2025 Volume 50 Issue 2 Pages 83-95
Attainment of vaginal patency is an endpoint for the onset of puberty in female animals in toxicity studies. It is widely acknowledged that certain substances with endocrine-modulating effects can influence the timing of puberty in female rats and that factors unrelated to endocrine mechanisms, such as malnutrition and stress, can also affect pubertal onset. Some epidemiological studies have also suggested a link between anemia and delay in pubertal onset in women, however, little is known regarding the relation between hematological changes and female pubertal onset in experimental animals. The purpose of this study was to examine the effects of anemia during the prepubertal period on pubertal onset and reproductive organs in female rats. In this study, anemia was induced by drawing a certain amount of blood from the jugular vein or by intraperitoneal administration of phenylhydrazine, a well-known inducer of hemolytic anemia. As a result, both treatment groups showed a transient anemia characterized by an approximately 20-35% decrease in hemoglobin levels compared to the control group. Anemia in these female rats produced no obvious changes in body weight on each postnatal day and had no effect on the weights and histopathology of reproductive organs after sexual differentiation, but the age at vaginal opening (VO) was delayed and the body weight at VO was higher than the same parameters in the control group. These results suggest that anemia in prepubertal females could cause a delay in pubertal onset.
Because the endocrine system regulates a large number of processes from gametogenesis to development and the function of tissues and organs, exposure to endocrine disruptors (EDs) raises concerns about their possible temporary or permanent adverse effects. The World Health Organization (WHO) defines an ED as “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, its progeny or (sub) populations” (WHO-IPCS, 2002). The growing concern for the adverse effects of EDs on humans and wildlife has led scientists to look for more rigorous and precise assessments of EDs. Developing these assessments is a significant challenge to those currently involved in regulation of chemicals. According to the European Chemical Agency (ECHA)/European Food Safety Authority (EFSA) guidance for ED assessment (Andersson et al., 2018) published in 2018, a substance shall be considered as having ED properties if it meets all of the following criteria: a) it shows an adverse effect in an intact organism or its progeny or in non-target organisms, b) it has an endocrine mode of action and c) the adverse effect is a consequence of the endocrine mode of action. Thus, it is quite important to scrutinize whether adverse effects observed in toxicity studies are a consequence of the endocrine mode of action.
Regarded as one of the endocrine-mediated parameters in the aforementioned EFSA/ECHA guidance, attainment of vaginal patency is an endpoint for onset of puberty in female animals and is usually assessed in some toxicity studies in compliance with U.S. Environmental Protection Agency (EPA) or Office of Economic Cooperation and Development (OECD) test guidelines (TGs) (U.S. EPA., 1998a, 1998b, 2009a, 2009b; OECD, 2001, 2007, 2018). The onset of puberty is controlled by the hypothalamic–pituitary–gonadal axis (HPG axis). Activation of the HPG axis leads to maturation of gonads and secretion of gonadotropins. It has been reported that some substances which have endocrine-modulating effects, such as estradiol, ketoconazole (known as a blocker of steroid biosynthesis pathway), fadrozole (known as a blocker of conversion from C19 androgens to C18 estrogens), and testosterone, alter the pubertal onset in rats (McDonald and Doughty, 1972; Marty et al., 1999). On the other hand, it has commonly been assumed that pubertal onset is also altered via non-endocrine modes of action, such as nutrition status and stress (Carney et al., 2004; Manzano Nieves et al., 2019; Li et al., 2019). For example, in reproductive toxicity studies, decreased body weight gain due to the general toxicity induces the delay in pubertal onset in female animals and is a well-known secondary effect of growth retardation (non-endocrine toxicity) (Carney et al., 2004). Furthermore, even in the absence of an effect on body weight, delayed vaginal opening (VO) due to factors such as deficiency of essential fatty acids (linoleic acid and linolenic acid) and vitamin B12, as well as accelerated VO in rats fed a high-fat diet, have been reported (Laws et al., 2007; Smith et al., 1989; Dryden et al., 1954; Frisch et al., 1975). As pubertal onset is affected not only by endocrine mode of action but also by non-endocrine mode of action, it is necessary for accurate evaluation to discern the mode of action (Stump et al., 2014). However, little is known about indirect mode of actions that induce alteration in pubertal onset.
