Effects of 4-Nonylphenol on reproduction of exposed females during puberty

4-Nonylphenol (NP) is considered an endocrine disruptor due to its capacity to interact with endocrine system, mimicking estrogen activity. It is a ubiquitous pollutant since it has been found in many human tissue samples and its precursor identified in sewage, sediments, rivers and drinking water. Thus, this work aimed to evaluate reproductive parameters in female Balb/C mice treated with 50 mg/kg body weight of NP for 21 days, a period in which they reach sexual maturity. Body weight during treatment, day of vaginal opening, organs weight, fertility rate and proportion of different follicle types were some of these parameters. Additionally, for analysis of some effect in littermates, a group of animals underwent two attempts at pregnancy after treatment. NP had no effect on body weight during treatment, nor did it alter the day of vaginal opening and fertility rate of females. The proportion of preantral, antral and degenerated ovarian follicles was not modified by treatment with NP, and no morphological or pathological alterations were observed. The number of pups did not differ between treatments in any attempt at pregnancy. Also the weight of pups on birth and female rate in littermates was similar among experimental groups after both trials of pregnancy. It is suggested that the NP available or accumulated during the period of sexual maturation (21 days) did not reach threshold levels capable of producing significant alterations in the reproductive parameters analyzed. Key-words: alkylphenol, endocrine disrupting chemicals, ovarian follicle, puberty, reproductive toxicology.


Introduction
In present, living organisms are exposed to a large amount and wide diversity of synthetic chemical compounds.These chemicals are found in the air (Onofrio et al., 2011), soil (Covaci et al., 2002), oceans and rivers (Josefsson et al., 2011) and in animal tissues (Ying et al., 2002;Jaspers et al., 2006).As a result of lack of strict regulations or proper toxicity tests when these compounds go on the market, the adverse effects of many compounds are discovered only after their widespread use in industry and by consumers (Rudel and Perovich, 2009;Knez, 2013).Some of these substances are known as endocrine disrupting chemicals (EDC), exogenous agents that can interfere with synthesis, secretion, transport, metabolism, binding, action, or elimination of natural hormones (Gore, 2008).EDC are found even in products considered inert, such as plastic (Hunt et al., 2003), and they are associated with some health problems in humans and animals, such as female and male subfertility (Annamalai and Namasivayam, 2015;Hond et al., 2015), certain types of cancers (Annamalai and Namasivayam, 2015), and neurological, developmental and reproductive disorders (Ness et al., 1993;Gore, 2008).These chemicals are claimed to be contributing to the fall in global fertility or be interacting with other causes for it, such as dietary factors (Petro et al., 2012).A worrying feature of EDC is that they usually have long half-lives, so they are persistent in environment (Bergeron et al., 1994).Also, the majority of them are lipophilic, accumulating in adipose tissue (Brevini et al., 2005).They have the capacity to cross the placental barrier (Barr et al., 2007) and be transmitted through suckling to the offspring of mammals, acting on developing embryos or newborns (Vos et al., 2003;Darbre and Harvey, 2008).Thus, EDC could affect not only exposed individuals, but subsequent generations (Dumesic et al., 2007).
Among EDCs, 4-nonylphenol (NP) is a chemical compound which mimics estrogen.NP is present in the environment mainly after degradation of nonylphenol ethoxylates (NPE; Soares et al., 2008).NPE is an alkylphenol widely utilized in the non-ionic surfactant industry, lubricants, antioxidants, detergents, paints, insecticides and herbicides, and also used as a stabilizer in plastic polymers (Oliveira-Filho et al., 2005;Rivero et al., 2008).Many studies show the presence of this pollutant in sewage, sediments, rivers and drinking water (Tsuda et al., 2000;Berryman et al., 2004;Shao et al., 2005).NP has been shown to be a global pollutant, frequently found in diverse human tissues.For example, it has been found in human adipose tissue samples (Lopez-Espinosa et al., 2009;Ferrara et al., 2011), breast milk (Ademollo et al., 2008) and blood samples from lactating women (Gyllenhammar et al., 2012).
Estrogenic action of NP is described for diverse animals, including birds (Razia et al., 2006), fish (Rivero et al., 2008) and mammals (Chapin et al., 1999;Tagaki et al., 2004).The median lethal dose of NP in male adult mice is 170 mg/kg body weight (b.w.) when administered intraperitoneally (El-Dakdoky and Helal, 2007), however effects can be observed at lower doses, such as 50 mg/kg b.w.(Nagao et al., 2001;Green et al., 2003).NP was capable of reducing the gene expression of the estrogen receptor in placental tissue and fetuses in pregnant female rats exposed to 50 mg/kg b.w.via oral for eight days or in a single dose intraperitoneally (Veld et al., 2009).Male mice treated with a lower dose of NP, 42.5 mg/kg b.w.via intraperitoneal for 35 days, showed a reduction in Anim.Reprod., v.13, n.4, p.795-805, Oct./Dec.2016 epididymis and testes weights, lower concentration of stored sperm and reduced motility.They also presented signs of oxidative stress and a reduction in antioxidant enzyme levels (El-Dakdoky and Helal, 2007).
Estrogens promote growth and development of female reproductive tract, regulating cell proliferation and gene expression in reproductive organs, pituitary and mammary glands (Soto et al. 1991;Costanzo, 2004).Primordial follicle formation is regulated by paracrine growth factors and local concentrations of steroid hormones, with small influence of folliclestimulating hormone (FSH) and luteinizing hormone (LH;Hunter et al., 2004;Uzumcu and Zachow, 2007).After the follicle enters the antral stage, it becomes dependent on FSH and LH to continue its maturation process (Hunter et al., 2004).From that point, growth is controlled mainly by FSH, whose production is modulated by the secretion of estradiol and inhibin A (Petro et al., 2012).As proper follicle and oocyte development relies on a well-orchestrated hormonal signaling, exposure to EDC in follicular environment could reduce oocyte quality or even jeopardize future embryo development (Petro et al., 2012).
Considering their characteristics, their ubiquitous presence in environment and the hazard posed by EDCs, especially estrogenic disruptors, it is important to carry out studies with these compounds, in order to identify those that affect reproductive function, at which concentrations, their mechanism of action and how to minimize damages.The objective of this work was to evaluate effects of NP on reproductive parameters in pubertal female mice, as well as to identify possible adverse effects on offspring.

