Serum Scavenging Capacity and Folliculogenesis Impact following Flaxseed Consumption in the First-Generation Mice Pups

Flaxseed is a source of antioxidants utilized for female infertility treatment in traditional medicine. This study investigated the effects of flax hydroalcoholic extract and flaxseeds during prenatal and postnatal (PND) periods on folliculogenesis and serum total antioxidant capacity (TAC). Pregnant NMRI mice received 500 and 1000 mg/kg of flax extract (LE) and the same doses of flaxseed (LS). Female pups received the same regimen for 56 days. The body, ovarian morphometry, follicle development, and TAC levels were evaluated. The ovarian weight significantly increased in the LE1000 group compared to the LS500 group. The LE500 group had a considerably lower number of primary and antral follicles compared to the CTL and LS1000 groups. The number of antral follicles significantly increased in the LE1000 group compared to the LS500 and LE500 groups. The number of preovulatory follicles was higher in the LE1000 group. A significant increase in the TAC levels was detected in the LS500, LS1000, and LE1000 groups. LE showed a dose-dependent protective effect on the folliculogenesis in F1, which is more evident with the dosage of 1000 mg/kg. This could be related to the strongest antioxidant property of LE1000, as shown by the highest levels of TAC.


Introduction
e ovary is a reservoir of follicles that, with the secretion of hormones influenced by the hypothalamic-pituitary axis, periodically induces follicular waves. Progression of small primordial follicles into large preovulatory follicles and, eventually, ovulation [1] can be affected by internal and external environmental factors, including free radicals [2]. During folliculogenesis, most of the follicles undergo atresia by apoptosis [3]. Antioxidants prevent follicular atresia by scavenging environmental free radicals, thereby improving folliculogenesis, maintaining effective ovulation [4], and women's health and fertility. erefore, the use of antioxidant supplementation in assisted reproductive technologies (ARTs) is increasing [5].
Due to the expensive and challenging access to modern medicine, especially in some areas, herbal medicine is expanding [6]. Linum usitatissimum, known as flaxseed or linseed, is one of the traditional plants with a high concentration of biologically active components such as α-linolenic acid, lignans, unique proteins, and also potent antioxidants including phenolic acids and flavonoids [7]. e efficiency of flaxseed on human health is attributed to its high content of antioxidants. e effect of flaxseed on many body organs, including the reproductive system, has been demonstrated by many researchers. Following flaxseed consumption, a significant increase in the mouse body weight and size of the uterus and ovary as the promotion of bovine ovarian cycle, blood progesterone level, ovarian follicle and corpus luteum growth, and developmental competence of oocytes was reported [8]. Evidence suggests a dose-dependent effect of the flaxseed, where a 5% dose delayed puberty onset and increased the ovarian weight and a 10% dose led to early puberty onset, ovarian weight gain, serum estradiol elevation, and lengthened estrous cycles in female rats [9].
In contrast to the above data, we previously reported a detrimental effect of flaxseed on the murine ovarian follicular reserve, with increased serum estrogen levels and apoptotic gene expression [10]. erefore, even if the effects of maternal diet during pregnancy and lactation on the proper development of the genital system and ovarian follicular reserve are well known [11], the present study aimed to evaluate the total antioxidant capacity of two doses of flaxseed (500 and 1000 mg/kg) on ovarian folliculogenesis in first-generation mice pups.

Preparation of Hydroalcoholic Extract of Flaxseed.
Fresh flaxseeds (Linum usitatissimum) were purchased from an herbal medicine store in Kerman, Iran. e plant was identified by a botanist of the Herbarium of Pharmacognosy Department, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran (voucher number: KF1628). Extraction was performed using a warm maceration method. Initially, a known amount (100 g) of the powder was prepared from the flaxseeds and then passed through a sieve. e powder was soaked with 80% ethanol for 72 h. Subsequently, the extract was concentrated on a rotary evaporator under vacuum conditions, dried in an oven at 40°C for 48 h, and stored at −20°C until use [12].

Animal
Procedures. Forty female and twenty male NMRI mice (weighing about 25-30 g; 6-8 weeks old) were purchased from the animal house affiliated with Afzalipour School of Medicine, Kerman, Iran (approval number: IR.KMU.REC.1397.148). Animals were kept under controlled conditions (lighting, humidity, and temperature) with standard mice chow and water ad libitum.
In each cage, one male and two female mice were placed for mating overnight. e female mice were monitored daily for vaginal plaque formation. Pregnant mice were randomly divided into five groups: control (CTL) and four experimental groups. Each group included four mice. Animals in the control group received rodent food. e mice in the experimental groups received 500 mg/kg/day [13] or 1000 mg/kg/day [14] of flax hydroalcoholic extract (LE) or flaxseed (LS) (LE500, LS500, LE1000, and LS1000, respectively). Extracts and seeds were added to the diet food of pregnant mothers until the end of lactation and then continued for the female offspring mice until postnatal day 56 (PND56) (n � 7) [15]. e large and small diameters were measured by using a digital caliper. e collected right ovaries were fixed in 10% formalin. e fixed tissue samples were dehydrated in ascending concentrations of alcohol and embedded in paraffin. Slices of tissue with 5 µm thickness were prepared and stained with hematoxylin and eosin and then evaluated under a light microscope (Olympus IX51, Japan) [16].