In epidemiological studies, pediatric patients with sickle cell anemia present with a delay of puberty, although they also show growth impairment (Gomes et al., 2017; Shah et al., 2021). Furthermore, many studies have shown that living in a high altitude environment with chronic hypoxia has effects on female reproduction, such as delayed age of menarche (Shaw et al., 2018), although there is a possibility that other concurrent factors, such as economic impoverishment and malnutrition, as well as behavioral and socio-cultural factors, also affect the determinants of female reproductive functions. These epidemiological data would suggest that anemia may be a non-endocrine mode of action for female pubertal onset, but a link between anemia and female pubertal onset is yet to be fully elucidated. On the other hand, anemia is one of the common toxicological endpoints for chemical safety assessment, however, little is known regarding the relation between anemia and female pubertal onset in experimental animals. In addition, hematology for assessing anemia is not a mandatory examination in the EPA or OECD TGs which include evaluation of VO. On the other hand, in OECD TG 443 (OECD, 2018), hematological examination is recommended in adult female rats after mating, pregnancy, parturition and nursing, but not in prepubertal females. It has been reported that some hematological parameters are changed after weaning, and the weaning should be an important turning point in hematopoiesis development in rats (Kojima et al., 1999). Hence, we hypothesized that compared to adult animals, prepubertal animals are more vulnerable to chemicals that cause anemia, and anemia during the prepubertal period affects the development of reproductive function.
The purpose of this study was to examine the effects of prepubertal anemia, independent of the effects of prepubertal growth retardation, on pubertal onset and the development of reproductive function in female rats. There are many forms of anemia, including iron deficiency anemia, aplastic anemia, hemolytic anemia, blood loss anemia, and megaloblastic anemia; anemia in our study was induced by two different methods, blood loss (phlebotomy) and hemolysis (Phenylhydrazine administration), in order to identify the differences between direct endocrine-modulating and non-endocrine effects. The findings of this study are expected to help us better understand the impact of prepubertal anemia on the development of female reproductive function in toxicity studies, differentiate endocrine from non-endocrine modes of action, and then provide more accurate evaluation of the potential for endocrine-disrupting effects in mammals.
Phenylhydrazine (PHZ) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan; Lot No. P3DCG-JP, purity; >98.0%). The test solution was prepared by dissolving PHZ in water for injection (Fuso Pharmaceutical Industries, Ltd., Osaka, Japan) at concentrations of 1 and 2 mg/mL on the days of administration.
AnimalsAll animal experiments were approved by the Institutional Animal Care and Use Committee of Sumitomo Chemical Co., Ltd., and performed in accordance with The Guide for the Care and Use of Laboratory Animals in the Environmental Health Science Laboratory of Sumitomo Chemical Co., Ltd. Crl:CD(SD) female rats at postnatal day 8 (PND 8) for Experiment I and PND 10 for Experiment II were purchased with foster dams from The Jackson Laboratory Japan, Inc. (Hino Breeding Center, Shiga, Japan). Each dam had 8 female pups. During the course of the study, the environmental conditions in the animal room were 22-26°C, 40-70% relative humidity, ventilated more than 10 times per hour, and illuminated by a 12-hr light (8:00-20:00) / 12-hr dark (20:00-8:00) cycle. All animals were provided ad libitum access to CRF-1 diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and filtered tap water. During the preweaning period, one dam and 8 litters were housed in each cage with nesting material (ALPHA-dri, Shepherd Specialty Papers, Inc., Massachusetts, USA). After weaning on PND 21, two pups were housed in each cage, and the dams were euthanized by carbon dioxide inhalation.
