Developmental, behavioral and endocrine alterations in male rats at early and late postnatal life following in utero exposure to low dose di-n-butylphthalate

Abstract

Environmental chemical pollutants that interfere with hormonal homeostasis or hormone signaling are the relevant agents inducing congenital or postnatally developed reproductive abnormalities in human beings, wild and domestic animals. In this study, we are examining reproductive effects of prenatal exposure of male rats to a low dose di-n-butylphthalate (DBP). Wistar female rats were given intragastrically DBP at a daily dose of 100 mg/kg b.w. during 15th–21st days of pregnancy. Anogenital distance (AGD) in male offspring decreased on postnatal day (PND) 2 followed by its normalization on PND 7 and 10. There were no other visible teratogenic lesions in the newborns. The testicle descent into scrotum of control males occurred on PND 38.5 ± 0.1, while in DBP group it accelerated by 5.3 days on the average. At the age of 6 months, DBP-exposed animals exhibited double increase of blood plasma testosterone level as compared to controls, and hyperactive male sexual behavior in the presence of receptive female. The duration of latent periods of the first mount and the first intromission, as well as post-ejaculatory refractory period, have been shortened; the number of mounts with intromission and the number of ejaculations increased significantly. Histological examination of the testes indicated activation of Leydig cells. The female-type sexual behavior as evaluated by appearance of lordosis of orchidectomized and primed with estradiol and progesterone 10-month-old males in response to mount or approach of sexually active normal male was enhanced in DBP-group. Both 10-month-old and aging males (18 months), castrated and hormone-primed, displayed homosexual type of behavior. Prenatal low dose DBP caused in 18-month-old males premature atrophy of the testes and accessory sexual glands, increased number of Leydig cell adenomas, a twice decrease of plasma testosterone level and exhausting of sexual potency. We concluded that prenatal exposition of male rats to low dose DBP determines epigenetic alterations of programming of sex brain differentiation and regulation of testicular steroidogenesis that leads to reproductive disorders and accelerated aging of reproductive system.

Introduction

The rising evidence of the human reproductive disorders is of a big concern worldwide. Environmental chemical pollutants that interfere with hormonal homeostasis or hormone signaling commonly named as endocrine disruptors. They used to be the relevant agents for congenital or postnatally developed reproductive abnormalities in human beings, wild and domestic animals. Endocrine disrupting chemicals (EDC) are to blame for the worsening of reproductive health and a decrease of fertility, which prevails in 9% of the world population [1], because reproductive system is extremely susceptible to their harmful impact. Mechanisms of endocrine disrupting effects are quite diverse including binding to nuclear and membrane hormone receptors, changes of hormone synthesis and metabolism, epigenetic modifications, direct effects on endocrine glands, oxidative stress etc [2–8].

The members of phthalate ester family are commonly used in the chemical industry as a plasticizer for the production of plastics, medical devices, cosmetics, drugs, and they are present ubiquitously in the environment and in human biological fluids. They are fixing on plastics by non-covalent bonds, which makes them easily separated and pollute air, water, food. Phthalates and their metabolites are found in 90–95% of the samples of human amniotic fluid as well as in blood and urine of children and adults. Di(n-butyl)phthalate is present in many consumer products. It produced in high volumes in the countries with developed chemical industry. Monobutylphthalate, the main metabolite of DBP, was found in very high concentrations in urine of pregnant women and other subjects taking medications, which contain DBP in their coating [9]. The concerns are rising about potential risks of DBP intrauterine exposure for human fetus development and reproductive disturbances in adulthood.

Due to antiandrogenic and other endocrine disrupting properties, di(2-ethylhexyl)phthalate (DEHP) and DBP are capable of exerting adverse effects on pregnancy outcomes and fetal gonads and other reproductive organs development. Association between phthalates prenatal and postnatal exposures and adverse reproductive effects has been demonstrated in epidemiological studies as well as on animal models [4, 7, 10–17]. Based on studies in rodents, Leydig cells are a target for phthalate esters reproductive toxicity. Developmental reproductive malformations in male rats include reduced anogenital distance (AGD), reduced accessory sexual glands, penile malformations (reduced size and hypospadias), cryptorchidism and impaired spermatogenesis (testicular dysgenesis syndrome), etc.

