, vegetable oil, tallow, salt, added vitamin and mineral premix. Essential dietary constituents of the rat feed are listed in Appendix 1. 55 3.1.2. Rabbit Model Being docile, rabbits as experimental animals have several advantages over other species with regard to basic studies. Furthermore, in this area of impotence research, the good structural and functional correlation identified between the rabbit corpus cavernosum (CC) and the human counterpart (Azadzoi et al., 1988; Bischoff, 2001) prompted the choice of rabbits to investigate the physiopharmacological aspects of the human erectile disorder in this animal model (Chapter 6). Twenty-four genetically pure-bred strain of New Zealand White (NZW) healthy male rabbits were procured for the study. At the initiation of the experimental procedure, the animals were sexually mature young adults (six months old). They were assigned at random to four experimental groups (n=6, each) and housed in individual steel cages (18’x16’x13’) under a stable air-conditioned atmosphere with adequate daily care and ready access to standard rabbit diet (pellets) and water. The pellets were procured in bulk quantities from Glen Forrest Stock Feeders of Western Australia. They were well balanced to meet the daily nutritional requirement of the animals. The constituents of the standard dietary supply included barley, oats, millmix, lupins, soya meal, lucerne, limestone, methionine, salt and added vitamin and mineral premix (Appendix 2). 3.2. Administration of Oestrogen and Phytoestrogen Individual rat groups were treated with 0.01mg or 0.1mg of oestradiol valerate (Progynova, Schering) prepared as a suspension in sterile water (in fixed volume of 0.3ml) and administered through oral gavage daily for one week (n=20, short-term study) and 12 weeks (n=20, long-term study) respectively. The valerate salt of oestradiol has optimal pharmacokinetic parameters for systemic effects / toxicity (Kuhnz and Putz, 1989) and the chosen doses were reported to produce significant modulations in body 56 weight and hormonal parameters in rats (Brewster et al., 1997). The control groups (n=10) for each duration of treatment received the same volume of vehicle (sterile water) through gavage. The treatment groups are described in Table 1. SHORT-TERM STUDY GROUPS NUMBER OF RATS DURATION Control 10 week Oestradiol valerate (0.01mg) 10 week Oestradiol valerate (0.1mg) 10 weeks LONG-TERM STUDY GROUPS NUMBER OF RATS DURATION Control 10 12 weeks Oestradiol valerate (0.01mg) 10 12 weeks Oestradiol valerate (0.1mg) 10 12 weeks Table 1: Experimental Groups of Rats Individual treatment groups (n=10) administered two doses of oestradiol valerate for week or 12 weeks and their respective untreated, time-matched control animals. Rabbits from the treatment groups (n=6, each) received 0.1mg of oestradiol valerate (this dose produced significant changes during investigation in the rat model) or phytoestrogen, soybean isoflavone daidzein (Sigma) 0.01mg or 0.1mg as suspensions in sterile water (fixed volume of 1ml) through oral gavage daily for 12 weeks. While soybean is the richest known source for phytoestrogen isoflavones daidzein and genistein (Nicholls et al, 2002), the choice of the specific isoflavone was based on earlier reports from other centres of better bioavailability (Xu et al., 1994) and functional efficacy (Picherit et al., 2000) of daidzein over genistein. Control rabbits of the study consumed the same volume of sterile water by gavage daily. The different treatment groups are listed in Table 2. 57 STUDY GROUPS NUMBER OF RABBITS DURATION Control 12 weeks Oestradiol valerate (0.1mg) 12 weeks Phytoestrogen Daidzein (0.01mg) 12 weeks Phytoestrogen Daidzein (0.1mg) 12 weeks Table 2: Experimental Groups of Rabbits Individual treatment groups (n=6) administered oestradiol valerate or two doses of phytoestrogen isoflavone daidzein for 12 weeks and the untreated, time-matched control animals. 3.3. Estimation of Body Weight and Food Intake Since oestrogen appears to have ubiquitous distribution and wide ranging physiological functions in males, several mechanistic and biochemical endpoints such as body weight, weight of peripheral androgen-dependent organs, serum hormone levels, sertoli cell morphology and sperm analysis (Brewster et al., 1997; Cook et al., 1998) may be evaluated to quantify the differential pharmacological effects of an administered oestrogen on these variables. Among these, reduction in body weight is favourably modulated by oestrogens through central mechanisms (Anderson et al., 1988; Brewster et al., 1997), peripheral pathways and metabolic parameters (Sanchis et al., 1997; LooseMitchell and Stancel, 2001). The objective of comparative body weight estimation of different experimental groups in the present investigation was to utilise this known physiological effect for the efficacy of the pharmacological administration of oestrogens in the rat and rabbit models. Using standard weighing scale (Shimadzu Corporation, Japan), rats of the short-term study group were weighed on days and and those of the long-term study group and rabbits at 0, and 12 weeks. Food consumption / animal / day were estimated at random 58 intervals throughout the treatment period, measured as the 24-hour difference in the weight of the pellets provided in the cage. 3.3.1. Data Analysis Statistical comparison was done using one way analysis of variance (ANOVA) from the Statistical Package for Social Sciences (SPSS) version 11.5 for windows. All the results were expressed as mean±SEM (standard error of the mean) and the level of significance set at p[...]