Effects of intrauterine exposure to betamethasone on reproduction of Wistar rats

Author(s)

Cibele dos Santos Borges

• Document type: Doctoral Thesis
• Press: Botucatu. 2017-02-15.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2017-02-07
• Advisor: Wilma De Grava Kempinas

Abstract

Betamethasone is the glucocorticoid of choice for fetal lung maturation, reducing the incidence of respiratory distress syndrome and neonatal mortality. Studies conducted in association with our laboratory have shown that prenatal exposure to this drug promoted changes in testosterone levels and sperm parameters of male rats offspring. Recently, effects on the hypothalamic-pituitary-adrenal axis were observed for generations. The aim of this study was to evaluatethe effects on reproductive parameters of male and female rats, caused by in utero exposure to betamethasone. Therefore, pregnant Wistar rats were divided into control group (n=11) and treated (n=13) with 0.1mg / kg betamethasone, intramuscularly, on gestational days 12, 13, 18 and 19. In the experiment 1, it was evaluated: the initial weight and the anogenital distance at PND 1, the puberty installation from PND 30, and from PND 70 as the estrus cyclicity. On PND 85, one female was euthanized in estrus and an aliquot of blood was collected for hormone levels and the reproductive organs were weighted. Ovary and uterus were fixed for histological and morphometric analysis. In DPN 90, sexual behavior was performed followed by fertility test, and in PND 95 an uterine segment from an animal per litter was used for pharmacological reactivity. In experiment 2, it was evaluated: the maternal food intake and weight gain during treatment, the body weight and anogenital distance of male offspring on PDN 1 and the puberty installation from PND 30. In DPN 45 and DPN110 an animal per litter was euthanized and an aliquot of blood was collected to hormone levels and the reproductive organs were weighted. The right testis was fixed in Bouin and processed: histology, morphometry and immunohistochemistry for PCNA and Cx43 protein. At PND 90 one animal from each litter was used to perform natural fertility test. In experiment 3, two males of F1 were used to obtain the following parameters: an animal from each litter on PND 90 was used to perform the sexual behavior test and after 30 days of recovery, it was euthanized and an aliquot of blood was obtained for hormone levels and the reproductive organs were weighted. The left testis had its tunica albuginea removed and used to determine daily sperm production. The left epididymis was used for the determination of sperm parameters (count, motility and morphology) as well as the fertility test by in utero artificial insemination. The other animal was euthanized in DPN 120 for pharmacological reactivity tests of male sex glands. In experiment 4, one male pup from F1 was used to mating with untreated females to obtain the second generation (F2). The offspring obtained from F2 was evaluated for the same parameters described in the experiments 2 and 3. In Experiment 5, the right epididymis from F1 and F2 animals, on PND 45 and PND 110 were processed for histopathology, immunostaining for Cx43 and p63 protein and stereology, and the left epididymis for pharmacology reactivity test. In utero exposure to betamethasone promoted a reduction in food intake and in the weight gain of mothers during the treatment. In the female pups were observed: a reduction on weight at birth, delayed on puberty onset, reduction in estrus numbers, increase in uterine weight, LH levels, estrous cycle length, uterine contractility, and area of the myometrium. In contrast, there was a reduction in FSH levels, lordosis quotient, endometrial area and fertility. In F1 male offspring, there was a reduction in the initial weight of male offspring, delayed puberty onset, low testosterone levels in PND 45, a gonadal weight increase and alteration in the pattern of Sertoli and germ cells organization on PND 45 and 110. Reduction on daily sperm production, in the diameter of the seminiferous tubules and also an alteration in the dynamics of spermatogenesis. In the epididymis, there was a delay in epithelial cells differentiation (PND 45), reduction on epithelium density volume, an increase in the number of immobile and abnormal spermatozoa and alteration in the expression of Cx43 and p63. The seminal vesicle showed a decrease in its weight and an increase in contractility activity. Fertility tests revealed reduction in the fertility potential of animals exposed to betamethasone, either by natural mating and by in utero artificial insemination. In F2, there was a similar pattern of changes on the sexual development of male offspring, as well as in sperm quality, characterized by reduced birth weight, delay in puberty onset and increase in weight of the testicles. Furthermore, there was a reduction in weight of the seminal vesicle, as well as their contractility activity. Hormone levels showed reduced FSH and increased LH. It was observed decrease in Leydig cells volume, reduction on sperm production, delay in the epithelial cells differentiation, alteration in the expression of Cx43 and reduction in the number of normal and mobile spermatozoa. There was a reduction in the ejaculatory frequency and fertility potential. From the foregoing, it is concluded that betamethasone exposure, in critical periods of intrauterine development in rats, promoted a reproductive multigenerational reprogramming. From the translational perspective these results warn of possible adverse reproductive outcomes of prenatal exposure to glucocorticoids in the human antenatal therapy. (AU)

• FAPESP's process: 12/25350-1 - Effects of in utero betamethasone exposure on male rats reproductive parameters, with emphasis on sperm quality and fertility: a multigenerational approach
• Grantee: Cibele dos Santos Borges
• Support Opportunities: Scholarships in Brazil - Doctorate