Many of male rats, which have been exposed to di(2-ethylhexyl)phthalate in utero and during lactation, were sexually inactive in the presence of receptive female, and it was not correlated with morphological abnormalities suggesting disturbance of sexual brain differentiation [18]. In mammals, sexual brain differentiation occurs under fetal testicular androgen control. In rat, critical period of this process corresponds to the last week of pregnancy (15th–21st gestation days) and lasts until 5th postnatal day (PND). Taking into consideration antiandrogen activity of DBP one of the risks can be associated with deviations in sexual behavior in male offspring that have been born to mothers exposed to DBP during critical period of sexual brain differentiation. We supposed that such a kind of behavioral abnormalities might occur even after exposure to low dose DBP that does not cause essential lesions in reproductive tract. Sexual behavior in male rats prenatally exposed to low doses of DBP that do not produce significant teratogenic effects were not studied. Earlier we reported an abnormally high sexual activity in male rats prenatally exposed to low dose DBP [19].

The objective of this study was to evaluate an impact of low dose DBP administered to rats during the last week of pregnancy on sexual development, behavior, and testicular morphology and function in male offspring at different ages with a special attitude to hormone-neurotransmitter imprinting of the sex brain differentiation conceptual framework [20]. Given the results of experiments on rats conducted by The U.S. National Toxicology Program, the lowest observed adverse effect level of DBP (NOAEL) that was administered via diet on gestation days 12–20 and induced reproductive lesions in male rat offspring was set at 66 mg/kg/day [11]. In order to reveal the long-term functional changes in the absence of significant teratogenic effects, we have chosen for this study DBP daily oral dose of 100 mg/kg.

Materials and methods

Materials and animals

Di(n-butyl)phthalate (dibutyl ether of phthalic acid) was from Alfarus (Kyiv, Ukraine). Estradiol-17β diacetate was purchased from Sigma (St. Louis, MO, USA). Progesterone, 1% olive oil solution, was from Biopharma (Kyiv, Ukraine). Immunoassay kit Testosterone ELISA was purchased from DRG (Germany).

Female Wistar rats (200–220 g) of the local Institute vivarium breeding housed in a temperature- (21–23 °C) and light-controlled room (darkness 10 h, light 14 h) with free access to tap water and standard dry food pellets. The experiments were carried out in accordance to European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 18 March 1986), and Recommendations of The First National Congress on Bioethics Issues (Kyiv, Ukraine, 20 September 2001). The experimental design and procedures were approved by the Bioethics Commission of the Institute (the Protocol No 13-KE from 25.05.2016). The total number of rats sacrificed in this work for measuring weights of reproductive organs, morphological study of gonads and ventral prostate and assessment of blood plasma testosterone levels was 42.

Design of the experiments

The rat dams with a regular 4–5 estrous cycle that have been estimated previously by everyday microscopic examination of the vaginal smears during 2 weeks were recruited for fertilization. They were placed in the cage together with sexually active male followed by the vaginal smear examination until sperms have been detected, and that day was considered the first day of gestation.

Pregnant dams were given by gastric intubation with cholesterol-free sunflower oil (control) or 10% DBP oil solution (DBP group) at 100 mg/kg/day during 15th–21st gestation days, 10 rats per group. Two replicates of the male offspring (n = 20 per replicate) from each pregnant group were formed by randomization, and males allowed to be kept in the vivarium untill 6 or 18 months of age.

The AGD was measured on PND 2, 7 and 10. The day of the testicle descent into scrotum was recorded as a feature of sex maturation. At 6 and 18 months of age, the males were tested for exhibition of male-type sexual behavior, and at 10.5 and 18 months for female-type sexual behaviors. At 6 and 18 months of age, they were sacrificed for taking blood for testosterone assay and reproductive organs for histology.

Hormone assay

After decapitation under light diethyl ether anesthesia, the trunk blood samples from control (n = 12) and DBP (n = 13) animal groups were collected in heparinized tubes. The plasma obtained by centrifugation and kept at − 20 °C prior was analyzed for testosterone level. Testosterone immunoassay was performed by Immunoenzyme assay kit Testosterone ELISA (DRG, Germany) followed by measurement at immunoenzyme analyzer Stat Fax (USA).