Experimental designIn Experiment I, on PND 15, all pups were weighed and the animals from each litter were randomly assigned to each group (control and two blood removal (BR) groups were 24 animals/group, and satellite control group was 23 animals/group) based on body weight to ensure that each group show similar mean body weight. To verify the appropriate volume and frequency of blood removal, a preliminary study was conducted prior to Experiment I, in which anemia was induced in female rats by repeated blood removal (1.5% of body weight) from the jugular vein twice a week from PND 18. As a result, juvenile animals recovered significantly faster from anemia induced by phlebotomy; therefore, in Experiment I, blood was drawn every 2 days from PND 16 to PND 28 at volumes of 1.0 and 1.5% of their body weight to continuously and stably induce anemia in the BR groups. In addition, to monitor hematological parameters, blood was also drawn on PND 35 and PND 57/58 in the BR groups. Blood samples for baseline data required to monitor the degree of anemia in the BR groups were taken from a satellite control group at the same time as the BR group to avoid the possible effects of blood sampling on sexual maturation in the control group, that is, the control group only subjected the same sampling manipulations, needle puncture, as the BR group, but no blood was removed. Instead, 23 animals assigned to the satellite control group were divided into three groups of seven or eight animals each, and blood samples for baseline data were collected on rotating days with two blood sampling intervals. An overview of the experimental design is shown in Fig. 1A. Clinical signs (once daily), body weight (PNDs 16, 18, 20, 21, 22, 24, 26, 28, 35, 42, 49, and 56), and food consumption (intervals PNDs 21-22, 22-24, 24-26, 26-28, 28-35, 35-42, 42-49, and 49-56) were monitored throughout the study.
Experimental designs for (A) Experiment I (blood removal) and (B) Experiment II (administration of phenylhydrazine). PND: postnatal day, VO: vaginal opening, PHZ: phenylhydrazine.
In Experiment II, on PND 17, pups were divided into 3 groups (PHZ 10 mg/kg/day, PHZ 20 mg/kg/day, and vehicle control) with 24 animals per group, in the same manner as Experiment I. A diagram of the experimental design is shown in Fig. 1B. The dosing volume was 10 mL/kg and PNZ was intraperitoneally administered to pups on PNDs 18 and 19. Water for injection was used as the vehicle and also administered to the control group. The dosage, method, and frequency of administration were determined based on the conditions of a prior research in which PHZ was administered to male albino rats (Gheith and El-Mahmoudy, 2018). To monitor hematological parameters, blood was drawn from the jugular vein periodically (PNDs 20, 24, 28, 35, and 42) and from the abdominal aorta on the day of necropsy (PND 55/56) from 8 animals/group. Clinical signs (once daily), body weight (PNDs 18, 19, 21, 24, 28, 35, 42, 49, and 55), and food consumption (intervals PNDs 21-24, 24-28, 28-35, 35-42, 42-49, and 49-55) were monitored throughout the study.
HematologyIn Experiment I, every 2 days from PND 16 to PND 28, blood samples were drawn from the jugular veins of rats in the two BR groups (volumes equivalent to 1.0 and 1.5% of body weight, respectively) and satellite control group (200 μL). In addition, to monitor hematological parameters after the completion of VO, 200 μL of blood was also drawn from the jugular vein on PND 35. On the day of necropsy (PND 57/58), blood samples were collected from the abdominal aorta under isoflurane (Isoflurane Inhalation solution, Mylan Inc., Tokyo, Japan) anesthesia without prior fasting. The blood samples were treated with EDTA-2K to prevent coagulation and subjected to automated counting using a multichannel blood cell counter XT-2000iV (Sysmex Corporation, Hyogo, Japan) to determine: Red blood cell count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Reticulocyte count Absolute (RET), and Reticulocyte count Percent (RET [%]). Samples that showed visible coagulation and items with measurement errors were excluded from the analysis.
