Anogenital distance plasticity in adulthood: implications for its use as a biomarker of fetal androgen action.

Androgen action during the fetal masculinization programming window (MPW) determines the maximum potential for growth of androgen-dependent organs (eg, seminal vesicles, prostate, penis, and perineum) and is reflected in anogenital distance (AGD). As such, determining AGD in postnatal life has potential as a lifelong easily accessible biomarker of overall androgen action during the MPW. However, whether the perineum remains androgen responsive in adulthood and thus responds plastically to perturbed androgen drive remains unexplored. To determine this, we treated adult male rats with either the antiandrogen flutamide or the estrogen diethylstilbestrol (DES) for 5 weeks, followed by a 4-week washout period of no treatment. We determined AGD and its correlate anogenital index (AGI) (AGD relative to body weight) at weekly intervals across this period and compared these with normal adult rats (male and female), castrated male rats, and appropriate vehicle controls. These data showed that, in addition to reducing circulating testosterone and seminal vesicle weight, castration significantly reduced AGD (by ∼17%), demonstrating that there is a degree of plasticity in AGD in adulthood. Flutamide treatment increased circulating testosterone yet also reduced seminal vesicle weight due to local antagonism of androgen receptor. Despite this suppression, surprisingly, flutamide treatment had no effect on AGD at any time point. In contrast, although DES treatment suppressed circulating testosterone and reduced seminal vesicle weight, it also induced a significant reduction in AGD (by ∼11%), which returned to normal 1 week after cessation of DES treatment. We conclude that AGD in adult rats exhibits a degree of plasticity, which may be mediated by modulation of local androgen/estrogen action. The implications of these findings regarding the use of AGD as a lifelong clinical biomarker of fetal androgen action are discussed.

Regeneration of the perineurium after microsurgical resection examined with immunolabeling for tenascin-C and alpha smooth muscle actin.

The regenerative process of the perineurium and nerve function were examined using an in vivo model of perineurium resection in the rat sciatic nerve. Our hypothesis is that the regenerative process of the perineurium can be demonstrated by immunolabeling for tenascin-C and alpha smooth muscle actin after microsurgical resection of the perineurium in vivo. A total of 38 Lewis rats were used. Eight-week-old animals were assigned to one of two groups: the epi-perineurium removal group or the sham group. Under operative microscopy, the sciatic nerve was dissected from surrounding tissues at the thigh level from the ischial tuberosity to the fossa poplitea. The epi-perineurium was carefully removed by cutting circumferentially and stripping distally for 15 mm. For CatWalk® dynamic gait analysis, only right sciatic nerves underwent surgery; the left sciatic nerves were left intact. For pathological and electrophysiological tests, both the right and left sciatic nerves underwent surgery. Analysis of data was performed at each time interval with a two-group t-test. P<0.05 was considered statistically significant. After resection of a 15-mm section of the epi-perineurium, immediate endoneurial swelling occurred in the outer portion and spread into the central portion. Although demyelination and axonal degeneration were found in the swollen area, remyelination and recovery of electrophysiological function were seen after regeneration of the perineurium. An immunohistological and electron microscopic study revealed that the perineurium regenerated via fusion of the residual interfascicular perineurium and endoneurial fibroblast-like cells of mesenchymal origin. CatWalk gait analysis showed not only motor paresis but also neuropathic pain during the early phases of this model.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the developing male Wistar(Han) rat. I: No decrease in epididymal sperm count after a single acute dose.