All procedures of this work were approved by
The Ethics Committee on Animal Use of the Institute of Biological Sciences at the University of Brasília, registered under number 98076/2012.
Male and female Balb/C mice were purchased from the Breeding Center of Laboratory Animals (Cecal) of the Oswaldo Cruz Foundation (Rio de Janeiro, Brazil) just after weaning, at post-natal day (PND) 21.They were maintained in the facility of the Institute of Biological Sciences with controlled temperature, food and water ad libitum and 12:12 h light/dark cycle.
Ninety-seven females at PND 25 were divided into four experimental groups: control group with animals receiving only tap water (n = 26); vehicle control group receiving only corn oil (n = 28); NP group with animals treated with 50 mg/kg b.w.NP diluted in corn oil (n = 22; Nagao et al., 2001;Kimura et al., 2007); and E2 group, treated with 1 mg/kg b.w.17βestradiol (E2) diluted in corn oil (n = 21;Veld et al., 2009).This E2 dose was chosen since it has more estrogenic activity according to in vitro studies compared to NP dose (Veld et al., 2009).
Treatments were given orally with the aid of a gavage needle according with experimental group.Treatments were given to females daily for 21 days, the period they reach sexual maturity (Paiva et al., 2005).Females were weighed weekly during treatment and observed daily for identification of vaginal opening, an evidence of sexual maturity (Chorilli et al., 2007).At the end of treatment, females of each experimental group were divided into two subgroups: nulliparous and a group to attempt pregnancy.A schematic diagram of the whole experimental design is showed in Fig. 1.

Nulliparous females
Eight females of each treatment group (except vehicle group with n = 10) did not cohabit with males.These females were kept in collective cages for 15 days after the end of treatments (until PND 61), the time spent to obtain gestations in the other female's subgroup, and then euthanized.Liver, kidneys, spleen, uterus and ovaries were collected and weighed.From these animals, data previous to pregnancy were obtained, i.e. an overall picture of the follicles and oocytes that would have become fertilized.