In Experiment II, 200 μL of blood was drawn from the jugular vein periodically (PNDs 20, 24, 28, 35, and 42) from 8 animals/group. On the day of necropsy (PND 55/56), blood samples obtained from 8 animals/group were collected from the abdominal aorta under isoflurane (Isoflurane Inhalation solution, Mylan Inc.) anesthesia without prior fasting. The blood samples were treated and analyzed in the same manner as in Experiment I.
Vaginal opening examinationAll animals were examined daily for VO status beginning on PND28, and the age (postnatal days) and body weight at VO were recorded. If any animal showed incomplete opening (persistent threads or a “pin hole”) for greater than three days, the day of VO completion was the age at which incomplete opening was first observed. However, this criterion did not apply to any animal in our study.
PathologyOn the day of necropsy (PND 57/58 for Experiment I, PND 55/56 for Experiment II), all animals were weighed and euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia and all of the thoracic and abdominal organs/tissues were macroscopically examined. Ovaries and uterus were weighed after removal. Ovaries, uterus, and vagina were fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, sectioned at 3 μm thickness, stained with hematoxylin and eosin, and then examined by light microscopy.
Statistical analysisDifferences in the study results between the treatment groups and control group were statistically analyzed at significance levels of 5% and 1%. For all analyses, a two-tailed test was performed. Regarding hematological parameters in Experiment I, the treatment groups were compared with the satellite control group. For body weights, food consumption, age, and body weights at the day of VO completion, hematological parameters, and organ weights, Bartlett’s test was used to evaluate homogeneity of variance. If the variance was homogenous, Dunnett’s multiple comparison test was used. If the variance was not homogenous, Steel’s multiple comparison test was used. The Wilcoxon rank sum test was used for assessment of the graded histopathological findings and the Fisher exact probability test was used for assessment of the non-graded histopathological findings.
No animals showed blood removal-related clinical signs in the 1.0% and 1.5% BR groups (data not shown). Slight decrease in mean body weight (up to 7.2% lower than the control group) was observed in the 1.5% BR group throughout blood removal period, and the difference was statistically significant on PNDs 20 to 24 (Fig. 2A). However, after the period, body weights recovered and were comparable to those of the control group on PNDs 35, 42, 49 and 56. No effect on food consumption was observed throughout the study (Fig. 2B).
Body weight (A) and food consumption (B) in Experiment I, and body weight (C) and food consumption (D) in Experiment II. Data are presented as mean of each group (n=22-24 for body weight, n=12 for food consumption, respectively). *p < 0.05, **p < 0.01: Significantly different from the control group. PND: postnatal day, PHZ: phenylhydrazine.
Pale extremities were noted in the 20 mg/kg PHZ group after first administration, especially on PNDs 19 and 20 (Pallor was observed in 24 out of 24 animals on PND19, in 23 out of 24 animals on PND20, and in 1 out of 24 animals from PND21 to PND23). No treatment-related clinical signs were observed in the 10 mg/kg PHZ group. In the 20 mg/kg PHZ group, body weight was significantly lower than in the control group (6.9% to 14.4%) on PNDs 21 to 28 (Fig. 2C); after PND 28, body weight remained slightly decreased without statistical significance. Food consumption was also significantly lower than in the control group (6.3%) during PNDs 21 to 24 (Fig. 2D). In the 10 mg/kg PHZ group, body weight and food consumption were not affected throughout the study.
