Reproductive tract lesions in male mice treated neonatally with tamoxifen

Male mice of the NMRI/Tg strain were treated with tamoxifen for the first 3 days after birth. The affected mice were sterile, with multiple reproductive tract lesions. These lesions included testicular hypoplasia, intraabdominal testes, epididymal cysts, and squamous metaplasia of accessory glands.     

Sex-differentiated enzyme levels and oestrogen uptake in adult rats treated neonatally with tamoxifen or CI-628

Serum cholinesterase, hepatic histidase and monoamine oxidase activity levels are higher in adult female rats than in adult male rats. Exposure of neonatal rats to antioestrogen (tamoxifen or CI-628) resulted in increased serum cholinesterase in adult females only and no effect on hepatic histidase and monoamine oxidase in both sexes. Neonatal tamoxifen or CI-628 treatment resulted in reduced body weights in adult male rats and reduced uterine wet weights in adult female rats. Circulating oestrogen levels measured in adult female rats treated neonatally with tamoxifen were not significantly different from controls. Specific oestrogen uptake in the brain of adult male and female rats was found to be higher in the pituitary than in the preoptic-anterior hypothalamic area and the median eminence-basal hypothalamus than in the cerebral cortex. There was higher uptake of [3H]oestradiol-17 beta in male pituitaries than in female pituitaries. No other sex-difference was observed. Neonatal tamoxifen treatment did not alter the capacity of these brain tissues to take up oestrogen. It is suggested that neonatal antioestrogen exposure has altered the endocrine expression of serum cholinesterase in adult female rats by interfering with normal imprinting mechanisms.     

Changes of immunoresponse in BALB/c mice neonatally treated with periodontopathic bacterial endotoxin

The aim of this investigation was to analyze the effects of early life exposure to periodontopathic bacterial lipopolysaccharides on immunoresponse. Newborn BALB/c mice were subcutaneously injected with 20 ng lipopolysaccharide of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans or Escherichia coli daily for 2 days, starting within 24 h after birth. The treated mice were given intraperitoneal injections of bovine serum albumin at 180 and 187 days of age. Seventeen hours after each injection, the mice were bled and sera were separated. Their sera were tested in an enzyme-linked immunosorbent assay system. The mean interleukin-4, interleukin-5, interleukin-6 and immunoglobulin E levels in the sera of mice treated neonatally with P. gingivalis lipopolysaccharide were significantly higher than those of the controls. However, in all cases, no significant difference was noted between mice treated neonatally with A. actinomycetemcomitans- or E. coli lipopolysaccharide and control mice. These data suggest that neonatal exposure to P. gingivalis lipopolysaccharide induces changes in immunological responses when the mice reach maturity.     

Plasma testosterone levels and ovarian testosterone content in adult mice treated with diethylstilbestrol neonatally

Neonatal female NMRI mice (n = 16) were treated with 5 micrograms diethylstilbestrol (DES) per day, for the first 5 days after birth and killed postpubertally. Control females (n = 52) were injected with vehicle only and killed in different stages of the estrous cycle. The plasma testosterone level was significantly lower in DES females than in control females in any of the estrous phases. Ovariectomy (n = 5), adrenalectomy (n = 5) or a combination of both ablations (n = 3) did not affect the plasma testosterone in DES treated females while it was significantly reduced in control females (ovariectomy n = 5; adrenalectomy n = 9); most effective was the combination ovariectomy-adrenalectomy (n = 7). Ovarian homogenates from DES treated females (n = 10) had a significantly lower testosterone content than homogenates from control females in any phase of the estrous cycle (6-10 females per phase), which held true on both a per ovary basis and when related to ovarian weight. After a 2 h incubation in vitro, the testosterone levels had increased significantly in DES homogenates (n = 6) and to a lower extent in homogenates from control females in estrus (n = 9). No similar effect was found in homogenates from diestrus (n = 10) or proestrus (n = 9) females. The results are discussed in relation to the special ovarian morphology of adult but neonatally DES treated females and also with respect to endocrine control mechanisms.     