Male-type sexual behavior

In order to test sexual behavior, five males delivered by different dams were included in DBP or control groups. Male-type sexual behavior was tested at age of 6 and 18 months according to [21] with some modification [22]. Before testing, the males were kept in darkness, and then they have been moved to an empty cage for a 5-min adaptation. A sexually receptive female that was ovariectomized 1 week before testing and then injected intramuscularly with 0.1 mg estradiol diacetate 48 h before the test, and 0.5 mg progesterone oil solution introduced 4 h prior to the test was placed for 15 min under dim red light in the cage with the male. The following indices of copulative behavior were recorded: duration of latent periods of the first mounting and the first intromission, the first ejaculation, post-ejaculatory refractory period, the number of ejaculations, mountings without intromissions, and the total number of intromissions. All procedures were conducted twice at 1-week interval taking into account that by the time of the second test they gained some sexual experience.

Female-type and homosexual behavior

To evaluate the sensitivity of the male brain’s behavioral centers to estrogen, which is obligate for expression of the female sexual behavior, the rat was orchidectomized 1 week before testing and treated with steroid hormones as mentioned above for preparing receptive females. The males were introduced to a sexually advanced male that was in the cage under red dim light for at least 5 min. The test lasted 10 min or up to 10 mounts of an active male. The number of lordosis reactions to approaches and mounts of normal male were recorded. The lordosis index was calculated as the percentage of the number of lordosis reactions relative to the total number of the active male's mounts. Additionally, manifestations of male-to-male sexual motivation and homosexual behavioral patterns were recorded at the presence of a sexually active normal male as approaches from the male’s rear, mounts and a shallow thrusts with its pelvis.

The reproductive organs morphology

After isolation, the gonads and accessory sex organs were weighed. The testes and ventral prostate were fixed in 4% paraformaldehyde, then paraffin-embedded, and 5 μm thick sections were prepared. The microscope sections were stained with hematoxylin–eosin and underwent histological examination using the Leica DME microscope (Leica Microsystems, Germany).

Statistical analysis

The results were averaged and compared with those of appropriate controls. They were presented as mean ± SEM and processed with Excel computer program by one-way analysis of independent experiments using the Student’s t criterion. The difference was set as significant at p ≤ 0.05. In the absence of a normal statistical distribution, the Wilcoxon–Mann–Whitney non-parametric U criterion was used. To compare the incidence values in two groups the sign pair test was used.

Results

The offspring development

Prenatal administration of DBP led to reduction of the litter: on average, 11.2 pups per mother in control group vs. 8.7 in DBP group. On the second PND, the weights of DBP exposed newborns was the same as those of controls. At this time point, an average AGD in DBP group was 3.35 ± 0.07 mm, compared to 3.63 ± 0.11 mm in the control group (p < 0.05). There was no statistical difference between groups on PND 7 and 10.

The testicle descends into the scrotum of control males occurred on PND 38.5 ± 0.1, while in DBP group it accelerated by 5.3 days (33.2 ± 0.1, p < 0.001), which indicates puberty praecox.

Observation of DBP exposed males after maturation up to 18 months has not found any difference in body weights, appearance, social and eating behavior, physical activity as compared to controls.

Male-type sexual behavior

Control 6-month-old males were tested in December, and demonstrated low sexual activity; in particular, they did not ejaculate for 15 min of observation. The low sexual activity may be caused by the seasonal fall of testosterone secretion and sex behavior [23]. At the same time, in the second test, after the animals have gained sexual experience, they have become more sexually active. That manifested in the appearance of ejaculation, a dramatic reduction in the duration of the latent periods of the first mount and the first intromission, and an increase in the number of mounts with intromission.

In contrast to the original sexual pattern in normal rats, in the DBP group of 6-month-old animals, in both sessions, there was found sexual hyperactivity, which covered all indicators characterizing the central and peripheral components of sexual behavior. As the result of the prenatal exposition of DBP, the duration of latent periods of the first mount in the first test decreased by an average of seven times, the first intromission—by six times, the number of mounts with intromission has doubled. In the second test, the prenatally administered DBP caused a 1.5 times increase in the number of ejaculations, and more than a threefold reduction of the post-ejaculatory refractory period, whereas the duration of the latent periods of the first mount increased (Table 1).