Hematology Experiment I: Blood removalSevere anemia was induced by repeated phlebotomy. Hematological parameter data are summarized in Fig. 3, Supplemental Fig. S1 and Table S1A. RBC and HCT were significantly lower in the 1.0% and 1.5% BR groups than in the control group (up to 16.3% and 26.2% for RBC and up to 18.5% and 23.3% for HCT, respectively) during PNDs 18 to 28 (Fig. 3A and B). Incidentally, the RBC was significantly higher in the 1.0% BR group than in the control group (3.7%) on PND 57 (Fig. 3A). HGB in the BR groups was significantly lower than that in the control group (up to 25.5% and 31.5%, respectively) during PNDs 18 to 28 for the 1.0% BR group and PNDs 18 to 35 for the 1.5% BR group (Fig. 3C). RET in the 1.0% and 1.5% BR groups was significantly higher than that in the control group (up to 61.2% and 71.1%, respectively) during PNDs 20 to 35 (Fig. 3D). RET (%) in the 1.0% and 1.5% BR groups was significantly higher than in the control group (up to 89.1% and 122.4%, respectively) during PNDs 18 to 35 (Fig. 3E). MCV was significantly higher than in the control group (up to 15.6%) during PNDs 22 to 28 for the 1.5% BR group and was significantly lower (3.6%) on PND 57 for the 1.0% BR group (Fig. 3F). MCH in the 1.0% BR group was significantly lower than that in the control group (up to 13.6%) on PND 20, 24, 26, 35 and 57, and MCH in the 1.5% BR group was significantly lower (up to 8.5%) on PND 20 and 24 (Fig. 3G). MCHC in the 1.0% and 1.5% BR groups was significantly lower than that in the control group (up to 11.3% and 14.4%, respectively) during PNDs 18 to 35 and PNDs 20 to 35, respectively (Fig. 3H).
Hematological parameters in Experiment I (Control group; n=7-8 [on the day of necropsy; n=23], 1.0% and 1.5% BR groups; n=15-23). *p < 0.05, **p < 0.01: Significantly different from the control group. PND: postnatal day.
It was confirmed that severe anemia was induced by PHZ administration. The hematological parameter data are shown in Fig. 4, Supplemental Fig. S2 and Table S1B. Compared to the control group, the 10 mg/kg and 20 mg/kg PHZ groups had significantly lower RBC (up to 16.5% and 33.6%, respectively) on PNDs 20 and 24 (Fig. 4A) and significantly lower HGB and HCT (19.5% and 34.5% for HGB, 11.8% and 25.1% for HCT, respectively) on PND 20. However, HGB in the 10 mg/kg group on PND 28 and HCT in the 10 mg/kg and 20 mg/kg PHZ groups on PNDs 24 and/or 28 were significantly higher than that in the control group (4.5% for HGB, up to 5.4% and 6.5% for HCT, respectively) (Fig. 4B and C). Compared to the control group, the 10 mg/kg and 20 mg/kg PHZ groups also had significantly higher RET (up to 90.9% and 113.2%, respectively) on PNDs 20 and/or 24 (Fig. 4D), significantly higher RET(%) (up to 127.0% and 152.2%, respectively) during PNDs 20 to 28 or 35 (Fig. 4E), significantly higher MCV (up to 23.7% and 25.9%, respectively) during PNDs 20 or 24 to 28 (Fig. 4F), significantly higher MCH (up to 15.1% and 18.3%, respectively) on PNDs 24 and/or 28 (Fig. 4G), and significantly lower MCHC (up to 8.9% and 12.1%, respectively) on PNDs 20 and 24 (Fig. 4H).
Hematological parameters in Experiment II (n=4-8). *p < 0.05, **p < 0.01: Significantly different from the control group. PND: postnatal day, PHZ: phenylhydrazine.
The age and body weight at VO, and organ weights are listed in Table 1. The age at VO in the 1.0% and 1.5% BR groups was significantly delayed by 1.8 days and 2.3 days, respectively (control: 32.5 ± 1.9 days, 1.0% BR: 34.3 ± 2.8 days, 1.5% BR: 34.8 ± 2.6 days). The body weight at VO was significantly higher in the 1.5% BR group than in the control group, but not statistically significantly higher in the 1.0% BR group (control: 120.7 ± 16.9 g, 1.0% BR: 127.9 ± 19.5 g, 1.5% BR: 133.1 ± 14.1 g).