Pregnant females
The other subgroup of each experimental group was put in cohabitation with males of the same age for 15 days to get pregnant.Two females from the same experimental group and one male were kept in cages with food and water ad libitum.This arrangement allowed monitoring of male fertility.After this period, females were put in individual cages and natural parturition was awaited.Offspring were weighed on their first day of life and had sex identified at 13th day of life.Offspring and dams were maintained until the day of sex identification.
Females that did not become pregnant were subjected to a second mating process as described above (2nd attempt at pregnancy).After this period, the occurrence of pregnancy was observed and natural delivery of offspring was awaited.Offspring were also weighed on their first day of life and had sex identified at 13th day of life.All animals were euthanized at PND 13 of litter, but dams had organs collected as described for the nulliparous females.Parameters analyzed in the litters comprised number of live offspring, weight at first day of life, female/male ratio and appearance of malformations.
The fertility rate was calculated by the proportion of females that got pregnant in relation to total females put in cohabitation with males.Different fertility rates were calculated for each attempt at pregnancy, in as much as animals were at different age brackets at the time of pregnancy assessment.

Euthanasia
Females were anesthetized with 100 µl of anesthetic solution (20% ketamine and 12% xilazine in saline solution 0.9%), via intraperitoneal injection.After euthanasia, ovaries, uterus, liver, kidneys and spleen were collected and weighed.Males utilized in the experiment and offspring were euthanized with an overdose of anesthetic solution (200 and 50 µl, respectively).

Histological analyses
For histological analyses, the right ovary of each animal was fixed in paraformaldehyde (4%) in phosphate buffer (0.1 M, pH 7.4), dehydrated in serial baths of increasing concentration of ethanol (70-100%), diaphanized with xylene and embedded in paraffin wax with aid of automatic tissue processor Oma DM-40.Sections of 5 µm thickness were obtained serially, discarding five sections between each, with microtome Leica RM 2235.Tissue sections were stained with hematoxilin-eosin to analyze ovarian follicles, classifying their stage and identifying morphological alterations.Microscope slides were analyzed under light microscope Axioskop 2 and images were obtained with Zen lite 2011 software.
Approximately 200 ovarian follicles were examined from each animal, counting only follicles presenting oocyte and granulosa cells, presenting the oocyte nucleus or not.Follicles were classified as preantral, antral and degenerated, considering as degenerated the follicles possessing oocytes with retracted cytoplasm or without contact with granulosa cells.The proportion of each type of follicle was calculated in relation to the total number of follicles observed.These proportions were calculated for every dam, and then, the mean for specific experimental group and age was calculated.
The uteri of three nulliparous animals from the control group and of six from the NP group were processed for histological analysis as described above.Sections with 5 µm thickness were obtained from a uterine horn region next to the oviduct.Microscopic slides were stained with hematoxilin-eosin and observed under light microscope Axioskop 2. Images were obtained with Zen lite 2011 software.

Statistical analyses
Normality of the data were tested by Kolmogorov-Smirnov or Shapiro-Wilk tests and, when proved, these data were compared by one way ANOVA parametric test and Tukey's post hoc test.Data not normally distributed were analyzed and compared with Kruskal-Wallis non-parametric test and Dunn's post hoc test.Differences were considered statistically significant when P < 0.05.These statistical analyses were performed using GraphPad Prism 5.00 software.
Data of age when 50% of females from a particular experimental group reached sexual maturity as accessed by vaginal opening, were analyzed by Mantel-Cox's test with significance level of 5%, performed in GraphPad Prism 5.00 software.Fertility rates in different attempts at pregnancy for each experimental group were analyzed using the Logistic Anim.Reprod., v.13, n.4, p.795-805, Oct./Dec.2016 procedure from SAS v.9.3 software, using the chi-square test with significance level of 5%.

Females' weight and sexual maturity
Figure 2 shows the mean weight of females from all experimental groups at the beginning and during the three weeks of treatment.Control and NP groups were significantly different from E2 group only at first day of treatment, which indicates treatment does not affect animals' growth.
Vaginal opening is a common indicator of sexual maturity in females (Fig. 3).The ages at which 50% of females of the same group were already sexually mature were 31, 30, 30, and 29 days, for control, vehicle, NP and E2 groups respectively.Vaginal opening curve was analyzed by Mantel-Cox's test and did not show differences among experimental groups (Fig. 4).

Fertility rate and parameters analyzed in offspring
The fertility rates in the first attempt at pregnancy and after the second attempt at pregnancy are shown in Table 1.There was no statistical difference (P > 0.05) between treatments concerning fertility rates.
All pups born had normal appearance with no signs of malformation.Mean number of live offspring, mean weight on first day of life, and female/male ratio of offspring born after both first and second attempts at pregnancy are shown in Table 2. Mean number of live offspring born after first and second attempt at pregnancy did not show an alteration in the experimental group to which dams pertained.In both attempts, the mean weight of offspring was not altered by any treatment received by their mothers.The female ratio of offspring from the first attempt at pregnancy did not differ among groups.Also, female ratio of offspring from the second attempt at pregnancy did not show differences between groups.