Table 1.

Effect of prenatal low dose DBP exposure on male-type sexual behavior in male rats of 6-month age

Feature	First test		Second test
	Control	DBP	Control	DBP
Latency period, s
First mount	109.4 (10–219)	15.2 (3–40)*	3.8 (2–9)**	20.4 (2–73)*
First intromission	125.2 (15–259)	21.2 (8–51)*	28.0 (3–113)**	30.8 (9–88)
First ejaculation	> 900	490.6 (315–743)*	361.4 (244–602)**	451.0 (273–658)
Post-ejaculatory refractory period	> 900	267.8 (3–585)*	416.2 (167–656)**	129.4 (22–255)*
Number
Mounts without intromission	3.8 (1–6)	2.4 (2–3)	4.0 (3–5)	2.8 (2–4)*,**
Mounts with intromission	9.8 (1–19)	18.8 (17–25)*	17.4 (11–35)	26.6 (20–32)**
Ejaculations	0 (0)	1.2 (1–2)*	1.2 (1–2)**	1.8 (1–2)*,**

Five rats in each group. The test lasted 15 min. The data are presented as mean values and minimum and maximum figures (in the brackets). Statistical analysis by Wilcoxon–Mann–Whitney non-parametric U-criterion

*p ≤ 0.01, p ≤ 0.05 compared to controls

**p ≤ 0.01, p ≤ 0.05 compared to the first test

As expected, sexual potency of 18-month-old control rats regressed with age. That was evidenced, at first, by the loss of the ability of one-third of males to mate with females and almost three times growth of latency time before the first intromission in the first test of male sexual behavior. Particularly revealing were the characteristics of the second test of the behavior: the duration of latent periods of the first mount increased seven times, the first intrusion—almost twice, the number of mounts without intromission—1.7 times. However, some indicators even improved, such as, for example, the latent period of the first ejaculation reduced 1.6 times; the number of ejaculation increased 1.5 times.

As it turned out in the study of sexual behavior of aging males, the increased activity of young animals was transient. Even against age involution of sexual activity of control animals, the DBP-group deteriorated significantly. It has changed for accelerated aging of the reproductive system, probably due to its exhaustion. That was evidenced by the dramatic decrease of sexual motivation and the ability of males to mate with females, and nearly tripling the time before the first intromission in the first test of male behavior as compared to aging control males. During the observation, ejaculations in both testings were absent, in the second test, the latent period of the first intromission lengthened 9.3 times, the number of the mounts without intromission decreased 2.4 times and with intromissions—9.5 times (Table 2).

Table 2.

Effect of prenatal low dose DBP exposure on male-type sexual behavior in male rats of 18-month age

Feature	First test		Second test
	Control (n = 6, 2 of them sexually passive)	DBP (n = 5, 1 of them sexually passive)	Control (n = 5)	DBP (n = 5)
Latency period, s
First mount	53.7 (7–100)	63.5 (11–147)	27.0 (1–60)	27.0 (18–39)
First intromission	299.7 (76–900)	101.0 (35–240)	51.4 (18–138)**	478.8 (42–878)*
First ejaculation	751.7 (307–900)	> 900*	223.4 (125–349)**	> 900*
Post-ejaculatory refractory period	823.2 (593–900)	> 900*	347.0 (205–530)**	> 900*
Number
Mounts without intromission	5.5 (4–8)	5.0 (4–7)	6.6 (5–7)	2.8 (1–5)*
Mounts with intromission	7.5 (0–19)	5.5 (1–9)	24.8 (20–32)**	2.6 (0–5)*
Ejaculations	0.2 (0–1)	0 (0)*	1.8 (1–3)**	0 (0)*

n number of rats in the group. The test lasted 15 min. Minimum and maximum figures are presented in the brackets. Statistical analysis by Wilcoxon–Mann–Whitney non-parametric U-criterion