Control | 1.0% blood removal | 1.5% blood removal | |||||||
---|---|---|---|---|---|---|---|---|---|
Age at VO (days) | 32.5 | ± | 1.9 | 34.3 | ± | 2.8* | 34.8 | ± | 2.6** |
Body weight at VO (g) | 120.7 | ± | 16.9 | 127.9 | ± | 19.5 | 133.1 | ± | 14.1* |
Ovaries (g) | 0.076 | ± | 0.011 | 0.080 | ± | 0.012 | 0.084 | ± | 0.014 |
Ovaries (g/100 g body weight) | 0.0354 | ± | 0.0037 | 0.0365 | ± | 0.0060 | 0.0374 | ± | 0.0047 |
Uterus (g) | 0.44 | ± | 0.14 | 0.43 | ± | 0.17 | 0.49 | ± | 0.23 |
Uterus (g/100 g body weight) | 0.21 | ± | 0.07 | 0.19 | ± | 0.08 | 0.22 | ± | 0.09 |
Body weight at necropsy (g) | 215.0 | ± | 25.2 | 220.3 | ± | 18.8 | 223.0 | ± | 15.9 |
Values are presented as mean ± S.D. (Control group; n=24, 1.0% BR group; n=23, 1.5% BR group; n=22)
*p < 0.05, **p < 0.01: Significantly different from the control group. VO: vaginal opening.
The age and body weight at VO, and organ weights are inventoried in Table 2. Age at VO in the 10 mg/kg and 20 mg/kg PHZ groups was delayed by 1.3 days and 2.5 days, respectively (control: 32.0 ± 0.8 days, PHZ 10 mg/kg: 33.3 ± 1.8 days, PHZ 20 mg/kg: 34.5 ± 4.9 days). The body weights at VO in the PHZ groups were higher than those in the control group (control: 124.5 ± 10.1 g, PHZ 10 mg/kg: 131.1 ± 11.8 g, PHZ 20 mg/kg: 131.7 ± 23.2 g).
Control | PHZ 10 mg/kg/day | PHZ 20 mg/kg/day | |||||||
---|---|---|---|---|---|---|---|---|---|
Age at VO (days) | 32.0 | ± | 0.8 | 33.3 | ± | 1.8** | 34.5 | ± | 4.9** |
Body weight at VO (g) | 124.5 | ± | 10.1 | 131.1 | ± | 11.8* | 131.7 | ± | 23.2 |
Ovaries (g) | 0.082 | ± | 0.013 | 0.076 | ± | 0.012 | 0.078 | ± | 0.012 |
Ovaries (g/100 g body weight) | 0.0359 | ± | 0.0051 | 0.0342 | ± | 0.0046 | 0.0360 | ± | 0.0055 |
Uterus (g) | 0.47 | ± | 0.14 | 0.49 | ± | 0.16 | 0.47 | ± | 0.18 |
Uterus (g/100 g body weight) | 0.21 | ± | 0.06 | 0.22 | ± | 0.08 | 0.21 | ± | 0.07 |
Body weight at necropsy (g) | 227.4 | ± | 19.0 | 222.9 | ± | 18.7 | 218.2 | ± | 20.0 |
Values are presented as mean ± S.D. (n=24)
*p < 0.05, **p < 0.01: Significantly different from the control group. VO: vaginal opening.
In Experiments I and II, the experimental treatment had no effect on the ovarian and uterine weights on PND 57/58 (Experiment I) and PND 55/56 (Experiment II), which was about three weeks after sexual maturation. In addition, no treatment-related histopathological changes were found in the vagina, ovaries, or uterus.