Histological Classification of Ovarian Follicles, Follicle
Count, and Stages. To count the number of follicles in the ovarian tissue, the prepared blocks were serially sliced, and for every ten serial sections, one slice was collected (8 sections for each sample, n � 7). e follicles were classified as (1) primordial if they contained an oocyte surrounded by one layer of flatted follicular cells; (2) primary if a relatively larger oocyte was surrounded by one layer of cuboidal follicular cells; (3) secondary if they contained two or three layers of cuboidal granulosa cells with no antral space; (4) preantral if they were enclosed in more than four layers of granulosa cells with one or more independent spaces; (5) antral if they contained multiple layers of cuboidal granulosa cells with a specified antral space; (6) preovulatory if an antral space divided the mural granulosa cells from the oocyte and its cumulus oophorus; (7) atretic if they contained fragmented oocyte, ruptured plasma membrane, and dropped granulosa cells into the antrum. Corpora lutea, forming large steroid-producing organs, were also counted [17].

Assessment of Serum Total Antioxidant Capacity (TAC)
Level.
e animals were anaesthetized with ketamine (5-10 mg/kg) and xylazine (5 mg/kg). eir blood samples were collected directly from the left ventricle of the heart and centrifuged at 2000-2500 rpm for 10 minutes. Serum samples were stored at −20°C until assay for the TAC level. e serum TAC level was measured by the ferric ion reducing antioxidant power (FRAP) method according to the TAC commercial kit (Cat no. NS-15012, Navand Salamat Company, Urmia, Iran) [18].

Statistical Analysis.
e normality of the distribution of continuous data was tested using the Kolmogorov-Smirnov test. In the comparison of continuous variables with more than two independent groups, the ANOVA test was used when the normal distribution condition was met, with the Tukey test as a post hoc. e Kruskal-Wallis test was used for nonparametric variables. Data were expressed as mean ± SEM. A P value ≤0.05 was considered statistically significant. Table 1, the body weight and ovarian size of mice that were daily treated with flaxseed and flax extract at different doses (500 and 1000 mg/kg) did not show significant changes. However, when comparing the ovarian weights among the groups, an increase of this parameter was observed in the LE1000 group compared to other groups, although this reached a significant difference when compared to the LS500 group (4.8 ± 0.5 vs. 3.7 ± 0.67, respectively, P < 0.05) ( Table 1).

Morphological and Morphometric Study of the Ovaries.
In controls, the ovarian cortex was densely populated by numerous primary, secondary, and tertiary (preantral) follicles interspersed among numerous big corpora lutea and corpora hemorrhagica (as evidenced by cells intensely stained in red). Small atretic follicles were also seen. e medulla had a highly vascularized and spongy stroma (Figures 1(a) and 1(b)). Ovaries from the experimental groups (LS500, LS1000, LE500, and LE1000) are rich in follicles at different developmental stages and corpora lutea. Numerous antral follicles with visible cumulus-oocyte complex are detectable (Figures 1(c)-1(j)). e medulla had a more compact aspect, probably connecting the cutting sections. Small atretic follicles were occasionally detected in all groups. e morphometric analysis evidenced that the number of primordial, secondary, preantral, and atretic follicles in the offspring ovaries did not show significant differences among all the experimental groups ( Figure 2), even if the lowest number of primordial follicles was found in the LE500 group (53.57 ± 12.87) and the highest in the LE1000 group (78.14 ± 7.91) (P > 0.05) (Figure 2(a)). e number of atretic follicles was contrariwise in these two groups (46.85 ± 5.38 and 26.85 ± 3.15 in the LE500 and LE1000 groups, respectively) ( Figure 2(d)). Treatment with the flax hydroalcoholic extract at a dose of 500 mg/kg (LE500) (31 ± 3.06) caused a significant reduction in the number of primary follicles in the ovarian parenchyma compared to the LS1000 group (43.28 ± 4.01) (P < 0.05) (Figure 2(a)).