*p ≤ 0.01, p ≤ 0.05 compared to controls

**p ≤ 0.01, p ≤ 0.05 compared to the first test

Female-type and homosexual behavior

Taking into account, the data we received about prenatal programming of hypersexual male behavior, somewhat unexpected were the results of the study of female sexual behavior of 10-month-old males, which indicate an increased feminization of their neuroendocrine reproductive phenotype compared to control. In conditions of removal by castration of androgenic factor of hormonal regulation of sexual behavior and estrogen–progestin priming, the sensitivity of neuroendocrine centers of experimental males to the stimulatory action of estrogens had been shown. Though the number of DBP-exposed males with lordosis behavior did not differ statistically from controls, those animals demonstrated a doubly increased number of lordosis responses in the presence of a normal male (10.0 at average vs 5.0 in controls); the lordosis index was 100 vs 65 in the control group. At the same time, in contrast to the control males, they performed mounts on normal males (on average 6.8 per the test time) (Fig. 1). The latter appeared in all tested males, while in the control group, homosexual manifestations were absent. Homosexual behavior lasted to 18 months of age (on average, 9.4 mounts comparatively to 0.8 in control), while lordosis responses were absent in both groups (Fig. 2).

Fig. 1.

Average indices of sexual behavior for homosexual and female types in male rats aged 10 months after prenatal administration of DBP. Notes: five rats in each group. Before testing, males were orchidectomized and primed with estradiol and progesterone (see text for details). The test lasted 10 min or up to 10 mounts of the active male. Left bars—controls, right bars—DBP. a The number of mounts of the experimental males on the active normal male, b the number of males with homosexual behavior, c the number of males with lordosis responses. Statistical analysis by Wilcoxon–Mann–Whitney non-parametric U-criterion for group A and by the sign test for groups B and C. *p < 0.05 compared with control

Fig. 2.

Average indices of homosexual behavior in male rats aged 18 months after prenatal administration of DBP. Notes: five rats in each group. Before testing, males were orchidectomized and primed with estradiol and progesterone (see text for details). The test lasted 10 min. Left bars—controls, right bars—DBP. a The number of mounts of the experimental males on the active normal male, b the number of males with homosexual behavior. Statistical analysis by Wilcoxon–Mann–Whitney non-parametric U-criterion for a and by the sign test for b. *p < 0.05 compared with the control

Testosterone levels

Blood plasma testosterone levels in DBP group (n = 6) at 6 months were significantly increased in comparison to controls (n = 6). In 18-months old experimental animals (n = 7), testosterone levels were more than twice less than those of controls (n = 6) (Table 3).

Table 3.

Plasma testosterone levels (ng/ml), body (g) and reproductive organs weights (mg/100 g b.w.) of control and prenatally DBP-exposed male rats (mean ± SEM)

Animal group	n	Body weight	VP	CG	SV	Epi	Testes	T
6-month-old rats
Control	9	310 ± 9	122.0 ± 12.8	59.0 ± 3.6	64.3 ± 3.2	355 ± 17	1064 ± 28	3.76 ± 0.85
DBP	9	330 ± 13	109.8 ± 6.3	50.9 ± 2.6	64.9 ± 2.3	319 ± 7	1024 ± 33	7.53 ± 1.45*
18-month-old rats
Control	11	418 ± 11	100.6 ± 5.5	44.4 ± 3.7**	63.1 ± 4.2	283 ± 7**	882 ± 24**	2.48 ± 0.71
DBP	13	435 ± 15	52.8 ± 6.1*	25.4 ± 2.6*	43.3 ± 2.2*	234 ± 10*	792 ± 38*	1.04 ± 0.17

VP ventral prostate, CG coagulative glands, SV seminal vesicles, Epi epididymis, T testosterone. Statistical analysis by Student’s t criterion

*p < 0.05 as compared to control of the same age; **p < 0.05 as compared to control of 6 months of age

The reproductive organs morphology

In 6-month-old descendants who were prenatally exposed to DBP the weights of gonads and accessory sexual glands did not change. Histology of the ventral prostate and spermatogenic layer of the testes in the DBP group did not differ from such in the control group. However, Leydig cells were enlarged and demonstrated the active phase of the hormone synthesis.