Relative weight of organs
Mean relative weight of organs from all treatment groups of nulliparous females are shown in Table 3. Group treated with E2 had lower relative weight of spleen compared with control.It is noteworthy that the standard deviation of the relative weight of uteri from NP group was quite high, above the mean weight of the group.This fact is probably due to three animals, in a total of eight, presenting notable augmented uteri in relation to others from this group.The mean weight of the uterus of these three animals was 2.35 g, while the other five animals had a mean weight of 0.45 g.
The relative weight of organs from females of all experimental groups euthanized after first and second attempts at pregnancy were also shown in Table 3

Ovarian follicles
Preantral, antral and degenerated follicles were identified in every animal studied, independently of experimental group or age (Fig. 5).The percentages of these types of follicles for each experimental group are shown in Fig. 6.They were compared among different treatments and confirmed that treatments did not alter percentages of different types of follicles in nulliparous females (Fig. 6A).In females that got pregnant at the first attempt, the percentage of antral follicles was shown to be lower in E2 than in NP group (Fig. 6B).Females that got pregnant only after the second attempt at pregnancy had their ovarian follicles classified too and no difference was detected in any type of ovarian follicle for distinct treatment groups (Fig. 6C).
Six females did not get pregnant at either attempt: two from control group, one from vehicle group and three from E2 group.Ovaries of these animals were analyzed to assess percentages of follicle types.Mean percentages of each type of ovarian follicle from nonpregnant females, independently of experimental group, were compared with the control group at different ages.There were no differences in preantral, antral and degenerated percentages between control and nonpregnant group (Fig. 6D).Thus, the absence of pregnancy cannot be explained by alterations in number of ovarian follicles.

Histology of uterus
After observing anatomically augmented uteri, a histological analysis was performed to verify the occurrence of any anomaly (Fig. 7).A uterus without alterations, with regular appearance, presents a large lumen in the center (Fig. 7A).This lumen is lined by one layer of cylindrical epithelium, which joins with connective tissue layer, found just beneath, forming the endometrium, the portion that undergoes most alterations during the estrous cycle of females.The layer of connective tissue has endometrial glands and is rich in extracellular material, particularly reticular fibers.These fibers become more abundant as distance from the lumen increases, while cells become rarer.Beneath connective tissue is localized the musculature.This description is in accordance with Junqueira and Carneiro (2004) and Kierszenbaum (2008).
A nulliparous female treated with NP, whose relative uterus weight was considered regular, had morphological alterations identified compared with control group.The epithelium was shorter, the endometrium was thicker and the lumen of the organ was reduced (Fig. 7B).In contrast, animals from NP group with relative uterus weight considered higher did not show histological modifications compared with control (Fig. 7C and 7D), except for one animal that presented glands with larger lumens, which may have contributed to increasing the diameter of the organ (Fig. 7D).