Congenital defects in the offspring of male mice treated with ethylnitrosourea

Daily doses of ENU (25-100 mg/kg) were injected intraperitoneally into ICR strain male mice for 5 days. The males were mated to untreated virgin females of the same strain on days 1-16 and 64-80 after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The uterine contents were examined on day 18 of pregnancy for evidence of dominant lethal effects. The fetuses were examined for external and skeletal abnormalities. ENU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in mg/kg by treating spermatogonial stem cells was estimated to be 1.0 X 10(-4), which is 3-fold lower than the rate previously estimated for the same endpoint at the same germ cell stage with MNU. Cleft palate was the most frequent external abnormality in the ENU-treated and the control series. Malformed vertebrae was the most frequent skeletal abnormality in the treated series. Rib fusion was the only skeletal malformation seen in the control series. Dominant lethals were clearly induced when germ cells were treated as postmeiotic cells.     

Abnormal anther development and high sporopollenin synthesis in benzotriazole treated male sterile Helianthus annuus L

Foliar application of 1.5% benzotriazole induced 100% pollen sterility in H. annuus. Pollen abortion in treated plants was mainly associated with abnormal behaviour of tapetum. A limited number of anther locule showed early degeneration of tapetum followed by disintegration of sporogenous tissues. On the other hand, some locules showed normal development of tapetum at initial stages. However, this tapetum exhibited degenerated and non-functional cell organelles. In both these situations tapetum failed to provide proper nourishment to developing microspores. The ultrastructure of both tapetum and microspores is different from that of control material with irregularities of exine deposition, endopolyploidy of tapetal nuclei and an alteration of organelle composition being correlated with sterility. Pollen grains thus developed were devoid of nucleus and cell organelles and were complete sterile.     

Pituitaries of rats neonatally treated with oestrogen

Summary Pituitaries of male and female rats neonatally treated with oestradiol dipropionate were examined with light and electron microscopy, disc electrophoresis and in vitro C¹⁴-leucine incorporation.Single dose of oestradiol administered shortly after birth to male rats has an prolonged stimulative effect on LTH synthesis and release. This is expressed in the cell hypertrophy, proliferation of ER, increase of corresponding band density and higher rate of ¹⁴C-leucine incorporation. In females this effect was less pronounced.STH cell in the treated male pituitaries were degranulated, corresponding hormone band density significantly lowered and incorporation of ¹⁴C-leucine was reduced. This could be considered as an inhibitory effect of oestradiol on STH synthesis and release. Such effect was more expressed two than four months after treatment and is more pronounced in male than in female rats.The technical assistence of Slobodanka Selimovi is gratefully acknowledged.     

Sexual behavior and penile reflexes of neonatally castrated male rats treated in infancy with estrogen and dihydrotestosterone

Male rats castrated neonatally and treated with a combination of 0.5 μg estradiol benzoate (EB) plus 50μg dihydrotestosterone propionate (DHTP) for the next 14 days displayed normal sexual behavior when injected with testosterone propionate (TP) in adulthood. Neither EB nor DHTP alone had this developmental effect inasmuch as only 20–25% of the neonatal castrates treated with just 0.1, 0.5, or 10 μg EB, or 50 μg DHTP, displayed ejaculatory responses. The periodic application of mildly painful electric shock, which has been previously shown to markedly facilitate ejaculatory responding in normal male rats, failed to improve sexual performance in these latter subjects. This was true even of the castrates treated neonatally with DHTP which frequently intromitted. Castrates treated with EB or DHTP alone neonatally were subjected to spinal transection (after testing of sexual behavior) for examination of penile reflexes. Those treated with DHTP showed normal reflexes, characterized by numerous erections and flips, indicating the presumably nonaromatizable DHTP has developmental effects on penile reflexes similar to those of testosterone. Subjects treated with EB, including four animals that had ejaculated at least once, displayed very few, if any, erections on reflex tests and no flips. These results show that sometimes intromissive and ejaculatory patterns can occur even though the animal appears to have little or no capacity for penile reflexes.     

Abnormal morphology of the penis in male rats exposed neonatally to diethylstilbestrol is associated with altered profile of estrogen receptor-alpha protein, but not of androgen receptor protein: a developmental and immunocytochemical study

Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)alpha in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 microg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERalpha-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERalpha.     