In the 18-month-old offspring of the experimental group, the weights of all studied organs were significantly decreased (1.7- to 2-fold) as compared with the control of the corresponding age (Table 3). Spermatogenesis was observed in control and in DBP-exposed animals only in the part of the seminiferous tubules, presumably because of apoptosis and reduction of the proliferation of Sertoli cells. The spermatogenic epithelium was desquamated in places; its structural elements chaotically migrated to the lumen of the tubule. These alterations were more expressed in the DBP-group.

In the interstitial space of the testes of control aging rats, there were cell conglomerates that have diagnosed as Leydig cell adenomas. These proliferative alterations were much more expressed in prenatally DBP-exposed males. A significant increase in the number of the Leydig cell adenomas was found compared to the control animals (4–7 vs 1–3 per rat). The Leydig cell pool was represented predominantly by small cells, indicating a decrease in their functional activity that corresponds to low plasma testosterone level.

Histology of the ventral prostate in DBP animal group did not differ considerably from that of control and corresponded to the age standard, with the exception of occurrence of more desquamated epithelial cells in the acinar lumina and apoptotic secretory cells, and an increase in the number of tissue basophils and leukocytes in the connective tissue.

Discussion

Low dose DBP exposure did not produce visible adverse effect on pregnant rats though reduced the number of newborns suggested pre- or postimplantation loss of embryos [11] noted such a kind of embryotoxicity.

Premature puberty and hyperactive male sexual behavior in young male rat offspring prenatally exposed to low dose DBP were unexpected findings in this study given the opposite results by other authors using higher doses of phthalates [10–12, 24, 25]. In order to clear up the pathogenesis of this phenomenon, as well as premature exhaustion of sexual potency in aged (18-month-old) animals, the levels of testosterone in blood plasma were measured. In particular, this was due to controversial literature data on testosterone level in adult males prenatally exposed to DBP at a daily dose of 100 mg/kg b.w. during the last week of maternal pregnancy and early postnatal life. They indicated both the lack of changes [26] and a decrease in the level of testosterone [27] or even an increase of that [28]. Two-fold increase of plasma testosterone level in our study is in line with analogous observations reported by the latter author and indicates a significant hyperandrogenization of animals. Presumably, this is the driving force behind DBP induced hypersexuality and, probably, premature puberty, because testosterone is a hormonal activator of the hypothalamic neuroendocrine centers and cortex. As a result, there is a stimulation of the sex drive to the female, which was revealed in young experimental males. As for the active metabolite of testosterone—5α-dihydrotestosterone—it activates mainly the sympathetic and parasympathetic centers of the spinal cord, providing sexual reflexes of erection, pairing, and ejaculation [29]. Morphological features of Leydig cells activation in this study correlated with an increased testosterone level in blood plasma.

Taken together, our behavioral data with androgen status of young animals, the results indicate an excessive masculinization of neuroendocrine system, which might be hypothetically attributed to DBP-induced hypersecretion of testosterone by intrauterine fetal testes during the time window of sexual brain differentiation. An argument in favor of this assumption is the information on the direct stimulating effect of low concentrations DBP and its metabolite, monobutylphthalate, on the synthesis of testosterone in the culture of mice Leydig cells MLTC-1 [30, 31]. The activation of steroidogenesis was due to an increase in the synthesis of mitochondrial steroidogenic regulatory protein StAR, which is involved in the transport of cholesterol from cytosol to the internal membrane of mitochondria. However, in high concentrations, both compounds inhibited steroidogenesis at the level of cleavage of the side chain of the cholesterol molecule and 17β- and 3β-hydroxysteroid dehydrogenase.

At the first glance, the results of experiments on the culture of Leydig cells contradict data on inhibition of steroidogenesis in the rat fetal testes against the background of the use of low doses of DBP in vivo in the so-called window of sexual differentiation of the brain, that is, during the last week of intrauterine development [28, 32–35]. Instead, in the paper by [36] there is a confirmation of the assumption about transient hypersecretion of testosterone by fetal rat testes under conditions of use of DBP. Feeding DBP to female rats at a dose of 100 mg/kg b.w. from 12 to 19 days of pregnancy caused the inhibition of testosterone secretion with fetal testes, followed by a rapid (within 24 h) restoration of the hormone level in the fetal blood plasma, and further—its growth above initial level due to rebound effect. We hypothesized that such a testosterone spike in the critical period of the male brain sexual differentiation could be the cause of its hypermasculinization.