Discussion
The stage of life at which exposure to endocrine disruptors occurs is crucial to determine the extension of injury.Developing tissues are more sensitive to endocrine signals, so deregulation of these signals may cause permanent damage to structure or function of tissues, which makes exposure during development (in utero, neonates or young) potentially more harmful (Markey et al., 2003;Rudel and Perovich, 2009).Considering this fact, the present work aimed to analyze whether NP given to recently weaned females during the period of sexual maturation provokes prejudicial effects on their reproductive function.
Animals' weight was not altered compared with control and vehicle groups during the entire treatment.This indicates NP and E2 do not affect the growth of animals at doses of 50 mg/kg b.w. and 1 mg/kg b.w., respectively.Although E2 group has initiated treatment with lower weight, these animals gained weight soon in the first week and maintained it in the normal pattern.Also, no sign of general toxicity was noted in female mice.
Administration of exogenous steroids, as estradiol and testosterone, to immature rodents disrupts the progression of puberty (Pak et al. 2003).Kim et al. (2002) have affirmed that NP modifies the estrous cycle in rats.Oral exposure to NP continuously for three generations of rats showed estrogenic activity, and the hastening of the vaginal opening by two days for the group exposed to 650 ppm NP (30-100 mg/kg b.w.), and by six days for the group exposed to 2000 ppm NP (100-350 mg/kg b.w.) in all generations (Chapin et al., 1999).In this same work, the estrous cycle of females was deregulated only in the group treated with the higher dose.However, in the present study, carried out with a lower and more realistic dose, there was no significant difference in the day of vaginal opening between experimental groups.
NP was not shown to impair mating or fecundation as fertility rates were similar for every group.The first attempt at pregnancy was less successful than the second due to the young age of females (between 46 and 60 days of life, just after sexual maturation), while in the second attempt at pregnancy females had between 84 and 98 days of life and were thus completely mature sexually (Chorilli et al., 2007).NP also did not influence the number of liveborn offspring after the two attempts at pregnancy, nor their weight registered on the first day of life.Nagao et al. (2001), studying two generations of rats, treating males and females with 50 mg/kg b.w NP, also did not find significant differences in the fertility rate and number of offspring from the parental generation when compared with control.Similarly, males treated with 42.5 mg/kg b.w. of NP intraperitoneally for 35 days, when mated with non-treated females, obtained fertility rate, number of implantations/litter, number of live fetuses, fetal body weight and occurrence of external abnormalities equal to the control group, apart from adverse effects noted in testes and sperm (El-Dakdoky and Helal, 2007).
Genetic sex, determined by chromosomes present in cells, is considered the major factor for development of sexual differences.However, it is currently known that many of them may occur as a result of variable exposure to sexual hormones during development (Majdic and Tobet, 2011).Besides, it is also known that intrauterine environment and the concentration of sexual hormones with which fetuses are in contact may modulate sexual differences, especially brain development and behavioral characteristics (Saal et al., 1999).For these reasons, this work evaluated if females' exposure to NP while they are young would be capable of producing an alteration in offspring sex.This effect was not observed, as the proportion of females in the litter was the same in all experimental groups, independent of the age of dams.This result demonstrated that 50 mg/kg b.w. of NP given to females for 21 days did not have an effect upon the sex of their offspring.All data issued from offspring suggested there was not any delayed action of estrogenic compounds, given to females during sexual maturation, upon development of their offspring.
The most consistent toxic effect described in works with NP is mineralization of kidneys in male mice, although there is a variation in doses at which this effect is observed (Green et al., 2003).NP produced an increase in the relative weight of kidney and liver, and a reduction in the weight of thymus in males treated with 50 mg/kg b.w.(Nagao et al., 2001), while in greater doses, such as 30-350 mg/kg b.w., alterations in the weight and also in the structure of kidneys were identified (Chapin et al., 1999).However, in the present work, which analyzed only organs from females, differences in relative weight of ovaries, uterus, liver and kidneys were not found, independently of experimental group and age of females undergoing euthanasia.Only the group of nulliparous females treated with E2 had a reduction in relative weight of spleen compared with control group.Estradiol and some endocrine disrupters have the capacity to inhibit proliferation of lymphocytes isolated from the spleen (Sakazaki et al., 2002) which is likely the cause of the observed effect in mice spleens.This effect has also been demonstrated in dogs treated with estrogen (Hart, 1990).Older animals did not show this effect on spleen.
A reduction in the proportion of antral follicles and a resulting increase in proportion of preantral follicles were expected (Zeleznik, 1981), due to increased amount of exogenous estrogen circulating, which is able to activate the negative feedback mechanism in the pituitary.This mechanism diminishes FSH secretion and interrupts the development of immature follicles (Zeleznik, 2004).Meanwhile, NP did not alter proportions among preantral, antral and degenerated follicles.The expected effect was observed just in the E2 group that got pregnant after the first attempt and was not too substantial (percentage of antral follicles only diverged from NP group).At the same time, alterations or deformities in ovarian follicles were not identified in any group.Mehranjani et al. (2010), working with offspring of rats submitted to an elevated dose of NP (250 mg/kg b.w.) in utero, found a reduction in the number of antral and preovulatory follicles and an increase in number of atretic ones.
Histological analyses of animal uteri did not confirm any adverse effect that might have been caused by NP.On the contrary, organs with relative weight diverging from the rest of the group were seen to be histologically similar to the control uterus.The sole alteration noticed was an augmentation of the secretory glands in the endometrium, which could be described as an estrogenic effect, since estrogens play a role in the development and proliferation of the uterine endometrium (Junqueira and Carneiro, 2004;Kierszenbaum, 2008).However, more studies should be performed to confirm if this effect is prejudicial to animal's health.Augmentation in uterine weight was previously reported for rats treated with 100 and 200 mg/kg b.w. of NP.This effect was comparable to that of diethylstilbestrol (DES), a well-known endocrine disruptor (Kim et al., 2002).Although in the present work NP did not produce anomalies, it was capable of promoting focal mucinous metaplasia in the endometrium and uterine glands in guinea pigs exposed to 40 mg/kg b.w. for 14 days (Danzo et al., 2002).Incongruity between these works may be the result of using different experimental animals, since different species have variable susceptibility to this compound.
When administered at a dose of 50 mg/kg b.w.during the period of sexual maturation in female mice, available or accumulated NP did not reach levels to produce significant alterations in the analyzed parameters, a few days after ending exposure, when females effectively initiate reproductive life.NP probably reaches a balanced state in tissue accumulation and bioavailable quantity after metabolization does not achieve the threshold for action (Green et al., 2003).The dose used in this work is equivalent to 3.5 g in a 70 kg person and do not had effect in the main outcome of reproduction: offspring birth.For comparison purpose, daily intake for humans is estimated between 0.37 μg/kg b.w (Gyllenhammar et al., 2012) and 0.43 μg/kg b.w (Lu et al., 2007).The absence of significant effects in E2 group, treated with a more estrogenic dose, corroborates these findings.
In conclusion, NP at 50 mg/kg b.w.do not cause damage to reproductive organs and function in female mice exposed for 21 days during puberty.Moreover, this work shows this length of treatment with NP does not have drastic effects on later fertility of females or birth weight and sex of offspring.