Discussion
Among herbs, those with antioxidant properties have attracted much attention worldwide. Diet is one way to get antioxidants. Dietary antioxidants can fight free radicals [19]. Recently, flaxseed has been shown to have therapeutic potential with anticancer, antiviral, bactericidal, anti-inflammatory, and antioxidant effects [20][21][22]. is medicinal herb contains numerous amounts of phenolic compounds with high antioxidant activities making it a potential source for treating degenerative diseases [23]. Flaxseed antioxidant effects on various organs such as the liver and kidney in rats exposed to thioacetamide have been reported. Administration of flaxseed could induce improvement in the liver function test, antioxidant status, and lipid profile. Also, this herb could reduce kidney necrosis, degeneration, and inflammatory cell infiltration [19,24]. In vitro, antioxidant activities of flaxseed extracted with different methods, including methanolic and butanolic extracts, in addition to those acquired using petroleum ether, benzene, and ethyl acetate, have been evaluated. e butanolic extract of flaxseed showed the highest levels of DPPH (α,α-diphenylβ-picryl hydroxyl) radical scavenging activity. In contrast, its petroleum ether and ethyl acetate extracts exhibited a significantly increased level of hydrogen peroxide scavenging activity. An increase in the ferric reducing antioxidant power (FRAP) was observed in its methanolic extract [25]. Since different extraction methods exhibited different scavenging activity levels, this study aimed to assess which, from the seed itself or the hydroalcoholic extract, has a higher ferric reducing antioxidant power (FRAP). e results of the study showed a significant difference between them based on FRAP; a daily intake of hydroalcoholic extract of flaxseed during intrauterine life, lactating, and then until puberty caused a significantly higher level of FRAP than the consumption of flaxseed did. Moreover, as mentioned earlier, this effect was dose-dependent, with the 1000 mg/kg dose showing the greatest effect compared to the 500 mg/kg dose. Previous reports have determined that the hydroalcoholic extract of flaxseed (200 mg/kg for 7 weeks) could improve the side effects of PCOS on the hormonal profile and histomorphometric features of the ovaries [26]. Others evidenced that when the bovine pregnant basal diet is supplemented with flaxseed (8% of the basal diet), there is an amelioration of the ovarian cycle, the number of ovarian follicles, and corpora lutea, as well as the oocyte developmental competence; differently, a higher dose of flaxseed (>8%) induced negative effects [27][28][29].
In line with the data available, we observed different effects of the extract and seed based on the exposure dose. According to histological data and TAC results, the dose of 1000 mg/kg of flax was more effective on folliculogenesis as compared to 500 mg/kg of flax in both the two forms of the extract and seed, as evidenced by a higher number of antral and preovulatory follicles. Among the tested treatments, animals receiving 1000 mg/kg of hydroalcoholic extract of flaxseed showed the highest increase in the number of ovarian follicles in comparison with the other treatment groups. Moreover, an increase in the number of corpora lutea, with respect to the LE500 group, was also observed, thus evidencing an improvement in ovarian physiology. Among the treatments, the lower dose (500 mg/kg) did not show significant differences with respect to the control and  other groups, except for a significant reduction of primary follicles and corpora lutea in the form of extract (LE500). In our recent study, it was observed that the dietary intake of 500 mg/kg of hydroalcoholic extract of flaxseed may affect ovarian reserve in the first-generation mouse pups by decreasing the number of primordial and growing follicles in addition to decreasing body and ovary weight. We also evaluated the expression of the anti-Müllerian (AMH) hormone as an ovarian reserve marker in the ovaries of adult female pups. Our data showed that flaxseed at a dosage of 500 mg/kg could decrease the AMH expression [30]. ese data were probably explained by the antiestrogenic property of the hydroalcoholic extract of flaxseed at the 500 mg/kg dosage [10].
In this study, we also reported that flaxseed at doses of 500 and 1000 mg/kg and also the hydroalcoholic extract of flaxseed only at 1000 mg/kg dosage can increase body and ovarian weight. is effect was not observed at 500 mg/kg of hydroalcoholic extract of flaxseed. In other studies, it has been suggested that body weight gain following flax intake is probably due to the estrogenic properties of this plant. Also, an increase in the number of growing follicles induced by flaxseed in different treated groups, except for the 500 mg/kg hydroalcoholic extract group, could lead to an increase in ovarian weight. is result is in agreement with previous observations that have reported that dietary flaxseed (10%) could cause a lower birth weight of mouse pups and female offspring with shorter anogenital distance, greater relative uterine, and ovarian weights, as well as earlier age, lower body weight at puberty, and a lengthened estrous cycle [9]. Another study determined that 5% dietary flaxseed led to a delay in the onset of puberty in females, whereas 10% dietary flaxseed could cause an earlier puberty onset, higher relative ovarian weight, and also a lengthened estrous cycle [9]. e novel data presented here about the improvement of folliculogenesis and the total antioxidant capacity (TAC) connected to the consumption of different forms (extract and seed) and diverse doses (500 and 1000 mg/kg) of flaxseed are the basis for future studies to confirm the potential applications of flaxseed supplementation to enhance the reproductive performance in physiological and pathological conditions. Data Availability e research data used to support the findings of this study are available. Request for access to these data should be made to Ezzatabadipour Massood (Corresponding author, m_eatabadi@kmu.ac.ir, ezzatabadim@gmail.com).

Ethical Approval
Animal studies were carried out according to the protocols approved by the Institutional Ethics Committee of Kerman

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Massood Ezzatabadipour, Tahereh Haghpanah, and Maria Grazia Palmerini designed the study and analyzed the data. Fahimeh Pourjafari performed the experiment and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.