It has been reported that fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat. We set out to replicate and extend these effects using a robust experimental design. Groups of 75 (control vehicle) or 55 (50, 200, or 1000 ng of TCDD/kg bodyweight) female Wistar(Han) rats were exposed to TCDD on gestational day (GD)15, then allowed to litter. The high-dose group dams showed no sustained weight loss compared to control, but four animals had total litter loss. Pups in the high-dose group showed reduced body weight up till day 21, and pups in the medium dose group showed reduced body weight in the first week postpartum. Balano-preputial separation was significantly delayed in the high-dose group male offspring. There were no significant effects of treatment when the offspring were subjected to a functional observational battery or mated with females to assess reproductive capability. Twenty-five males per group were killed on postnatal day (PND) 70, and approximately 60 animals per group (approximately 30 for the high-dose group) on PND120 to assess seminology and other end points. At PND120, the two highest dose groups showed a statistically significant elevation of sperm counts, compared to control; however, this effect was small (approximately 30%), within the normal range of sperm counts for this strain of rat, was not reflected in testicular spermatid counts nor PND70 data, and is therefore postulated to have no biological significance. Although there was an increase in the proportion of abnormal sperm at PND70, seminology parameters were otherwise unremarkable. Testis weights in the high-dose group were slightly decreased at PND70 and 120, and at PND120, brain weights were decreased in the high-dose group, liver to body weight ratios were increased for all three dose groups, with an increase in inflammatory cell foci in the epididymis in the high-dose group. These data show that TCDD is a potent developmental toxin after exposure of the developing fetus but that acute developmental exposure to TCDD on GD15 caused no decrease in sperm counts.

Effects of dietary phytoestrogen exposure during perinatal period.

Developmental effects of phytoestrogens were studied in offspring from pregnant rats who received a free-feeding diet of either rat chow that was very low in phytoestrogens (low phyto), rat chow low in phytoestrogens and given a genistein and diadzein supplement tablet (high phyto), or normal rat chow (normal) from the second week of pregnancy to weaning (postnatal day 21). Measurements of anogenital distance, daily weights, righting reflex and ultrasonic vocalizations were made on neonatal pups and plasma testosterone and corticosterone were assessed in adult males. There was a significant effect of phytoestrogen treatment on USV for all male and female offspring. Differences between groups in daily weights and anogenital distance were attributed to the micronutrient levels of the two rat chow types employed in this study. No differences in righting reflex test, corticosterone levels or testosterone levels were found among treatment conditions. These results are the first demonstration of phytoestrogens affecting USVs and underscore the complexity of the effects of these substances on biobehavioral development.

Normal ontogeny of perineal muscles and testosterone levels in Mongolian gerbils; response to testosterone in developing females.

The spinal nucleus of the bulbocavernosus (SNB) of Mongolian gerbils (Meriones unguiculatus) becomes sexually dimorphic during postnatal life, rather than prenatally as in rats. We therefore examined the early postnatal ontogeny of Mongolian gerbils, focusing on growth, serum testosterone (T) levels, and the sexually dimorphic perineal musculature innervated by the SNB. Serum T levels were higher in males than in females from birth through adulthood, with several early postnatal peaks and a large increase in T occurring during puberty in males. The SNB target muscles-the bulbocavernosus (BC) and levator ani (LA)-were present in both sexes on postnatal day 1 (PND1). Cross-sectional areas of BC fibers in males increased with age, and concurrently the myofibers of the BC became more fully developed and organized. In PND10 female pups, the BC muscle was virtually absent, while the LA muscle remained (although it was reduced in size). Postnatal treatment of female gerbils with androgen caused the BC muscle to remain and the LA muscle to become larger by PND10. Sexual dimorphism of the SNB develops differently in gerbils compared to other species, although its target muscles appear to respond to androgen in a manner similar to that in rats.

The androgenic induction of spinal sexual dimorphism is independent of supraspinal afferents.

On the day of birth, female rats received either a thoraco-lumbar spinal transection or sham operation, followed by administration of either testosterone propionate (TP) or oil immediately after surgery and again on the third day of life. Upon sacrifice at 30 days of age examination of spinal cords revealed that TP masculinized the spinal nucleus of the bulbocavernosus (SNB) in terms of cell number, soma size, and nuclei size. The perineal muscles innervated by the SNB were present only in those rats which received TP. Neonatal transection did not alter any of these effects of androgen treatment. Thus, supraspinal afferents are unnecessary for androgen induction of sexual dimorphism in the SNB. Remaining candidates for the site of androgen action include the SNB motoneurons and/or muscles themselves.