Acknowledgments
We are grateful to Dr. César Koppe Grisólia for the use of equipment in his laboratory, to Dr.

Figure 1 .
Figure 1.Experimental design with timeline representing post-natal day (PND) of treated females.Treatments are in blue, steps of the first attempt at pregnancy are in green and steps of the second attempt at pregnancy are in red.

Figure 2 .
Figure 2. Mean weight of females from all experimental groups at the beginning and during three weeks of treatment.The only difference appears between control and NP group compared with E2 group, but just on the first day of treatment.This difference was rapidly surpassed, indicating that treatment did not have effect on females' growth.ANOVA and Tukey's post-test, α = 0.05.Different letters indicate statistical difference between values.

Figure 3 .Figure 4 .
Figure 3. Identification of vaginal opening indicating sexual maturity in female mice.Left: female before vaginal opening.Right: female after vaginal opening, when protuberant external genitalia (arrow) and vaginal canal lumen can be observed.

Figure 5 .
Figure 5. Diverse examples of ovarian follicle types.A: General view of ovary from a female pregnant after first attempt at pregnancy treated with NP.B: Ovary's general view of female pregnant after first attempt from control group.C: Antral follicle found in female pregnant after first attempt treated with E2.D: Degenerated follicle, near to an antral, from a female pregnant after first attempt from NP group.E: Detail of degenerated follicle showing lack of contact of oocyte with granulosa cells, found in female pregnant after second attempt treated with NP.A: antrum; AF: antral follicle; CL: corpus luteum; Cu: cumulus cells; DF: degenerated follicle; GC: granulosa cells; Mg: mural granulosa cells; O: oocyte; ON: oocyte nucleus; PAF: preantral follicle.

Figure 6 .Figure 7 .
Figure 6.Percentage of follicle types identified in animals.A: Nulliparous females.B: Pregnant females after first attempt.C: Pregnant females after second attempt.D: Percentage of follicular types of control groups with different ages and non-pregnant females.ANOVA with Tukey's post hoc test or Kruskal-Wallis with Dunn's post hoc test, α = 0.05.Different letters indicate statistical difference between values.

Table 1 .
Fertility rate of females after first and second attempts at pregnancy.

Table 2 .
Data obtained from pups born after mating treated females in two pregnancy tries.

Table 3 .
. No difference was observed in any organ between experimental groups at that age.Mean relative weight of organs from nulliparous and pregnant females after both tries.Different letters indicate statistical difference between values in same column.Numbers in parenthesis represent number of animals analyzed for each group.
Concepta McManus for assistance with some statistical analyses, to Dr. Cláudio Afonso Pinho Lopes for valuable considerations to this work and to Coordenação de Aperfeiçoamento de Pessoal de Nível