While several studies have suggested the relation between anemia or hypoxia and pubertal delay in humans, to the best of our knowledge, this is the first report to suggest that anemia causes a delay in pubertal onset in female rats. There are many forms of anemia, including iron deficiency anemia, aplastic anemia, hemolytic anemia, blood loss anemia, and megaloblastic anemia. In our study, anemia was induced by two different methods, blood loss (phlebotomy) and hemolysis (PHZ administration), in order to identify the differences between direct endocrine-modulating and non-endocrine effects. In Experiment I, 1.0% and 1.5% blood removal during PNDs 18-28 induced 20-30% decreases in hemoglobin and this anemic condition was stably maintained during the phlebotomy treatment. After the completion of treatment, hemoglobin level remained slightly decreased on PND 35, however, it fully recovered to the control level at the end of the study (PND 55). Anemia induced by repeated blood removal resulted in delayed VO and an increased body weight at the time of VO in the 1.0% and 1.5% BR groups following a slight and temporary low body weight during the initial phase of the anemia-inducing 1.5% blood removal. However, body weight recovered by the time of VO initiation (PND29), and there were no statistical differences in body weight after recovery between the 1.5% BR and control groups. Laws et al. (2007) conducted a feed restriction study in rats and reported that VO is insensitive to changes in growth, as decreases in body weight of approximately 20% did not significantly affect the timing of VO in rats. In addition, Carney et al. (2004) reported that the timing of pubertal onset was not affected in animals whose body weight was 6–19% lower than controls, but that the timing of VO was delayed in animals whose body weight was reduced by 30–43% compared to controls, and these animals had lower body weight at VO. In contrast, our study showed that the animals with slight and temporary low body weight (1.5% BR group; up to 7.2% lower than control between PND 20 and 24) due to blood removal anemia exhibited VO delay accompanied by a higher body weight at VO (1.5% BR group; 10.3%). Thus, the delayed VO observed in the BR groups was not attributable to growth impairment as generally known. Regarding reproductive organs, anemia had no effects on the weights of the uterus and ovaries, or the histopathology of uterus, ovaries, and vagina. Thus, Experiment I showed that prepubertal anemia causes delayed pubertal onset, but does not affect the reproductive organs of female rats after puberty, nor does it impact reproductive function in adulthood.
In Experiment II, we investigated whether anemia induced by chemical administration leads to delayed pubertal onset. PHZ, which was selected as the test substance for this experiment, is known to induce reactive oxygen species formation, peroxidation of lipids and oxidative degradation of spectrin in the membrane skeleton. The hemolytic anemia caused by this compound is thought to be due to oxidative alterations in red blood cell proteins (Berger, 2007). Ten or 20 mg/kg of PHZ was administered on PNDs 18 and 19. Although anemic changes, such as an approximately 20-35% reduction in hemoglobin, were observed on PND 20, these changes were almost completely abrogated on PND 24 in the 10 and 20 mg/kg groups. In contrast, low body weights (14.4% lower than control on PND 21) were observed in the 20 mg/kg PHZ group and continued until the day of necropsy (4.3% lower than control on PND 55). The delayed VO and higher body weight at VO were observed in the 10 and 20 mg/kg PHZ groups. As with Experiment I, the results of Experiment II (i.e., no effects on body weight in the 10 mg/kg group, slight effects on body weight in the 20 mg/kg group [on PND28, it recovered to -6.9% compared to the control group], and higher body weight at VO than that of the control group) suggested that delayed VO is not attributable to growth impairment. Weights and the histopathology of female reproductive organs were unchanged by PHZ administration. It is generally agreed that EDs can give rise to histological changes in the vagina, uterus, and ovary (OECD, 2009). Besides, administration of the compounds known for having endocrine-modulating effects also decrease ovary and uterus weights and cause VO delay in female rats (Ashby et al., 2002; Kim et al., 2002; Marty et al., 1999). Taken together, these observations rule out a direct relationship between endocrine mode of action and PHZ-induced delayed VO. Experiment II demonstrated that the administration of an anemia-inducing chemical could also cause delay in pubertal onset without producing changes in weights and the histopathology of female reproductive organs.