Abnormal development of cardiovascular systems in rat embryos treated with bisdiamine

Administration of N,N'-bis(dichloroacetyl)-1,8-octamethylenediamine, bisdiamine, in pregnant Donryu rats on day 10 of gestation induces a high incidence of cardiovascular anomalies in fetuses. Bisdiamine administration induced aplasia of the sixth aortic arch artery, with both the right and left primitive pulmonary arteries being directly linked to the truncus, and resulting in four types of malformation of pulmonary arteries (PAs). When two primitive PAs shared a single root, the consequence was either pulmonary trunk hypoplasia, as is seen in tetralogy of Fallot, or type I persistent truncus arteriosus (PTA) as classified by Collet and Edwards. When root portions of two PAs did not fuse, either type II or type III PTA resulted. In controls, the right dorsal aorta (DA) between the right seventh intersegmental artery (IA) and the site where both DAs fuse degenerated and the left aortic arch (AA) and the right subclavian artery (SA) were formed. Bisdiamine administration induced two additional types of vascular anomalies. In one of these, the right DA between the right 4AA and the right 7IA degenerated and a left AA accompanied by an aberrant right SA resulted. In the other type, the left DA between the left 4AA and the left 7IA degenerated and a right AA accompanied by an aberrant left SA resulted. These results indicate that administration of bisdiamine induces malformation in the great blood vessels by disturbing persistency and degeneration of aortic arch arteries and DAs.     

Ultrastructure of the developing fibrocartilage of the os penis of rat

Development of the fibrocartilage of the os penis of rat was studied by transmission electron microscopy. Prepubertal (0-4 weeks of development) and pubertal (4-8 weeks of development) males were examined. Effects of castration on the development of the fibrocartilage were also examined. During the first 0-4 weeks of development, cells in the primordium of the fibrocartilage became large and the cytoplasm had well-developed rough endoplasmic reticulum (rER) and many intermediate filaments. Collagen fibers increased markedly in amount in the extracellular matrix (ECM) during the period. For 4-6 weeks, when gonadal secretion of androgens increases, the cells developed into mature chondrocytes with lacunae. Collagenous bundles were pushed away from the lacunae, resulting in a characteristic appearance of this fibrocartilage. The cytoplasm of the mature chondrocytes of the fibrocartilage was characterized by many intermediate filaments, oil droplets, glycogen granules, and well-developed rER. At 6 weeks, calcification started on the cell membrane of the mature chondrocytes. At 8 weeks, a large part of the cartilage matrix was calcified. Matrix vesicles that originate from degenerated chondrocytes were found in the ECM of decalcified samples. In castrated males, cells of the primordium of the fibrocartilage ceased further development after castration. Intermediate filaments were still abundant in the cytoplasm and collagen fibers increased even after castration, but mature chondrocytes never differentiated. There were no signs of matrix vesicle formation, calcification, or cell degeneration in the fibrocartilage primordium. The developmental process of the fibrocartilage can be subdivided into two phases: collagenous matrix formation during the prepubertal period (0-4 weeks), and maturation of chondrocytes and calcification after puberty (4-8 weeks).     

Increased bone mass in male and female mice following tamoxifen administration

Tamoxifen is capable of preserving bone mass in gonadectomized rodents as well as intact female mice; however, a detailed 3D quantitative analysis of the structural changes produced in the growing skeleton of intact mice of both genders by this agent is lacking. Employing quantitative microcomputed tomography (muCT), we assessed the effects of 4-hydroxytamoxifen (OHT) on the femora of C57BL/6J mice administered this agent either for 12 (males and females) or 2 (females) weeks. In mice of either gender, but especially in females, 12 weeks of OHT exposure led to dramatic increases in both cortical and trabecular bone. Females exposed to OHT for either 2 or 12 weeks demonstrated significantly increased cortical wall thickness, trabecular bone volume, connectivity, and number, as well as decreased trabecular separation. Significant increases in several of these parameters were also evident in males after 12 weeks of OHT administration. In view of the expanding use of OHT to induce Cre-mediated recombination events, our findings suggest that care should be exercised when interpreting the skeletal phenotypes of mice exposed this agent, particularly in situations where the effects of OHT might synergize with the phenotypic outcome of a specific genetic alteration.     

Age determination of the male os pubis

An extensive (n = 739) sample of well-documented male pubic bones was examined for age-related features. Using the traditional aging methods, Todd's system was found to over-age and both the Todd and the McKern-Stewart systems did not account for age variability seen in advanced pubic bone patterns. Based on a 5-year interobserver error study, certain categories in each system were collapsed because observers could not consistently differentiate between them by morphological pattern. The traditional and the modified versions were than analyzed using linear regression analysis. All approaches perform poorly when the entire sample is used. Truncation strategies to eliminate older individuals result in substantial improvement. The traditional Todd system and its modified form are found to be the best systems. When implementation is considered, a modified Todd six-phase system is recommended.