In male rats, AGD is longer compared to that in females, and it is one of the signs of sexual dimorphism. Developmental testosterone deficiency in the rat male fetuses leads to retardation of the perineum growth. Reduction of AGD in male offspring was the result of exposure of mothers to DBP at doses higher than 50 mg/kg over the pregnancy [11]. It seems that a slight, but statistically significant decrease in AGD in the first days after birth in the rats prenatally exposed to low dose DBP indicates antiandrogenic activity of the studied compound and contradicts the assumption of hypersecretion of testosterone by the intrauterine fetal testes. However this contradiction could be discarded if one takes into account the ability of DBP to interact with androgen receptors in vivo tests [37], but not in vitro [32], and because they are not involved in the androgen-dependent brain differentiation that is induced by estrogenic metabolites of testosterone [20]. Perhaps, DBP must be converted to its active metabolite, monobutylphthalate, before exerting in vivo antiandrogenic effect at the tissue receptor level. This suggestion is supported with data on a significant decrease in AGD in the male rat fetuses which have been exposed to monobutylphthalate during the treatment of their mothers intragastrically on the 15th–17th days of gestation [38].

In this study, hyperactive male sexual behavior and hyperandrogenism in DBP-exposed 6-month-old male rats have switched to accelerated aging of the reproductive system in 18-month-old ones. Reduction in plasma testosterone in comparison with that of control animals of the same age was not caused by loss of Leydig cells but rather by decrease in steroidogenesis as it occurs in normal aging rats [39]. This is in line with the morphology of testes in low dose DBP-exposed males and completely corresponds to the premature regression of reproductive potential. The presence of Leydig cell adenomas in the testes of 18-month-old Wistar rats exposed prenatally to DBP at daily dose of 100 mg/kg b.w. during mother’s gestation days 12–21 was reported earlier [10]. However, in that work, DBP and control groups did differ in size of adenomas but not in their number, while in our study there was an increase of the number of Leydig cell adenomas in rats of the same age.

Taking into account the data we received about prenatal programming of hypersexual male behavior, the results of the study of female sexual behavior of 10-month males, which indicate an enhanced feminization of their neuroendocrine reproductive phenotype compared to control were somewhat unexpected (Fig. 1). In conditions of elimination by castration of androgenic factor of hormonal regulation of sexual behavior and estrogen–progestin pre-treatment, increased sensitivity of neuroendocrine centers of experimental males to the stimulating effect of estrogens was revealed. Animals demonstrated a doubly increased number of lordosis reactions when the normal male approached. In contrast to the control males, all DBF-exposed rats demonstrated mounts on normal males, which is considered a homosexual behavior maintained at 18 months of age (on average, 9.4 mounts vs 0.8 in control), and lordosis responses were not observed in both experimental and control groups.

Because of the excessive masculinization of the brain during its early programming, the probable explanation for simultaneous feminization might be lesions of neuroendocrine structures with damaging products of DBP-induced oxidative stress [40, 41]. According to the literature, both the use of phthalates and aging increase in Leydig cells the production of reactive oxygen species that damage the steroidogenic pathways [39]. Probably, in the aging animals of the DBP group, there is an additive effect caused by DBP and age-enhancing oxidative stress, which is manifested in the accelerated aging of the reproductive system. However, pathogenesis of feminization phenomenon warrants further investigation.

Thus, due to the administration to rats in the last week of pregnancy of threshold (in relation to the teratogenic effect) dose of DBP, male offspring developed alterations of sexual behavior and hormonal function of the gonads, which could be called "prenatal DBP syndrome". It manifests itself as follows: (a) premature puberty; (b) elevated levels of testosterone in the blood plasma; (c) hyperactive male sexual behavior; (d) homosexual behavior; (e) ability to display homosexual and female-type sexual behavior after castration and estrogen-progestin priming. There is premature exhaustion of sexual potency with age while simultaneously preserving homosexual behavior.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.