... the level of testosterone is a dominant determinant of frequency of coitus, indicating its role in improvement of libido (Adaikan et al., 1999) Androgen deficiency often precipitates loss of libido and erectile impairment; testosterone replacement-mediated improvements in these men include increased interest in sexual activity and other measures of sexual function (Tenover, 1998) Erectile dysfunction... series leading to ejaculation and the beginning of the next series with mounting 67 4 .2. 5.1 Parameters of Sexual Receptivity in Female Rats Oestrous / hormonally primed female rats usually initiate mating behaviour through scent-marking and orientation towards the male rats This will be followed by an initial period of mutual anogenital investigation and abrupt run away by the female causing the male to... possible to obtain insights and information on certain components which cannot be studied in patients in a scientifically objective way due to ethical constraints (Barfield, 1993) Hence male rats were the subjects of this investigation which concerns with the delineation of erectile function/ dysfunction in the presence of oestradiol induced hormonal changes 4 .2 Materials and Methods 4 .2. 1 Animal Groups... completion of long term in vivo studies within a shorter duration owing to their narrow life span In addition to determination of erectile capabilities of rats by standard mating behaviour tests using parameters of mounting, intromission and ejaculation described in Chapter 4, a direct measure of intracavernous pressure (ICP) in treated rats will give an accurate and objective experimental index of penile... oestrogens once implicated positively in ejaculation (Wood and Williams, 20 01) are recently shown to improve mounting as well as intromission patterns in rats (Huddleston et al., 20 03; Cooke et al., 20 03) Taken together, these findings suggest that the central effects of E2 may be proerectile, regulating both the erectile as well as motivational aspects of male sexual behavior similar to the role of. .. subcutaneously 4 hours before the initiation of copulation experiments The female rats with evidences for good proceptive induction in the form of hopping and darting movements, voluntary approaches to the male and lordosis were used in the study 4 .2. 5 Mating Experiments Rats being nocturnal animals are best studied during the day by maintaining them under reversed lighting (Wilson CA, 1993) Copulation... typical male behavioural pattern (MacLusky and Naftolin, 1981; Baum et al., 1991); some authors consider this effect to be a function of E2 resulting from local aromatisation (McCarthy et al., 1993; Gorski, 1993) In this context, it may be believed that the oestrogenic influence on the male brain is somewhat more than its role in the female brain at that stage of development since in the female foetus,... sexual dimorphism of the hypothalamus has been shown to function in humans (Allen et al., 1989) Despite these advances, the precise role of oestrogens at the end organ level of male sexual behaviour is under evaluated Interestingly, studies in a line of 84 transgenic ERKO mice lacking functional α receptors showed detrimental changes in the male sexual behaviour due to the absence of oestrogen action... dismounts and a period of rest following ejaculation 68 d Post-Ejaculation Interval or Refractory Period (PEI / PERP): The time interval of refractoriness between the first ejaculation and the subsequent mounting of the female rat by the male rat e Mount Frequency: (MF): The number of mounts without intromission from the time of introduction of female and the ejaculation by the male rat f Intromission Frequency:... Figure 21 : Intromission Frequency in Long-Term Treatment Group Total number of intromissions by the male rats in three experimental groups at 0, 6 and 12 weeks (n=10, each) The decrease in counts at 6 and 12 weeks in the oestradiol treated rats was not statistically significant 80 EJACULATION LATENCY TIME (MINUTES) 25 20 15 Control E2:0.01mg 10 E2:0.1mg 5 0 0 6 12 DURATION (WEEKS) Figure 22 : Ejaculation . constraints (Barfield, 1993). Hence male rats were the subjects of this investigation which concerns with the delineation of erectile function/ dysfunction in the presence of oestradiol induced. between the introduction of female rat to the first mount by the male rat. b. Intromission Latency: (IL): The interval from the time of introduction of female to the first intromission. Intromissions. leading to ejaculation and the beginning of the next series with mounting. 4 .2. 5.1. Parameters of Sexual Receptivity in Female Rats Oestrous / hormonally primed female rats usually initiate