Based on the results of Experiments I and II, it was suggested that transient anemia during the developmental period can cause a delay in VO without any serious reproductive impairment. Of note, Grill et al. (2001) demonstrated that iron deficiency anemia after weaning did not affect the onset of sexual maturity in female rats, so anemia, especially before weaning, may be involved in delay in pubertal onset. In humans, it is generally known that the major risk groups for anemia (especially iron deficiency anemia) are young children, adolescent females who grow rapidly and in addition have their first menstruations with iron losses, women of reproductive age who lose iron with their menstrual periods, pregnant women with an increased need for iron, and lactating women (Milman, 2011; WHO, 2011). Hence, there is a possibility that pubertal female rats show more severe effects of anemia, potentially causing a delay in VO, compared to adult rats. Therefore, monitoring blood parameters during the growth period could be useful in understanding the mechanisms behind VO delay. Having found the relation between anemia and pubertal onset in this study, the remaining question to discuss is what mechanism underlies anemia-related delay in pubertal onset. Puberty is a transition period between childhood and adulthood characterized by secondary sexual development, genital maturation, and attainment of reproductive ability, and these are controlled by the HPG axis (Abreu and Kaiser, 2016). Especially, the pubertal onset in mammals is triggered by increase in gonadotropin releasing hormone (GnRH) secretion (Sisk and Zehr, 2005; Uenoyama et al., 2019). It is well known that GnRH secretion is suppressed by stress (Rivier and Rivest, 1991; Li et al., 2010), while stress-induced reproductive disorders are usually reversible in humans (Perkins et al., 2001; Falsetti et al., 2002). The anemia-induced VO delay observed in this study is considered to be a reversible change, as no effects on the reproductive organs were noted after recovery from anemia. Therefore, it is possible that this occurs through a mechanism similar to that of reproductive effects induced by stress. Anemia is a physiological cause of tissue hypoxia due to the reduced ability of HGB to deliver enough oxygen to tissues. It is also demonstrated that intermittent hypoxia increases corticosterone production in rats (Hwang et al., 2017) and that high altitude hypoxia enhances corticotropin-releasing hormone (CRH) release in rats (Chen et al., 2004). Furthermore, there have been reports indicating that continuous administration of CRH to female rats on PND 28 results in a delay in VO (Kinsey-Jones et al., 2010). A recent study demonstrated that intermittent exposure to hypoxia increased the levels of rfrp3 mRNA, a negative regulator of GnRH in rats (Terrizzi et al., 2021). From this evidence, it can be hypothesized that anemia induces a state of hypoxia in the body, leading to a stress response (increased production of corticosterone and increased release of CRH). This, in turn, may elevate the gene expression levels of rfrp3, thereby suppressing the release of GnRH necessary for the onset of puberty, ultimately causing a delay in pubertal onset. In the present study, anemia, which is strongly believed to impose burdens such as hypoxic stress on the organism, was induced in juvenile rats in two different forms: blood loss anemia and hemolytic anemia, to a degree that did not significantly affect their mortality or body weight. The association between anemia and the onset of puberty was investigated. As a result, it was observed that under conditions where blood loss anemia and hemolytic anemia caused a maximum decrease in RBC by 26.2% and 33.6%, respectively, and a maximum decrease in HGB by 31.5% and 34.5%, respectively, anemia was shown to be associated with a delay in VO, which may be accompanied by an increase in body weight at VO. However, the mechanism of anemia-induced delay in pubertal onset underlying these causes and results requires further research to verify the above hypothesis.
In conclusion, we determined that transient anemia during the prepubertal period can delay pubertal onset without affecting reproductive function (weight and histopathology of reproductive organs) in female rats. In toxicity studies, when pubertal onset is altered without any effects on reproductive organs, hematological examination before sexual maturation might help determine whether the delay is caused via endocrine mode of action or is secondary to anemia.
Authors would like to thank Kenta Minami, Misaki Matsumoto, Masahiro Izumi, Maki Yamaguchi, Keiko Tanaka for technical support. We also would like to thank ASCA Corporation (https://www.asca-co.com/english_site/index.html) that provided proofreading services funded by Sumitomo Chemical Co., Ltd.
Conflict of interestThe authors declare that there is no conflict of interest.